Citations for Dr Belmonte

I posted a comment at The Autism Crisis which referred to a recent talk given by Dr Matthew Belmonte. Ms Dawson felt the comments were off topic and so I have moved the discussion to my blog.

I am still in the process of trying to understand the comment that Dr Belmonte left, in particular the concept of the veridical percept. When I do understand it I will post a reply.

List of citations for Dr Belmonte. They are listed in approximate order of relevance. Also posted at

http://daedalus2u.blogspot.com/index.html

I have added some commentary. I have tried to use as few citations as possible. This is more to show that NO physiology is sufficiently complex to support the many complex physiological effects observed in autism and ASDs.

Thomas DD, Ridnour LA, Isenberg JS, Flores-Santana W, Switzer CH, Donzelli S, Hussain P, Vecoli C, Paolocci N, Ambs S, Colton CA, Harris CC, Roberts DD, Wink DA. The chemical biology of nitric oxide: implications in cellular signaling. Free Radic Biol Med. 2008 Jul 1;45(1):18-31. PubMed link

This is quite a good review paper on signaling by NO. Dave Wink is a senior researcher in the field, he just chaired the Gordon Conference on NO last week. Most of the higher concentration effects are relevant for metabolic physiology (and his specialty cancer), not so much neurological effects. The levels that are most relevant for neuronal effects are in the 1-30 nM/L range (0.03 to 0.9 ppb by weight). That is the basal level is around 1 nM/L up to a few times the EC50 of sGC in cells (~10 nM/L). There are no techniques to measure those levels in vivo on the time and length scales that are known to be important. We know that levels can't be that high (10 nM/L) long term in the endothelium because if they were, there would be systemic hypotension (as in septic shock). This is the first review that really puts together all the different concentration scales that NO is important at.

Because NO sources and sinks are small, and NO is highly diffusible, there are large gradients in NO concentration. Because NO is ~10x more soluble in isotropic lipid, lipid membranes have a large effect on the diffusion. Many lipid membranes are not isotropic. What effect that has on NO diffusion has not been investigated.

Sources are important and sinks are important. Superoxide is an important sink (depending on its concentration). I have a slight quibble (actually very slight). They mention that SOD levels are high enough that superoxide shouldn't affect NO levels in the nM/L level. If everything were uniform and isotropic I would agree, but SOD levels are not isotropic, and superoxide is usually generated vectorally to the inside of vesicles, mitochondria and microsomes. Superoxide is confined by lipid membranes so levels can (in theory) get high inside of those vesicles. Superoxide is an anion, so it will be affected by local electric fields and be held up against the inside of the lipid membrane that is confining it. The SOD would be floating around in the bulk. NO is ~10x more soluble in the lipid membrane, so depending on the precise locations of the NO source, the superoxide source, the SOD, the local electric field and the lipid volumes in the vicinity the effects could be complex.

The major take home message is that because NO can diffuse everywhere, and each NO sensor only senses the sum of NO from all sources, all sources and all sinks matter, including the basal level. The basal level is more important the lower the levels are, the lowest levels mediate things acutely through sGC, as in the brain. There can be effects from lower NO levels which don't activate sGC, for example when NO combines with superoxide and forms peroxynitrite and nitrates proteins. These proteins can accumulate NOx over long periods and integrate a NO/ROS signal over that time. I think this is important in regulating mitochondria number in neurons where mitochondria biogenesis (regulated by NO) must be matched to mitochondria need for the entire neuron (which can vary by 3 or more orders of magnitude depending on axon length). My hypothesis is that mitochondria accumulate nitrated proteins over their metabolic lifetime and that integrates the metabolic load on those mitochondria. When those mitochondria are recycled via autophagy, the NOx is released and produces a NO signal that triggers the biogenesis of the appropriate number of mitochondria to support the needed metabolic load.

Garthwaite J. Concepts of neural nitric oxide-mediated transmission. Eur J Neurosci. 2008 Jun;27(11):2783-802. PubMed link

This is a good review on neuronal signaling via NO, pointing out that NO does cause both LTP and LTD and that the quantities needed for activity are very small in the nM/L range. That is the same level that activates sGC and causes vasodilation.

Many neuropeptides and growth factors, when they are endocytosed by their receptor, activate nitric oxide synthase, and the receptor/NOS complex is conveyed back to the cell body while it is producing NO. I suspect that the NO that the endocytosed receptor complex produces is important in the LTP produced by many of these growth factors as that NO from inside the axon adds to the local NO on the outside. Myelin is transparent to NO, but the alternating layers of lipid and aqueous would certainly modulate its diffusion in complex ways.

NOS binds to the serotonin transporter which couples NO release to serotonin uptake.

Wang S, Paton JF, Kasparov S. Differential sensitivity of excitatory and inhibitory synaptic transmission to modulation by nitric oxide in rat nucleus tractus solitarii. Exp Physiol. 2007 Mar;92(2):371-82. PubMed link

This paper shows changes in membrane potential following exposure to ~ nM/L levels of NO. My conceptualization is that the NO release that causes the vasodilation observed in fMRI BOLD also changes the sensitivity of neurons in that affected volume element to be triggered.

It has been shown (Goense & Logothetis, 2008) that there can be vasodilation without neuronal activation. But we already knew that because even in a volume element that is observed to be highly activated, not every neuron is in a state of discharge. The NO primes the neurons to discharge, but which ones do discharge depends on which ones the action potentials propagate into.

Krause DN, Duckles SP, Pelligrino DA. Influence of sex steroid hormones on cerebrovascular function. J Appl Physiol. 2006 Oct;101(4):1252-61. PubMed link

This paper is quite interesting because it relates to the "extreme male brain" hypothesis of ASDs. Testosterone decreases NO levels and estrogen increases NO levels. This paper relates that primarily to blood flow in adults, I think the effects on fetal neurodevelopment in utero would be important. NO inhibits Leydig cell production of testosterone, so low NO causes high testosterone levels. High testosterone levels cause a more hirsute phenotype, which expands the niche for the bacteria I am studying, causing increased NO/NOx production by them, exerting feedback control on androgen synthesis. My hypothesis is that the association of in utero testosterone levels with ASDs and ASD-like behaviors is real, but that both of them are secondary to low NO status. Many conditions associated with hyperandrogenic effects in adults are also associated with low NO, as in polycystic ovarian syndrome. Stress causes low NO and stress tends to cause high androgen levels.

I think a better characterization of the autism would be as the "extremely stressed brain" because most of the differences are not gender specific.

In utero levels of testosterone and pathways by which that can affect neurodevelopment lead into the next papers, on epigenetic programming of adult physiology.

Racasan S, Braam B, Koomans HA, Joles JA. Programming blood pressure in adult SHR by shifting perinatal balance of NO and reactive oxygen species toward NO: the inverted Barker phenomenon. Am J Physiol Renal Physiol. 2005 Apr;288(4):F626-36. Epub 2004 Nov 16. Review. PubMed link

This paper relates to the epigenetic programming of adult physiology by NO/ROS balance in utero. The mechanism(s) for epigenetic programming by NO/ROS balance are not fully understood. The mechanisms for epigenetic modification (changes to DNA methylation for example) are mechanisms that could pertain in essentially every cell type and in essentially every tissue compartment. If NO/ROS mediated epigenetic programming modified adult function in some organs it would be surprising if that same mechanism(s) did not pertain in many organs in that the substrate (genomic DNA) is the same.

Stress in utero in animals is known to change adult brain size (some types of stress increase it) and adult behaviors. Stress in utero is also known to increase the incidence of ASDs. People with ASDs are exquisitely sensitive to stress. Chronic stress does increase acute sensitivity to stress even after the chronic stress is removed, even in adults.

MeCP2 deletion is known to cause autism-like symptoms (Rett Syndrome). MeCP2 codes for the gene that allows for differential expression of methylated DNA, DNA that is methylated as a consequence of the epigenetic programming of those cells. Presumably if aberrant readout of methylated DNA can "cause" autism-like symptoms, then sufficient changes to methylation of DNA at any step along the way could conceivably do similar things.

I don't think of Rett Syndrome as "autism", rather I see it as "autism-like". I see "autism" and the ASDs as being due to neuroanatomy and epigenetic programming of that neuroanatomy most of which occurs in utero (due to low NO (my hypothesis)). I see "autism-like" as being due to acute decreases in NO brought about by a variety of mechanisms. An important mechanism is epigenetic programming of the brain in utero to be in a lower NO state. Social isolation does this also, so can metabolic stress as in mitochondrial disorders (which generate lots of superoxide) and things like Rett Syndrome where there is metabolic stress due to tissue compartments being mosaic (with different MeCP2 status) resulting in cells not working "in sync". I think that raising NO levels can help "autism-like" symptoms, changing the neuroanatomy is (I think) not possible. I think that raising NO levels soon enough in neurodevelopment can switch an individual from development on an ASD trajectory to an NT trajectory (to some extent).

I think the acute resolution of autism symptoms with fever (Zimmerman 2007) is due to increased NO from iNOS. I have an extensive blog about what I see as the physiology behind it.

Godfrey KM, Barker DJ. Fetal nutrition and adult disease. Am J Clin Nutr. 2000 May;71(5 Suppl):1344S-52S. Review. PubMed link

See table 1, a list of 10 tissue compartments where there is evidence of in utero programming in humans. The physiology of many adult human organs is known to be epigenetically programmed in utero. It would be beyond surprising if the most important organ, the brain, was not.

Blogging milestone; 10,000 visits

I passed the 10,000 visit mark yesterday and have implemented some changes. I will now be accepting anonymous comments, so those of you who were worried that my black helicopters would track you down can now post anonymously.

I didn't want anonymous comments at first because I blog a lot about autism and there are some very nasty characters out there spreading disinformation on mercury, vaccines and such. I have enough material now in my archive that people can know where I am coming from and the level of discourse expected on this blog. I welcome people who disagree with me, but you have to back up your disagreement with facts and logic.

I welcome all comments that add to the discussion, or which ask questions. If I can answer them I will try to do so. All comments get emailed to me, even the ones on posts that are quite old. I am happy to answer question on old posts too. It may be old to me, but not old to anyone else.

Theory of Mind vs. Theory of Reality: The tradeoff along the Autism Spectrum

In any great organization it is far, far safer to be wrong with the majority than to be right alone. -- John Kenneth Galbraith

If the only tool you have is a hammer, you tend to see every problem as a nail.
-- Abraham Maslow

In the land of the blind, the one-eyed man is king.

How autism provides resistance to the delusional thinking of groupthink (aka drinking the Kool-Aid).

When your primary competition is with other people, life is a zero sum game. When your primary competition is with reality, there are no limits.

The cause(s) of Autism Spectrum Disorders (ASDs) remain unknown. The complex genetic disorder hypothesis posits ASDs are an emergent disorder of multiple genes, perhaps dozens or more. How a common disorder of so many genes could evolve has not been suggested. One hypothesis is that genes associated with ASDs did confer some advantage resulting in their selection in the past, but are now detrimental. I suggest ASDs are not disorders at all, but normal (even human defining) developmental responses, particularly to stress in utero and early childhood. This fundamental human neurodevelopmental paradigm programs the brain and behaviors mediated by that brain so as to optimize survival and reproduction depending on maternal and infant stress; invoking abilities to understand, interact with and manipulate other humans when times are good, and abilities to understand, interact with and manipulate the environment via tool production when times are hard. It is hypothesized that this trade-off between Theory of Mind (ToM) and Theory of Reality (ToR) is the quintessential trade-off along the Autism Spectrum. Implications for interaction difficulties between individuals with ASDs and Neurologically Typical individuals (NTs) are discussed. Prevention and treatment are also discussed.

Background ASDs

The Autism Spectrum Disorders (ASDs) are defined and diagnosed by difficulties in communication, social interaction and by repetitive behaviors. In ASDs, there is a high (but not absolute) concordance between monozygous twins, [1] moderate concordance between dizygous twins, [2], [3] and lesser concordance between siblings. With no generally accepted environmental cause, ASDs are thought to be primarily genetic in origin with associations of perhaps 135 genes.[4] No doubt the complex effects on brain structure and behavior observed in ASDs are not mediated via a single pathway, but calls to abandon a search for a single explanation are premature. [5]

A number of single mutations have been associated with multiple cases of autism-like symptoms. I call these "autism-like" because it is not clear if the cause and sequelae of these autism-like syndromes are identical to or even similar to the common cases of autism and ASDs (which remain unknown). A good example is Rett Syndrome which is known to be caused by a loss in the MeCP2 gene which is on the X chromosome. Females have 2 copies of the X chromosome, one of which is silenced. Active MeCP2 in some cells rescues the organisms from the fatal loss of MeCP2 which afflicts males (who have only one X chromosome). RS females develop seemingly normally until 6-18 months when they develop the characteristic RS phenotype which includes autism-like symptoms, but is also characterized by non-autistic symptoms of small head size, breathing abnormalities, vascular abnormalities, scoliosis, growth retardation and others. Because the effects of MeCP2 deletion are mediated through aberrant transcription of methylated DNA, then aberrant transcription of methylated DNA is sufficient to lead to autism-like symptoms. Perhaps the symptoms of common ASDs are also caused by DNA methylation, or perhaps via a shared final common pathway triggered by aberrant DNA methylation.

Nitric oxide is a pleiotropic signaling molecule used in thousands of metabolic pathways where it regulates, ATP supply, O2 consumption, steroid physiology, transcription, axon targeting, the cell cycle, epigenetic programming and many other aspects of physiology, development and neurodevelopment. Many of the pathways observed to be abnormal in ASDs are mediated through NO signaling.

I suggest that the final common pathway mediating autism and autism-like symptoms is low NO in utero, during neurodevelopment and as an adult. Low NO is the archetypal stress response. Virtually any type of stress ultimately results in low NO which physiology uses to trigger compensatory responses. For example, oxidative stress occurs when superoxide levels are increased. Superoxide consumes NO at near diffusion limited kinetics so a high superoxide state is necessarily a low NO state. NO inhibits cytochrome c oxidase. To release that inhibition and increase O2 consumption to maximize aerobic ATP production, the NO level must be lowered. Psychosocial stress increases oxidative stress through catecholamine oxidation, ATP stress increases oxidative stress through increased mitochondrial potential necessary to increase ATP flux, xenobiotic stress increases superoxide through the cytochrome P450 pathway, immune system stress increases superoxide through the respiratory burst. Just about any type of metabolic stress would decrease NO levels and according to the present hypothesis would tend to produce a more autistic-like phenotype. This may be the mechanism for the association of copy number variations with autism. This may also be the mechanism behind the ASD-like symptoms produced by MeCP2 deletion. Females with MeCP2 deletion are mosaic. Some of their cells do have appropriate methylation readout and some do not. Presumably this differential regulation of DNA expression causes metabolic inefficiencies and metabolic "stress" due to cells being out of "sync". In a mosaic organ with non-synchronous regulation, some cells would be working harder than others, perhaps even working at cross purposes. The final common pathway of essentially every kind of metabolic stress is decreased NO.

Virtually all ASD physical symptoms are consistent with pathways regulated by NO being skewed in a low basal NO direction. I suggest that the ASD phenotype is a stress compensatory pathway mediated by low NO.

NO/ROS Balance Programs adult physiology in utero

The physiology of virtually all adult organs is known to be programmed in utero in response to a number of fetal stressors including nutrition[6] stress and hormonal factors. [7] NO/ROS balance in utero does lead to epigenetic programming of adult blood pressure in rats. [8] Stress is a low NO state. [9] The most characteristic physical feature of ASD children is a larger brain[10], with smaller and more numerous minicolumns. [11] Low NO is suggested to cause the characteristic minicolumn structure associated with autism[12] and the timing of stressors may be crucial to the development of the autism phenotype. [13] In guinea pigs, brief prenatal stress increases brain/body mass ratio, and changes adult behavior. [14] Prenatal stress increases learning ability in rats. [15] Prenatal stress does program hypothalamo-pituitary-adrenal function. [16] Low NO does cause neuronal hyperplasia. [17] The patterning of many neural structures is determined in part by gradients in NO mediating proliferation, differentiation, or apoptosis. [18] There are increased asymmetries in the brains of ASD individuals[19], suggesting differential regulation of neuronal growth when the sizes of those structures are formed in utero. Stress in utero causes adaptive changes in the adult physiology of multiple organs; it would be beyond surprising if it did not exert adaptive influences on the most important organ, the brain.

I suggest that low NO in utero, brought about by maternal stress leads to the ASD phenotype in affected individuals, and the genotype that leads to the ASD phenotype was adaptive under conditions where humans evolved, in the “wild”, but is perhaps now less adaptive due to environmental change(s). What possible advantages could the ASD phenotype hold?

The Brain is fundamentally the most Human organ

Humans evolved large and complex brains only because such brains conferred survival and reproductive benefits. Human evolution was shaped mostly by events 100k or more years ago. Humans are social animals, as are all primates. Humans are unique in their use of language with syntax and grammar to convey complex ideas. Humans are the only extant hominin that manufactures and uses tools. The first instances of manufactured stone tools date to about 2.5 to 2.7 MYA (million years ago), and was near universal by 2 MYA[20]. Manufactured tools of perishable materials perhaps were earlier. Modern humans are good at tool manufacture and tool use. Tool use has profoundly shaped human evolution and those parts of the human genome that affect brain structures important for tool creation and use. Similarly, communication and language has profoundly shaped human evolution and those parts of the genome that affect brain structures important for language acquisition and use. The major structures of the brain are formed in utero and early childhood, and are then largely fixed throughout adult life. It is only in utero and early childhood that neurons can be epigenetically programmed to form the major structures in the brain with the characteristic neuroanatomy observed in ASDs such as increased asymmetries, larger numbers of neurons[21] and larger brains.

Brain size at birth limited by female pelvis: Brain optimization requires tradeoffs

The size of the newborn brain is limited by the size of the mother's pelvis through which it can be successfully born. In the absence of medical C-section, cephalopelvic disproportion results in significant infant and maternal mortality. What ever advantages a large brain at birth provides it comes to naught if the infant or mother dies. The structure and size of the brain at birth must be an evolved trade-off between the multiple tasks that brain will be called upon to perform at birth and over the life of the individual and the substantial risk during a natural birth. The only time the most fundamental aspects of brain structure can be modified is while those structures are being formed. Much of the formation of those brain structures occurs in utero, much of it in the first trimester following closure of the neural tube (which is when teratogens such as thalidomide can cause autism). Epigenetic programming of cells occurs when those cells are dividing and undergoing differentiation. Much of the differentiation in early human development occurs in the first trimester. Patterns of methylation modify DNA expression and modify the phenotype of that differentiated cell for the lifetime of that cell. Many neurons do not divide over the lifetime of the individual. NO does modify methylation through the folate pathway and so modifies DNA methylation in ways that are quite complex (for at least this one gene system). Presumably multiple genes are epigenetically modified in complex ways by this same mechanism.

Oxidative stress and low NO cause changes in DNA methylation. Presumably this is part of the normal mechanism by which stress (which results in oxidative stress and low NO) causes global epigenetic reprogramming of diverse genes in diverse tissue compartments under diverse circumstances. Psychological stress causes long lasting changes in neurological functioning as for example PTSD. The details of how psychological stresses of what types cause the characteristic neurological changes that manifest as PTSD are mostly unknown. We know it happens, so there must be physiology that supports those characteristic changes.

DNA methylation mediated through NO does influence expression of genes that are involved in some autism-like syndromes, such as Fragile-X mental retardation gene (FMR1). Aberrant readout of DNA methylation is implicated in the autism-like symptoms of Rett Syndrome (RS). Many of the symptoms of RS are characterized by physiology being skewed in the direction of low basal NO.

Fundamental brain optimization tradeoff: Theory of Mind (ToM) for Theory of Reality (ToR)

Humans are unique among animals for their abilities at communication; language making and language using and tools; tool making and tool using. These abilities are highly dependent on a large brain with substantial plasticity for self modification via learning throughout life. The relative importance of these two extremely important human behavioral characteristics is dependant upon the environment the infant is born into. The major neuroanatomy of the brain originates from structures arising during early neuron proliferation and differentiation during and after neurulation in the first trimester. These structures are then elaborated on later in utero. It would be beyond surprising if the relative aptitude of the brain for communication and/or tool making/using (including manual dexterity for manipulating objects) were not to some extent programmed in utero.

That trade-off would show up as a trade-off between abilities to understand and manipulate other humans (Theory of Mind), and abilities to understand and manipulate reality (Theory of Reality). These are the differences that are seen in people along the autism spectrum. I will discuss how a less developed ToM interferes with ASDs communicating with neurologically typical individuals (NTs) and their very well developed ToM. For the most part ASDs don't pick up the nuances of communication, particularly how it relates to motivations, beliefs and other mental states. The NT ToM forces NTs to think in anthropomorphic terms, even when it is inappropriate because they lack a robust ToR.

I spend a lot of time trying to explain this in several different ways using several different analogies. What I am trying to describe is exceedingly complex, as complex as an entire human brain. Something that complex cannot be described simply except in simplistic terms. It would be like trying to describe a library in a single paragraph.

Good times --> Need Good ToM

One hypothesis of this paper is that when times are good, and a woman's first trimester of pregnancy is characterized by low stress, then the "optimum" infant brain will be one optimized for better communication. If times are good, there will be plenty of other humans around, and the infant's primary competition for food and mates will be with other humans. Because times are good, the cultural information the adults have is working well to produce those good times. Copying that good cultural information with high fidelity is important. Good communication, a good ToM with the ability to understand and manipulate other humans is the best strategy when times are good.

Hard times --> Need Good ToR

When times are hard, a woman's first trimester will be characterized by high stress. The optimum infant brain will be skewed away from communication because during evolutionary time, hard times meant fewer humans in the territory. With fewer other humans around the need for communication is reduced. The cultural practices of the adults are not working well to produce good times. If the cultural practices are not working well, they need to be modified until they are working well. When times are hard, there won't be many other humans around because they will die in infancy. Competition will be against reality for food, shelter, and to stay alive. The adults don't know how to make good times, so that is something the infant will have to figure out for him/herself.

Theory of mind, theory of reality and theory of cognition

I will make a distinction between being able to think about something (cognition) and being able to think about that thinking process (meta-cognition) and coin a term Theory of Cognition (ToC), to denote the ability to think about and emulate different types of cognition. The term is an attempt to be analogous to ToM and ToR, which are meta-abilities to think about and compare multiple models of other minds (necessary for communication by emulating other mental states), and multiple models of potential realities. I am making this distinction because the different types of computation that humans do are not necessarily mapable onto each other.

Analogy:
Word processing ToM Theory of Mind Emulating other minds
Spread sheet software ToR Theory of Reality Emulating Reality
Operating system ToC Theory of Cognition Choosing Emulations

Trying to understand human communication with a ToR might be like trying to write a document not with word processing software but with a spread sheet. A document could be written in a spread sheet. It would be slow, cumbersome and the document wouldn't have the right formatting and wouldn't be as polished as something done in a word processor. On the other hand, a large spread sheet calculation simply can not be done using word processing software. The word processing software doesn't support the primitive functions, addition, subtraction, multiplication, etc that a spread sheet calculation requires. Some of the complex word processing functions can be emulated on a spread sheet but spell checking and grammar correction would be very cumbersome and difficult.

This is the sense that I am trying to convey, that people with a robust ToM can do good and robust communication that is nuanced, and well understood by others with a matching and robust ToM. Their shared ToM is analogous to the word processing software, and a well formatted document is analogous to human communication between individuals with a shared ToM. The ToR is analogous to the spread sheet software and the large spread sheet analogous to a highly technical ToR. A theory of cognition (ToC) would be the selection of the proper software type to do the required computations (ToM(English), ToM(French), ToM(ASL), ToR). There are multiple ToMs, each language is different to some extent, although there are other communication modes, body language, cultural signals, gestures. There is only one ToR, the one which accurately describes reality as it actually is. Individuals may have a ToR that is highly specialized and individualistic, physics or medicine for example. But all of the different ToRs all mesh into one (or should) because they all describe a single reality.

In this analogy I am trying to illustrate that a specialized piece of computation machinery may work very well for one task (word processing) and not at all for another (spread sheet calculation). If you tried to input a spread sheet into a word processor, you would get many error codes, many misspelled words; the word processing software would reject it as badly formed. A very well formed spread sheet cannot be read on a word processor. This is analogous the problem that some NTs have with understanding people with ASDs.

Normal background "housekeeping" features can impede communication. Many types of different word-processing software have automatic spell checking. If a word is spelled wrong, the software will change the spelling to match the spelling to one of the words in its dictionary. If the word is not misspelled, but is simply not in the dictionary, the software can't recognize it and will change it anyway. This is a type 1 error, a false positive. The software falsely identifies a character string and modifies it to match its default identity. This is an inherent property of specialized pattern recognition systems. There is a trade off of type 1 errors (false positive) for type 2 errors (non detection). If you don't have access to modify the "error correction function", it may be impossible to type certain strings because the error correction keeps changing them. In certain word processing software this can be extremely annoying and make writing outside the scope of the software impossible. If the software won't let you have certain character strings in your document, you can't write about them. If your ToM won't let you have certain ideas, you can't think about them. Appreciating that your ToM doesn't have the capacity to think certain thoughts is extremely difficult.

Being unable to conceptualize ideas is not uncommon. There are people who believe in the literal truth of ancient texts and are unable to conceive that they do not accurately describe reality, irrespective of what data can be collected today. These beliefs are from a ToM, shared with others of their community. Such beliefs did not arise from observations of reality, they were told to individuals by other individuals who believed them. Those false beliefs are derived by those false beliefs being communicated to the individual and so shaping their ToM. Such beliefs are often extremely resistant to change.

Learning can be looked at as modification of the brain's neural network so the neuroanatomy can support what ever new idea it is that is being learned. Usually this takes a long time and is quite difficult. Learning physics or mathematics is difficult because the normally developing neural patterning doesn't support that type of thinking the way it supports language. I will discuss this more later.

A mother's necessity makes her child an inventor.

Maternal stress --> fetal development along autism spectrum --> Asperger phenotype

The hypothesis of this paper is that low NO in utero causes development along the autism spectrum so as to program the brain in utero to one that supports a better ToR. Precisely the phenotype that is needed when mothers are stressed and so times are hard. What ever technology and cultural practices are being used, they are not working well enough and so new ones need to be developed.

Which individuals are most adept at tool use today? It is people with Asperger’s, people with ASDs. Many scientists and engineers have Asperger’s, and it is suggested that Einstein, Newton, and many brilliant scientists had Asperger’s. [22] Asperger even said “It seems that for success in science or art a dash of autism is essential.” [23] The stereotypical nerd is someone with facility at math, science and with characteristically poor social skills[24]. The mirror neuron system[25] (responsible for understanding the actions of other individuals) exhibits dysfunction proportional to ASD severity. [26]

The major barrier to revolutionary scientific innovation is conventional thinking and existing paradigms. [27] What Kuhn calls “normal science”. Ideas transmitted culturally are difficult to displace even when wrong. It is nearly 150 years since Darwin’s “Origin of Species”, with overwhelming data supporting and no datum inconsistent with evolution, yet in the USA, 40% of the population believes evolution is false. [28] Some on the Nobel Committee were unable to accept relativity as valid and so Einstein received the Nobel Prize for the photoelectric effect, not relativity. [29]

Cultural notions of what is appropriate affect abilities (i.e. what people think they or anyone can do). Women exposed to a hypothesis wrongly attributing mathematical ability to genes on the Y chromosome have impaired mathematics performance. [30] A degree of social isolation from disrupted mirror neurons may insulate ASD individuals from incorrect paradigms of science, technology and the peer pressure associated with cultural practices which must be abandoned to overcome hard times. No doubt 2.8 MYA everyone “knew” stones didn’t make good tools. The first stone tools were not developed after committees of peers reviewed proposals and selected the highest scoring for implementation; they were developed by the “Einsteins” of the time working alone. Acquisition of nut cracking skill by capuchin monkeys (Cebus apella) using stones as hammer and anvil takes about 2 years and requires considerable repetitive nonproductive effort while watching proficient individuals. [31] No doubt repetitive trial and error was needed to acquire de novo skill(s) to manufacture stone tools 2 MYA, and such individuals had to ignore criticism that they were bizarre for “uselessly” banging stones together.

These culture notions are transmitted through the robust NT ToM. To avoid being adversely influenced by potentially incorrect ToMs, ASDs require a weaker ToM, a ToM that perhaps allows some communication, but one that can easily be ignored, resulting in the ability to ignore the "Kool-Aid" which is a stronger version of groupthink. The delusional world views that some NTs have can become extremely compelling to them,, such that they are unable to perceive that it is delusional, particularly when a charismatic leader with a strong ToM (essentially the definition of a charismatic leader) imposes it on his/her followers. More on this later in the discussion of cargo cult science.

ASD individuals developing skills unrecognized as useful by NTs must possess a compulsion to acquire those skills despite peer pressure that such skills are useless. Many ASDs acquire skills that NTs think are useless, fascination with train spotting, bird watching, collecting, virtually every savant ability is acquired to a degree that NTs do not find useful (if they did, then NTs would acquire such skills to that degree and it would not be thought of as savant). Language and communication is the "savant" skill of NTs. NTs possess a skill at communication (with other NTs) that ASDs cannot hope to master. Just as ASDs sometimes have skills that NTs cannot hope to master. Just as elite athletes have skills that non-athletes cannot hope to master. A society with the ability to use the best skills of a highly variable and diverse group would be better able to cope with adversity than a society where all individuals had the same average abilities. Not every individual in the village needs to be proficient at making stone tools, provided there are enough proficient individuals and the tools their skills produce can be traded for other things.

Cognition: Non-algorithmic calculation

Cognition in human brains is done by neural networks, the fundamental details of which are mostly unknown. Some cognitive abilities (such as savant calendar) are known to be non-algorithmic because the errors are not always the same, and the time for performing the calculation is not asymmetric depending on calculation direction the way a computation performed using an algorithm would be. [32] It is likely that most if not all other types of human cognition are non-algorithmic.

I am using algorithm in the sense that an algorithm is what a Turing machine executes. An algorithm is a series of instructions that when acted upon manipulate data and perform a calculation. The computers that people are familiar with are algorithmic. Calculators use a calculating engine (the processor) to operate an algorithm (the software) to manipulate the data. In general the data does not modify the software or the processor while the computation is in process, and given the same data, the same processor running the same software will produce the same output each and every time the calculation is performed.

Neural networks are inherently non-algorithmic in the sense that there is no "algorithm" explicitly being implemented by the neural network. A neural network may be used to implement an algorithm. For example, humans and other animals have the ability to do approximate mathematical operations such as comparison. Two groups of objects can be compared and the one with the larger number can be selected even when the members of each group have not been counted. This selection can be made by individuals unable to count and even by animals. This selection is non-algorithmic. An individual able to count is also able to count the members of each group and then tell which group is larger by comparing the two values. The person, who can count, knows that the counting algorithm produces a more reliable comparison than the non-algorithmic visual comparison. The person who cannot count does not know how to implement the counting algorithm.

Human brains are not optimally configured to run algorithms. A processor that can run algorithms is in essence emulated in a human brain to run the algorithm under consideration, such as counting or multiplication. The form that the data is in may greatly limit what algorithms that data can be manipulated with. For example multiplication using Arabic numerals is easy. Multiplication using Roman numerals is exceedingly difficult. There is essentially no algorithm for multiplying Roman numerals, individuals use a look-up table. Learning algorithms takes considerable time and effort for many individuals.

Communication requires a Theory of Mind

Exactly how neural networks in the brain configure and reconfigure themselves to do the computations that certain cognitive tasks require is unknown. Presumably there is some type of feedback that modifies the network when sub-optimal results are achieved so as to configure the network to produce better results. How this occurs is unknown, but for language acquisition, some conclusions as to how this optimization works can be made which I discuss below. What I want to emphasize the compulsive aspects of language acquisition. People do not choose to acquire the language they acquire as children, their brains acquire it (or synthesize it de novo) for them.

All communication requires two parties, a sender and a receiver. The sender must have a mental concept, translate that mental concept into a communication medium, transmit that message to the receiver, who must receive and then translate that message back into a mental concept. In that sense, all communication is only the transmission of representations of internal mental states. For there to be communication, the mental concept must necessarily be mapable onto the neural structures of both individuals. If one party is not able to represent the mental concept in their brain, the concept cannot be communicated either from them or to them. In a sense, communication is the transmission of data that allows the receiver to identify and map that concept into a mental representation, in effect the receiver is doing pattern recognition on the data stream and generating a mental representation, in effect a pattern of thought either generated de novo, or a familiar pattern previously used.

In this sense, communication can only occur between individuals with a shared Theory of Mind. This is the sense that I am using ToM in this paper, the emulation of the cognition of another individual to achieve a mapping of the mental state of one individual with the mental state of another individual. The possible fidelity of that mapping determines the possible fidelity of that communication. If a mental state cannot be mapped onto another individual's ToM, then that mental state cannot be communicated to that individual.

Pattern recognition is a well recognized ability. All systems encoding pattern recognition are subject to different forms of error. There is the type 1 error, the false positive, the error in wrongly identifying a false instance as positive. There is also the type 2 error, the false negative, the error in missing the correct identification of a correct instance. In a general sense any pattern recognition system can be made more sensitive, that is with a reduced type 2 error, but then there is an increased type 1 error and there are more false positives.

A type 1 error is getting the attempted message wrong; a type 2 error is missing the attempted message. Since communication is a two-party interaction, the "fault" of miscommunication cannot be attributed to either party, the "fault" lies in their interaction.

Many human interactions engender other types of error. There is no generally accepted definition of what is a Type 3 error, but one definition is "the error committed by giving the right answer to the wrong problem". When times are hard, and the culturally transmitted traditional information isn't capable of solving the hard times, that is an example of the right answer to the wrong problem. The time of adolescence and early adulthood is often a time of rebellion against authority, against conventional wisdom, against cultural norms. Young people are testing the limits of their culturally acquired information; testing to see what works and what doesn't. This is somewhat speculative but this might be a mechanism to reduce the cultural transmission of obsolete or dysfunctional practices. In the absence of a written language, the only cultural practices that can be transmitted are those adopted by the next generation. If the older members of the tribe live long enough to transfer their wisdom to adults past their adolescent rebellion period, perhaps the wisdom is worth transferring. If not, then perhaps it isn't and the tribe should try new approaches until that happens.

Communication and language acquisition

Social animals communicate with each other. In humans the ability to develop language is innate and the brain structures to support language and language development must be coded for genetically. Language itself must be learned or is synthesized de novo during certain periods of brain development. This point is quite important. When humans are growing up in a culture, they adopt the language of the culture, provided that the language is "well formed". If the language the adults are using is not "well formed", the children synthesize a new language that is "well formed". That is, when the children of immigrant parents grow up, they do not adopt the pigeon language their parents are speaking, they either adopt the "well formed" dominant language, or synthesize a "well formed" Creole. The various sign languages did not become "well formed" until children grew up with signing as their first language, which they modified into a "well formed" language.

The acquisition of language in this way tells us several things; that the ability to acquire language is innate, that there is a more "primitive" cognitive structure underlying language (by that I mean that the structure of "thought" has a component that is simpler than the linguistic components humans communicate with). Without a simpler and more primitive cognitive structure, the Creole could not be analyzed as it is being formed to ensure the resulting Creole has a "well formed" grammar. However, the ability to form a Creole is lost at a certain age. The immigrant parents of the Creole synthesizing children continue to speak their pigeon language. This implies that the cognitive structure that analyzes language as it is being learned and forces it to be "well formed", i.e. to conform to standard human linguistic patterns is lost (to some extent) with age. It also implies a compulsion to learn the "standard" language and a compulsion to force others to comply with the "standard".

The development of a de novo language, such as a Creole, is a collective outcome produced by a population. It is not produced by a single individual. Another way of describing it is that the population developing the language acquires a shared neural mapping of the medium of the language (sounds, gestures, etc) to neural structures producing the mental states that are the ultimate outcome of communication (that is the ideas being communicated). In this context, there is no arbitrarily correct mapping. The mapping is correct so long as it is the mapping shared by the group. In the sense of the Galbraith quote at the start what ever the majority adopts as the linguistic mapping is the correct mapping. This is a very important point. What ever the majority adopts as correct is correct; everything else is wrong.

For a single majority linguistic mapping to arise spontaneously there must be very powerful mechanism(s) to eliminate deviation from the mapping acquired by the majority. The majority acquire a shared Theory of Mind with respect to linguistic mapping. In other words, the differences between the shared Theory of Mind and that of any individuals in the population are reduced. The deviation is not reduced by changes to the shared theory of mind; the deviation is reduced by individuals adopting the shared ToM as their own. This is an important point. There is no "shared" ToM. There are only individual ToMs which correspond to the shared ToM more or less. The shared TOM can only be shared to the extent that all individuals have the same components and the same structural relationships between those components. The shared ToM reflects the "lowest common denominator"; the ToM that overlaps with everyone else's ToM is all that can be shared. I think this relates to the importance of "peer pressure" in the age group capable of forming a Creole language. If peer pressure were not so compelling, a single coherent language would be difficult to achieve.

The rigidity of an inflexible ToM maintains stability of communication, of information transmitted culturally to the next generation. If your ToM doesn't support an idea, you cannot transmit it, receive it, understand it, or even think it. When times are easy, transmitting the cultural information that led to those easy times is important. It is important to do so with high fidelity because it worked. When times are hard, the culturally transmitted information isn't working, and so needs to be abandoned or modified. The fidelity of transmission must be reduced so what ever is wrong and/or isn't working can be eliminated.

The ToM of NTs that allows them to communicate so easily with each other limits what they can communicate to ideas that are within the shared ToM. This is an extremely important point, but it is a point that NTs have an extremely difficult time understanding because they can only think using ideas that are within their shared ToM. If an individual's ToM is insufficiently flexible to map an idea, that idea cannot be understood unless the ToM changes. But there is tremendous peer pressure to maintain the shared ToM of the group and to not change it.

This rigidity of the NT ToM is what causes ideas to persist even when those ideas are wrong and the rejection of correct ideas even when well supported by incontrovertible data. Many religious ideas have no supporting evidence and are in fact demonstrably wrong. For example the idea that the Earth is less than 10,000 years old and was created in 6 days as described in Genesis. Similarly the idea of evolution is rejected without a single piece of data inconsistent with it.

Most ToM ideas are transmitted from other individuals, not generated de novo.

Conflicting compulsions for ToM and ToR

A specific ToM is only useful for communication in the context of the group of individuals that share it. The mapping of a data stream (i.e. speech or gestures) into ideas and mental states is arbitrary and the only correct mapping is the one that everyone else in the group shares. There must be a tremendous compulsion to modify one's ToM to conform to that of the group. It is this compulsion that forces the emergence of a single language in a group.

In contrast, a ToR is only useful in so far as it actually corresponds to reality. To eventually develop a robust ToR, the individual must have a compulsion to modify his/her own ToR until it does correspond with reality, irrespective of the ToR of others in the group.

Thus developing and maintaining a good ToR is in conflict with developing a good ToM. A ToM needs to remain static for individuals to be able to communicate with each other. A ToR needs to be dynamic and change when ever it is found to be in error or to be dysfunctional.

I think this is the source of much of the resistance to new ideas in human culture but also in the scientific community. These concepts are laid out by Thomas Kuhn in his book, The Structure of Scientific Revolutions. Most scientists do what Kuhn calls "ordinary science", where they work within the paradigm of their scientific field. It is difficult to work outside the paradigms of a scientific field. Any contradiction of an existing paradigm is considered extraordinary and so requires extraordinary evidence. Some individuals are unable to reject paradigms even when they have been shown to be wrong. In these individuals, their rigid ToM has locked them into a perpetual state of error, and they don't have a sufficiently robust ToC to appreciate that their thinking is faulty and in error. It is mechanisms similar to the mechanisms that enforce a rigid ToM during language acquisition that compel adherence to the faulty ToM in later life, peer pressure, appeal to authority, tradition.

Communication and ASDs

Communication in humans encompasses a number of modalities including speech, sign language, body language, written language, music, artistic expression and perhaps pheromones. Most of these have components that are learned, improve with practice and degrade with disuse demonstrating the involvement of neural structures which retain plasticity (positive and negative) even in adulthood.

Autism is defined by behaviors, behaviors related to social interactions where autistic individuals have what are called characteristic deficits which can be reliably measured. However what constitutes a deficit is a matter of perspective. One example is a "deficit" in the ability to impute anthropomorphic motivation and emotion to inanimate objects as in the work of Frith. In this research, triangles were animated and made to move in three different ways, randomly, goal directed and moving interactively with implied intentions. The two sets of purposeful motions were designed to evoke anthropomorphic responses, e.g. chasing, fighting and coaxing, tricking. Individuals were scored on how closely they matched the scripts the animators of the triangles were trying to portray.

The ASD individuals scored lower than the NTs did, and this was described as a "mentalizing dysfunction". This was taken as a confirmation that people with ASDs have an impairment in attribution of mental states. However, whose "mental state" did the ASDs have an impairment in recognizing? The "mental state" of the triangles? Was this error a type 1 error (false positive), or type 2 error (false negative)? One might say the ASDs had a type 2 error, failure to recognize the "mental state" of the triangles, but one could (more correctly I think) say that the NTs had a type 1 error of falsely attributing a "mental state" to obviously inanimate triangles.

There is no intrinsically correct representation of the mental state of triangles. Triangles do not have mental states. The only way that a mental state can be attributed to triangles is via anthropomorphic projection of human-type intentions onto inanimate objects. In most circumstances this would be a Type 1 error; falsely observing anthropomorphic attributes in inanimate objects. It could also be thought of as a type 3 error, wrongly using a human based anthropomorphic model where it is inappropriate. This type of projection is not uncommon. Imputation of motivation and intentions to inanimate objects was at one time the basis for the religious belief that demons and spirits inhabit and animate virtually every object.

Inappropriate invocation of anthropomorphic feelings is a large part of the entertainment industry. Many cartoons are stylized after humans and many humans develop grossly and dangerously wrong ToR based on these erroneous ideas. In regions where bears are endemic, campers feeding bears is a serious problem. People assume that the anthropomorphic representations they have seen on TV are representative of how bears will react in real life. There have even been cases where a parent has applied peanut butter to a child's face so a bear cub would lick it off to obtain cute pictures.

This relates to the second quote, "If the only tool you have is a hammer, you tend to see every problem as a nail." If the only cognitive structures you have to think with are the cognitive structures of human emotions and communication, trying to figure out the properties of inanimate objects would consist of trying to ascribe human motivations and intentions to those inanimate objects and trying to figure out what they would do next in human terms.

Savant cognitive abilities

A striking feature of some people on the autism spectrum is that in some instances they have narrow and highly superior cognitive abilities. Human mental abilities have distributions in the population, with "normal" abilities being distributed "normally". Usually people with autism are somewhat lower on intelligence tests such as WISC, but with somewhat higher scores in block design. When intelligence tests without a communication component such as Raven's Progressive Matricies are used, some autistic individuals score much higher, in some cases as much as 70 percentile points higher (n=7). [33] That is 70 percentile points higher. Such a lack of congruence between tests is sufficient to show they are not measuring the same thing and we shouldn't use the same label to denote what the different tests are measuring even if there is good correlation among NTs. That correlation can only be spurious.

The distribution of intellectual abilities is "normalized", that is differences are measured and then scaled to fit on a distribution. That scale is arbitrary, and does not reflect any sort of absolute scale of difficulty.

As social animals, humans live in societies, larger communities of humans where there can be specialization and division of labor. It is this specialization and division of labor that has allowed humans to collectively master many technologies. Presumably different mental tasks are optimized by different neural structures. Dispersion in mental abilities requires dispersion in neural structures.

Savant abilities are not rare among people on the autism spectrum, and sometimes occur in individuals with profound disruptions in other cognitive abilities. This shows that to some extent, some cognitive abilities are independent of each other. Presumably superior performance in some cognitive tasks and inferior performance in others represents a trade-off of abilities along multiple spectra. The brain is limited in size, its computation capacity is limited, relative cognitive abilities of individuals will depend on the myriad details of the neurodevelopmental path that individual took.

Communication is "savant" ability of NTs

Many ASDs have savant abilities, which demonstrate that what ever part of the brain is providing those cognitive abilities has superior performance to the corresponding part of NT brains with lesser performance. The one area where NTs are universally better than ASDs is in communication. I suggest that communication is the savant ability of NTs, and that NTs have traded reduced abilities on ToR and ToC for enhanced ability in ToM.

The difficulty in relations between ASDs and NTs is that NTs don't appreciate that the ToM they are using for communication is a savant ability that ASDs don't share, and shouldn't be expected to be able to emulate. An ASD can't emulate the NT savant ability to communicate any more than an NT can emulate an ASD savant ability at mathematics. If you don't have the brain structures that can do the computations, you can't emulate the behavior. You might be able to fake how it sounds, but because the fundamental neural structures are not present, it is just an act and can't have the actual content.

Trying to think about Reality with a ToM is like doing Cargo Cult Science

Richard Feynman coined a term, Cargo Cult Science, to describe the practices of people who may be doing what they call experiments, but they are missing the fundamental intellectual honesty to be actually doing science. The term comes from the observation that tribes in the South Pacific would observe westerners arrive and set up landing strips which would bring aircraft laden with cargo, all sorts of goods that seemed to appear like magic. Along the lines of Arthur C. Clarke's observation that "any sufficiently advanced technology is indistinguishable from magic". They tried to understand the source of this cargo and how to get cargo for themselves using their understanding of reality. They generated a Cargo Cult, and proceeded to adopt rituals to try and cause cargo to appear.

This is really an excellent metaphor for trying to think about a subject with the wrong approach. Their thinking was that such good cargo had to come from the Ancestors, but the Ancestors would only bring such good cargo if they were communicated with in the right way, which the westerners knew how to do, so copy them and the cargo would appear. They built landing strips, control towers and populated them with radio control operators, but to no avail.

Explaining that their approach was wrong would fall on deaf ears. They don't have the background to understand where the cargo actually came from. They had anthropomorphized their observations and reduced them to the human terms they could understand using their ToM. They didn't come to their beliefs via facts and logic, facts and logic won't dissuade them from their beliefs.

Obviously there are multiple individuals involved, a leader and followers and the leader may achieve lots of things even if no cargo shows up. Presumably it is the charismatic persuasion of the leader using the leader's ToM that causes the followers to believe the leader. Simply by leading the effort to obtain cargo the leader achieves status over the others. Even when doing something completely useless and wrong, the society holds together if all of the members share the same ToM. Individuals not sharing the conceptualization of obtaining cargo by building airstrips would not fit in.

To people who have savant mathematical ability, those without it who are trying to emulate mathematical abilities can be seen as trying to do cargo cult mathematics. They can go through the motions, but don't have the ability to generate the content. Similarly, ASDs who try to communicate with NTs are doing cargo cult communication. They can go through the motions, but there is a lot of stuff that is being missed.

Neurological structures required to support an idea

The only ideas that an individual can think about are ideas that can be mapped into that individual's neural network. To learn a new idea, either the neural structure present is sufficiently flexible that the new idea can be mapped into it, or the neural structure must be modified until the new idea can be mapped onto it.

The process of learning a new idea must include as the first steps, the process of modifying the neural networks of the brain such that they can support the new idea being learned. Often the first step is "unlearning" ideas that are wrong. I think that this modification of the brain to support new ideas is why learning takes such a long time. New neural structures need to be made.

All mental representations require some level of neuronal "overhead" to be sustained. The details of how the brain does that are not understood. While the capacities of the brain are large, they are not infinite, and at some point trade-offs must be made.

Socially isolated individuals develop on an autism-like pathway.

All important physiological systems are under feedback control (that would be all physiological systems). Presumably an organ as important as the brain is also under feedback control, and this is reflected in the improved efficiency obtained through practice at certain mental tasks.

Presumably if there is a trade-off of ToM vs. ToR, then isolated individuals with no need for a ToM would develop a more robust ToR. This does appear to be the case in multiple organisms including humans, monkeys and rodents.

The classic work on socially isolated monkeys was done by Harlow in the 1960's, [34] and present animal welfare regulations would make such experiments problematic. Some monkeys were raised with no social contact at all, even with their mothers. Such monkeys were profoundly affected and exhibited rocking behaviors, self-injurious behaviors and profound disruption in abilities to interact with other monkeys. They were termed autistic by the experimenters.

Surprisingly, some of these socially isolated monkeys exhibited superior cognitive abilities. What is especially interesting is that these superior abilities were termed "deficits" by the experimenters. [35] Socially reared monkeys were conditioned with a tone and a startle stimulus. A redundant lamp was then paired with the tone. Socially isolated monkeys conditioned to the redundant light, the socially reared monkeys did not. The experimenters characterized the non-conditioning of the socialized monkeys to the redundant signal of the light as "blocking" the isolated monkeys then exhibited what was termed a "deficit" in blocking. Why the experimenters chose to use the term "deficit" to refer to a superior ability tells us something about the experimenters and their expectations about the socially deprived monkeys, not the monkeys.

Rhesus monkeys raised in social isolation have superior learning performance to those raised in social environments. [36]

Involvement of nitric oxide in social interactions and communication

The archetypal social interaction in mammals is the bonding of the mother to her infant. All mammals exhibit this behavior and have exhibited it for as long as mammals have suckled their young. The first social interaction all mammals have is with their mother. Even mammals thought of as primarily non-social do have this social interaction.

NO is involved in the development of the bonding and smell recognition that occurs in ewes within 2 hour of giving birth. Inhibition of nNOS blocks formation of that olfactory memory, and this blockage can be reversed by infusion of NO into the olfactory bulb. [37] Oxytocin is essential in the formation of normal social attachment in mice. [38] Reduction in oxytocin release following epidural anesthesia in heifers preceded a reduction in maternal bonding type behaviors[39]. Activation of the oxytocin receptor causes activation of nitric oxide synthase. [40] The connections that mediate maternal bonding can occur in the space of a few hours[41], limiting the distance over which axons must migrate to form these new connections.

Why NO is the signaling molecule that mediated the neural remodeling to cause maternal bonding makes evolutionary sense. Lactation is extremely energy intensive. If a mother does not have the metabolic resources to generate sufficient milk of sufficient nutritional quality to sustain her infant until it is weaned, she (and her infant) is better off not bonding to her infant and abandoning it. Spending metabolic resources on a reproductive attempt that will fail will reduce the success of potential future reproductive attempts. A failed reproductive attempt has no advantage either to the mother, or to the infant. An infant's best reproductive strategy in those circumstances is to do what ever increases the likelihood that the infant's mother will have a successful reproductive event later, so that the non-surviving infant may have a surviving sibling.

I discuss this at length in my blog on infanticide. Using NO as the positive signaling molecule to mediate maternal bonding directly couples maternal bonding to energy status. The low NO of metabolic stress directly reduces the degree and fidelity of maternal bonding. In extreme metabolic stress (the most important states for maternal bonding to be blocked) the maternal instinct turns from nurturing to infanticide. It needs to be appreciated that infanticide under conditions of extreme metabolic stress is as much a "maternal" instinct as is nurturing when times are better. I see infanticide as the brutally hard state that desperate metabolic stress induces in postpartum women.

Social isolation reduces NO generating neurons in the brain

When rodents are raised in a socially deprived setting, the numbers of NO producing neurons in some parts of their brains are reduced. [42] A reduction in basal NO in the brain due to development under socially isolated circumstances makes sense. Many social interactions are mediated via NO mediated pathways, including bonding and other pathways mediated through oxytocin. If the environment one is growing into is non-social, social neural pathways have little or no survival benefit. Better to develop the neural structures that will be useful.

Socially isolated individuals retain sufficient neural plasticity to partially recover

Social isolation at birth produced monkeys with profoundly disrupted social abilities. Experiments demonstrated that some of the disrupted social abilities could be restored. This involved the use of "therapist" monkeys, usually socially raised normal monkeys that were substantially younger than the isolated monkeys. [43] In females, a socially isolated female could recover somewhat and be an improved mother following pregnancy and raising an infant however many times the first born infants did not do very well but mothering did improve with subsequence births[44] demonstrating plasticity in neural networks mediating mothering behaviors during pregnancy and/or mothering activities. Since maternal bonding is the archetypal communication pathway for mammals, this suggests that other fundamental communication pathways have plasticity also.

With NO being involved in bonding, improved bonding and mothering interactions with subsequent births is consistent with increased neurogenic nitric oxide as a causal mechanism. If a non-social environment becomes social, reconfiguring neural structures to cope with social interactions would be advantageous.

Potential for treatment

I suggest an analogous treatment for ASD individuals may be to incorporate them into play groups with significantly younger NT children that are at similar developmental stages, but with sufficient adult supervision that nothing untoward can happen.

Doing this in the context of increasing NO levels via the techniques I am working on my have important therapeutic effects.

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Role of low basal NO in capillary and vascular abnormalities

Vascular remodeling under low NO conditions.

I have really just skimmed the surface in what I have presented here. There are a lot more details that reinforce the chain of facts and logic that tie all of this together. This is already quite long and making it longer still isn't going to help you readers very much. Read what I have linked to, and if you have questions or want clarification of certain points, ask me.

There are many chronic degenerative diseases that are associated with what is termed vascular damage. The ultimate cause(s) of this vascular damage remains obscure. I suggest that much of what is perceived to be damage is not "damage" per se, but is simply the consequence of normal active remodeling of the vasculature under chronic conditions of low NO which results in the characteristic and dysfunctional morphology observed. In other words, the vascular state is the consequence of the low NO, and that vascular remodeling processes remain intact, active and essentially fully functional. These processes are simply operating in a low NO environment where the remodeling eventually result in dysfunction vascular morphology. Correcting the low NO environment should restore normal vascular remodeling and restore normal vascular function once there has been sufficient remodeling under conditions of normal NO levels.

In other words, to some extent the "damage" is not acute or even chronic "damage", rather it is simply a consequence of long term remodeling and is to a large extent reversible if caught early enough depending on age and the tissue compartment affected. This is an important point. I think it is not appropriate to call it "damage", if the normal remodeling pathways are working properly. Secondary damage to the tissues perfused by that dysfunctional vasculature is more problematic. There may be regeneration in some tissues such as muscle and liver, but less in others such as the brain or retina. Regeneration and regulation of vascularization is a critical aspect of wound healing. If the vasculature could not regenerate itself, wounds could not heal and death would occur fairly quickly. Even in the elderly, wounds do heal, demonstrating that the regulation of vascularization is intact even if it proceeds at a slow rate.

Vascularization is a complex process requiring many coupled and interacting pathways to function successfully. When vascularization "goes bad", how many pathways are affected and in how many different tissue compartments? A few, dozens, hundreds, thousands? Presumably each endothelial cell regulates itself from internal and external signals. For many endothelial cells to "go bad" simultaneously and in characteristic ways such that characteristic abnormalities develop over macroscopic spatial dimensions the regulation in millions of cells would have to simultaneously "go bad" in precisely "the same" way to achieve "the same" pathology over "the entire" tissue compartment (such as the retina) or systemically throughout the organism. That seems implausibly unlikely.

My hypothesis is that the observed vascular dysfunction is actually good regulation around a bad setpoint and that the bad setpoint is set external (to the endothelial cells) (the "setpoint" has to be external because what physiology is trying to "fix" is external levels of O2 and other nutrients in the local vicinity by modulating vascular perfusion) by the local NO level. Mostly the local level of NO is determined by the local NO production "source", eNOS in the endothelium and then taken up by the major "sink", hemoglobin in red blood cells. There is nothing simple or simplistic about this. The control system must have enough degrees of freedom to regulate the vasculature under each and every physiological condition that occurs, or vascular regulation fails and the organism dies. If the organism is still alive, then vascular regulation has not completely failed… yet.

My focus will be more on how low NO generates the characteristic morphology and what the long term consequences of those changes are rather than on other factors. I am not going to go into the detailed mechanisms of what regulates vascularization. I am going to more focus on the macroscopic control system which must exist even though the details are not very well understood. The experimental techniques necessary to measure and understand those details are quite challenging. They must involve gradients of diffusible (and so necessarily small) molecules over the smallest length scales of the vasculature. There are essentially no sensors to measure the small diffusible molecules that must be involved on the length, time and concentration scales involved. So long as those control systems use NO as a signaling molecule, a change in the basal level of NO will affect that control loop by skewing it in a characteristic direction.

The characteristic vascular remodeling I am most concerned about occurs "at rest", that is under the physiological state that organisms are in most of the time, and during sleep. During non-rest there are other pathways that provide additional regulation of the vasculature but because remodeling at rest occurs, the pathways that are involved at rest must be sufficient to mediate the remodeling.

I spend a lot of time talking in generalities, talking of the philosophy of design and control and how that must be applied in physiology. This is not the standard "hypothesis-experiment-conclusion" that is the standard methodology in science. The reason I don't take that approach is that the systems are too complex; they consist of too many coupled non-linear parameters for which there are no techniques to measure, let alone measure on the length and time scales that must be important to regulate vascularization. If we limit ourselves to what is possible to measure, the vast majority of physiology is completely inaccessible. Even though we can't measure parameters, we know that they must have certain properties due to stability considerations; certain properties because they evolved; certain properties because mammals gestate in utero under very different O2 levels and so on. The degree of certainty from such inferences isn't as high as from actual measurement, but the point of this argument is not to "prove" that low NO causes vascular abnormalities, but rather that NO is involved in "enough" pathways that having the "right" basal NO level must be important. How important? That is a good question and one that can only be answered experimentally. My purpose is to demonstrate that there is sufficient a priori justification to test the hypothesis that raising basal NO will improve vascular abnormalities. If my hypothesis about basal NO is correct, then raising basal NO will have no adverse effects because it simply restores a more normal state where all regulatory pathways work better.

We don't have the ability to measure basal NO levels, and even if we did measuring them in vivo would be to invasive and cause too much injury to be done ethically. There are so many different tissue compartments and so many different pathways that utilize NO as a signaling molecule, that the idea of external control through artificial means is completely preposterous. Because there are so many NO mediated pathways, it is very likely that different individuals will have different and to some extent idiosyncratic thresholds for some of them. What this means is that while there may be some generic symptoms from the effects of low basal NO, there will very likely also be idiosyncratic symptoms. Eventually as basal NO gets lower and lower, more and more NO mediated physiological pathways become marginal and eventually will "fail". As more and more NO mediated pathways become disrupted the course of many degenerative diseases gets closer and more similar. For example, as end stage kidney failure gets worse, vascular disease gets worse too and kidney failure is a common complication of vascular disease. I think the commonality of vascular abnormalities with many degenerative diseases is due to low NO being one of the final common pathways involved in them all.

I am a chemical engineer, so I will talk about and outline my reasoning about this in engineering terms, sensors, control systems, feedback, and that sort of thing. This is not to imply any type of design as ID proponents consider it. That is simply how I think of and understand physiology, just the same as any other chemical plant, a very complex chemical plant with exquisite controls most of which we have no idea how they work, what the design parameters are, what parameter is being controlled to do what and under what circumstances. Hacking into the control system of a complex chemical plant and perhaps bypassing the safety systems (when you don't know what is/is not a safety system) is not something to be done casually. That is how I see a lot of medicine, the same as trying to hack into a chemical plant where none of the pipes or wires are labeled as to what they are carrying, where they are coming from or where they are going, you don't know what the control system is based on, or even what is being controlled. Is that high pressure gas line carrying substrate for a process, a pneumatic control line, a pneumatic power line, a heat transfer line, or does it carry pneumatic messages inside tubes or some of all of these? We know that there is a gigantic amount of redundancy, and that there are so many different levels and layers of control that disabling all of them is difficult. Difficult but not impossible. The most important thing to remember in all of this is that these control systems have one and only one goal, the survival and reproduction of the organism. If for some reason we think a control system looks like it is doing something else that is probably a mistake on our part. A mistake based on the arrogance of our virtually complete ignorance of physiology.

I make a big point of how complicated physiology is, and how little we know about it because most people don't appreciate how complex it really is. The human genome has been sequenced (for some individuals), but the function of the vast majority of the DNA remains completely unknown. We don't know the complete function of any single protein; let alone how it interacts with thousands of other proteins under diverse circumstances.

A lot of what I cite is animal and in vitro research. I appreciate that in vitro and animal studies are not sufficient to base treatment in humans on. However there are no clinical trials where NO levels have been measured (there are no techniques that are sufficiently non-injurious to do in humans in vivo). There are no clinical trials where NO levels have been increased systemically (there are no generally recognized techniques to do so). With no techniques to measure basal NO and no techniques to increase it, some might say it can not be important. That would be wrong. 100 years ago there were no techniques to measure insulin levels in blood and no techniques to increase insulin levels either. Diabetes had been known for millennia. Eventually it was determined to be caused by insufficient insulin and that it could be effectively treated by supplying insulin from an external source. Diabetes in ancient Greece was caused by the same lack of insulin that modern diabetes is caused by.

Most of what I am talking about is quite generic to mammals. The "details" might be different, but the broad brush regulatory pathways are essentially the same. Those details are important for developing therapies specific to humans, but that is not my goal here. My goal is to illustrate how improved regulation of basal NO would improve regulation of vascularization. I don’t need to know the precise basal NO setpoint for a particular individual to know that bringing it closer to "normal" will improve the regulation of pathways mediated by NO.

We know that vascularization has not completely "failed". Even in the worst of circumstances wounds still heal although very slowly. The vascularization pathways are still working, still trying to maintain function, but they are having a very difficult time doing so. What ever is causing that must be a systemic agent that is at the heart of the regulation of vascularization. That agent is basal NO. Increasing basal NO won't act as a drug; it will simply allow normal physiology to reassert the control it evolved to do.

Something as important as basal NO is regulated, it is highly regulated. The problem is that our modern lifestyle has broken a critical component of that regulation. That critical pathway in NO/NOx physiology is the generation of NO and nitrite on the skin by a commensal biofilm of ammonia oxidizing bacteria due to release of ammonia in sweat.

Types of vascular effects covered.

General regulation of the vasculature via NO: vascular tone, blood and lymph flow, hypoxia, anemia, vascularization
Capillary rarefaction: hypertension, Raynaud's Rheumatoid arthritis and systemic sclerosis, Fibromyalgia
Acute changes: triggering of ischemic preconditioning à long term remodeling Retinopathy: Diabetic, hypertensive, tortuous vessels
Brain: Migraine, White matter hyperintensities, vascular dementia, reduced perfusion secondary to migraine, brain atrophy secondary to reduced perfusion
Diabetic vasculopathy: Diabetes type 1, metabolic syndrome, cause of vascular damage, cause of peripheral injury, nerve, poor healing, infections.

General Regulation

I have covered white matter hyperintensities in an earlier blog. I see that as the shut down of long range axonal transport due to low ATP as regulated by low NO. I will write about fibromyalgia in a later post. I did write about Morgellons, which I see as very similar to fibromyalgia except more in the skin and with greater systemic effects (likely because the skin is a big organ, and when a big organ is subjected to low NO stress, it affects a lot of things). I plan to write a future blog focused on fibromyalgia, so I won't spend as much effort on it as it deserves.

The engineering truism, "Good, Fast, Cheap; pick any two" is how evolution has configured physiology. Good only means good enough to survive and reproduce. Fast relates to the time scale of the organism's needs, when running from a bear, speed is of the essence. Cheap relates to the overhead in terms of metabolic cost and forgone reproduction. Every aspect of physiology has evolved as a compromise and trade-off between immediate costs and how that affects ultimate reproductive capacity. Regulation of blood flow is no exception. Every different aspect of vascular regulation is part of a unified physiology. The regulation of blood flow occurs over many different time scales (from seconds to months), under each and every different physiological state that a living organism can achieve (if regulation of blood flow ever "failed", the organism would die).

It is wrong to try and think about different aspects of that regulation in isolation, but only as part of the entire system. We know that the regulatory system evolved, and that every ancestor with a vasculature had a viable regulatory system (that would be every vertebrate ancestor). That constrains its properties considerably. This evolution probably explains a lot of the redundancy and robustness of physiological systems. When a new and improved pathway develops, the DNA encoding it becomes common in the gene pool because it provides improved survival and reproduction. The old pathway doesn't get removed; it stays there and is turned off, turned down, or inhibited, or relegated to a secondary, tertiary or later role. The "cost" of maintaining the DNA associated with a pathway now made obsolete is very small, the metabolic cost of maintaining DNA is that of the organic molecules that make it up, the metabolic cost of the enzymes that keep it repaired, the risk that a mutation in it will lead to something bad. Tiny risks compared to the risk that the newly evolved pathway won't always work in every situation that the old pathways worked in and which might be needed in some extreme circumstance, just in case. This is one of the difficulties in studying systems such as the vasculature. They are highly redundant and can tolerate quite large perturbations until they "break". When they do "break" the organisms dies. The transition between seemingly normal function and dysfunction leading to death can appear to be quite abrupt. It may be difficult to tell just how abrupt those changes actually are without knowing in detail exactly what is going on.

It is also wrong to try and apply any kind of "linear" model to physiology. Some times you can, but usually only for a small part of the actual dynamic range that physiology actually works in. Nothing in physiology is linear or continuous. Genes are expressed or not expressed, and discrete numbers of molecules of proteins are produced. Those proteins interact (or not) with discrete other proteins. Sometimes you can use a continuum model, but there are only 2 DNA molecules that the transcription factors can regulate. At the level of gene expression physiology is quite discrete.

The Good, Fast, Cheap tradeoff in human designed systems is somewhat different. Human labor in the design is often the largest part of the cost. Simplifying the design is done to minimize the design labor component. That always increases the cost and the response time. A typical heuristic for reducing design cost is to apply a "factor of safety". It is cheaper to simply use 3 times more structural material (as the ASME Boiler code calls for) than to calculate "exactly" what is needed, to make the system controls sufficiently precise and reliable such that a lower factor of safety is tolerable, or to tolerate occasional failure. Modular design is good engineering practice because it makes for easier design, easier debugging and easier coordination of human design efforts. None of those tradeoffs are important in living organisms, so what is perceived to be modular design is actually an artifact of systems that have evolved. Adopting a "physiology is modular" heuristic in examining evolved systems is fraught with potential error. There is no "evolutionary pressure" for organisms to evolve physiology in modules, only to evolve physiology that preserves the life and reproductive capacity of the organisms. Physiology may have the illusion of being modular because existing pathways can become duplicated and the now redundant pathways can diverge to accomplish different but similar tasks.

As human designed systems "evolve" down their "learning curve", more effort gets put into design. The design effort per unit may go down, but as the number of units goes up, the total design effort can become enormous. For a technology that is fully mature, the major cost is the raw materials. Evolved systems such as living organisms can in some ways be considered a "fully mature" design, that is where the engineering trade-off of "good, fast, cheap" has been "optimized" to a particular evolved value. That value may not be the value that we want, because it represents the optimization that occurred over many thousands of generations, mostly under conditions quite different than modern life. Our DNA didn't evolve for us to live happy lives. It evolved only because each and every one of our ancestors survived and reproduced, sometimes under quite horrific conditions.

General regulation of the vasculature via NO

There is an ok review that covers some of the basics.[1] There are a few misconceptions in this review. They make the very common error that too much NO is bad and that too much NO causes the formation of peroxynitrite (from NO and superoxide). This is simply incorrect. Peroxynitrite only occurs in vitro when there is near stoichiometric formation of NO and superoxide. [2] This is virtually certainly the case in vivo, but in vivo is considerably more complicated because superoxide (and peroxynitrite) is always confined by lipid membranes. They are both anions, and lipid membranes are impermeable to anions except through anion channels. Superoxide from mitochondria is confined to the mitochondrial inner matrix. Peroxynitrite is similarly confined. Peroxynitrite can decompose into NO2, and NO2 can diffuse through lipid membranes.

They are correct that it is a balance. They don't seem to appreciate how much feedback and crosstalk there is between NO and oxidative stress. NO and superoxide are very much complementary physiological principles. They are analogous to the conjugate variables of quantum mechanics, to the complementary principles of Yin and Yang, male and female, hot and cold, light and dark. These are only analogies, and shouldn't be taken literally. NO and superoxide react at near diffusion limited kinetics, as fast as it is possible for chemical species to react with each other. It is not possible to have both NO and superoxide present simultaneously. Which ever one is in excess will destroy the other. Because NO is lipid soluble (~10x over aqueous in isotropic lipid (lipid membranes are not isotropic, so that is more complicated still)) and superoxide is not, lipid membranes confine superoxide but not NO. This allows NO and superoxide to (nearly) co-exist in close proximity, provided the enzymes providing the NO and superoxide are kept separate (although nitric oxide synthase does make both NO and superoxide as discussed below).

A great many sources of both NO and superoxide are co-regulated by NO, superoxide and peroxynitrite. For example nitric oxide synthase generates both NO and superoxide. As the L-arginine level gets low, then NOS generates superoxide and forms peroxynitrite which has the effect of modifying NOS by oxidizing a critical zinc thiol complex so that NOS becomes uncoupled, and instead of making NO and superoxide makes only superoxide. This can be thought of as the "off" switch for NOS generating NO. A pulse of superoxide from another source can drop the NO level, accelerating the production of NO, locally depleting L-arginine, superoxide is formed by NOS, peroxynitrite is generated, this uncouples NOS which generates more superoxide until the NOS in the vicinity is all irreversibly switched to making superoxide instead of NO. This "switch" changes physiology from a low oxidative stress state dominated by NO to a high oxidative stress state dominated by superoxide. This is the generic "stress" response; lower NO levels switch physiology to respond to stress. This is how mitochondria respond, this is how ischemic preconditioning is triggered, this is how the respiratory burst is triggered, this is what mast cells do, release proteases to switch xanthine oxidoreductase to generate only superoxide. Low NO makes the threshold for all of these switches lower. In the limit, the threshold becomes so low the cells are only in the oxidative stress state. This can be sustained for considerable time, depending on the tissue compartment. It cannot be sustained indefinitely in all tissue compartments without adverse effects in multiple systems. Characteristic vascular remodeling is one of those adverse effects in the vasculature.

There is hysteresis when an organisms or tissue compartment enters an oxidative stress state. Usually stress states are conditions of high metabolic demand, it is advantageous to minimize the metabolic resources necessary to maintain the organism in that state, to free up those resources for productive use.

Peroxynitrite damage occurs due to slow turn off of oxidative stress

Peroxynitrite is a normal signaling compound. Peroxynitrite only occurs at near stoichiometric levels of NO and superoxide. Peroxynitrite effects are not observed in healthy individuals. Peroxynitrite damage doesn't occur in low NO states, it also doesn't occur in high NO states. Presumably peroxynitrite effects occur during the transitions of physiology, during the switching transients; from a superoxide dominated state to a NO dominated state and/or from a NO dominated state to a superoxide dominated state. We know the transition from a NO dominated state to a superoxide state is rapid and exhibits hysteresis. The main NO generating enzymes are turned off by peroxynitrite. The zinc thiolate couple in NOS becomes oxidized which decouples NOS so it produces only superoxide, similarly the Mo-thiol couple in xanthine oxidoreductase becomes oxidized so it no longer reduces nitrite to NO but only generates superoxide from O2 and reducing equivalents.

Presumably the damage observed and attributed to peroxynitrite occurs while physiology is attempting to switch from a superoxide dominated state to a NO dominated state. This requires sufficient NO to overcome the hysteresis of the low NO state. If there isn't enough NO, the transition cannot occur crisply, and physiology stays longer in the state where it generates the near stoichiometric levels of NO and superoxide that cause peroxynitrite damage. The solution to this ineffective and slow switching is to increase basal NO levels so that the transition can occur more rapidly and more robustly.

This is an extremely important point. The presence of peroxynitrite damage is not due to too much NO. Peroxynitrite damage is due to there being not enough NO (except under very rare those circumstances and the problem then isn't too much NO but not enough ATP see the blog on mitochondria damage).

The switching from the NO state to the superoxide state can occur very quickly. If you need to run from a bear, release of epinephrine causes acute oxidative stress. Revving up metabolism takes some time. Mitochondria need to disinhibit cytochrome c oxidase, the heart needs to start pumping blood at a high rate, get the liver putting out glucose at a high rate, the pancreas putting out insulin at a high rate and the lungs supplying O2 at a high rate. ATP cannot be stored. There is hysteresis in systems supplying ATP; ATP must be generated as fast as it is used. An analogy would be a pipeline which has inertia. You can't turn on and off a large pipeline instantaneously. The same is true of ATP. When it isn't needed, but might be in a few seconds, mitochondria get ready to generate it but dissipate the mitochondria potential as heat instead of generating ATP. This wastes substrate, but it is more important to be able to ramp up ATP production in a few seconds and escape with injury than ramp it up slowly and get caught. This readying of physiology to supply ATP at high rates is known as the "fight or flight" state.

When ATP is needed at high rates as in "fight or flight", an optimized organism would shut off ATP consuming pathways that are not needed during that time. If that time is brief, those longer term systems can be turned back on. If the time is prolonged, then what ever those pathways are supplying is lost until they are turned back on. Physiology can turn on the fight or flight state in a few seconds, it takes much longer to stand down from it.

A fundamental aspect of the damage that occurs from chronic low NO occurs because of the chronic activation of the "fight or flight" state. The fight or flight state evolved to be a temporary state. An emergency overload state where some necessary metabolic functions are put off until later to save ATP for immediate consumption. A state where damage is tolerable to save the life of the organism.

Modulating ATP demand over time is an extremely important physiological process, and one which is insufficiently appreciated because it is so automatic, so universal, and goes to such deep evolutionary time that all organisms exhibit it. The reason all organisms exhibit it is because is reduces the "overhead" associated with the production of ATP. That overhead includes the molecules that make up the ATP generating apparatus, the additional muscle to carry those extra muscles around. "Just in time" ATP generation allows those extra molecules to be used for reproduction instead. Over evolutionary time ATP allocation has evolved to be very efficient. This allocation of ATP is what occurs during fight or flight, it is what occurs during ischemic preconditioning.

Because peroxynitrite is a normal signaling compound, there will always be peroxynitrite effects, there will always be peroxynitrite "damage". Some amount of "damage" is unavoidable and physiology has evolved systems to deal with unavoidable amounts of damage. That damage isn't repaired during the low NO state because physiology is doing other things with the ATP, such as running from a bear. Repairing damage has too low a priority. The damage accumulates until there is a high NO state during which it can be repaired. Chronic low NO prevents the repair of peroxynitrite and other damage. When ever the damage rate exceeds the repair rate, damage will accumulate. The absolute rates don't matter, only that the damage exceeds the repair. The problem isn't too much damage, the problem is insufficient repair.

Vascular tone, blood flow, and lymph flow

The vasculature is active tissue. Arterial and venous blood is under pressure, the pressure drop between the heart outlet at the aorta and the heart inlet at the vena cava drives the flow of blood. The cross section of the vessels is regulated locally along their length, in (very) complex ways to regulate that flow. Red blood cells carry O2 from the lungs to the peripheral tissues and carry CO2 back to the lungs. Red blood cells are confined to the vasculature. O2 diffuses from red blood cells into the peripheral tissues. All tissues obtain O2 from the blood except for the external skin. The outer few hundred microns of the external skin derive O2 from the external air. All O2 diffusion is passive diffusion down a chemical potential gradient (more on this later).

The usual lack of blood flow in the skin is easily observed because non-pigmented skin is transparent and is not seen to be red except under conditions of hyperemia. Only a small fraction of the body is in direct contact with blood, only the endothelium. All other cells derive nutrients (other than O2) from extravascular fluid, that is from fluid that has "leaked out" of the vasculature (though it is not leakage per se, it is absolutely necessary extravascular flow). It is this extravascular fluid that carries glucose to the cells, other nutrients including protein (mostly as albumin), insulin, and all other nutrients and signaling components of blood. The extravascular fluid moves much slower than blood. Virtually all cells derive glucose from this extravascular fluid. Necessarily the glucose and insulin levels in plasma in contact with cells is lower than in bulk blood because intervening cells have consumed some of it. How much lower is a good question which is difficult to answer because getting samples to analyze is extremely difficult. The glucose level in the extravascular space next to the cells that are taking that glucose up is of course a much more important parameter than what the glucose level is in bulk blood remote from the cells that are using it.

Adequate flow of extravascular fluid is just as important as adequate flow of blood. The time constant for extravascular fluid flow is longer, but is obviously important and so obviously is actively regulated by physiology. If it were not actively regulated either there would be too much, or too little, or both in different tissue compartments.

The importance of extravascular flow of lymph is not always appreciated. Because it cannot be measured easily and is different in every tissue compartment (or even in the same tissue compartment due to gradients between capillaries), it is not as well mixed as blood is, it is not routinely measured and there are no clinical correlates with it. The fluid must "leak" out of capillaries at the proper rate, and then be transported along through the lymph vessels at the proper rate and then fed back into the circulation at the proper rate.

Accumulation of extravascular fluid is known as edema. This occurs for a variety of reasons, because the flow channels are blocked (as in filarial diseases such as elephantiasis), when there is too much fluid because the kidneys can't get rid of it and it has to go somewhere (the edema of congestive heart failure) and for things such as ascites (in the abdominal cavity).

CO2 must be carried back to the lungs also. CO2 transport is pretty complicated and won't be discussed in detail. CO2 as an uncharged gas diffuses pretty well. It is water soluble and forms carbonic acid, H2CO3. There are significant kinetic impediments to the formation of H2CO3, and so there are enzymes, carbonic anhydrase that catalyze it. H2CO3 disproportionates into H+, HCO3-, and CO3(2-) depending on pH. These are charged, and so cannot penetrate lipid membranes except through ion channels. Some of these are actively ported through cell membranes. Other ions must be co-ported to maintain ion neutrality. Chloride is the ion that does that in red blood cells, but nitrate and nitrate are similar to chloride ion in a lot of ways. They are not considered that important in ion channels, so the conductance of ion channels for nitrate and nitrite are not always measured along with other ions. CO2 can diffuse from tissue compartments containing carbonic anhydrase through intervening tissue compartments that don't, and into tissue compartments that do.

Regulation of vascular tone, blood flow, and lymph flow

The primary regulation of blood flow is via regulation of the cross section of vessels carrying that blood. The heart can pump more blood, and at a higher pressure, but for that blood to go where it is important for it to go the vessels have to modulate their cross section. Vessels are dilated where blood is being regulated to go, and constricted where blood is being regulated to not go. There is limited blood and also limited blood pumping capacity, so both types of control are needed, local to increase local blood flow, and non-local to decrease other blood flow.

The major regulator of vascular diameter and vascular tone is nitric oxide. NO is produced in the endothelium by eNOS. It is the NO that diffused into the vessel wall that regulates its tone. NO activates sGC which makes cGMP which relaxes smooth muscle. NO also diffuses into the blood and is taken up by red blood cells via kinetics that are first order in NO and first order in red blood cell concentration. The major passive sink for NO in the body is hemoglobin. Hemoglobin has a very high affinity for NO, and metabolizes it to either nitrite plus nitrate or to nitrosyl heme. Hemoglobin is normally confined to erythrocytes. Free hemoglobin destroys NO ~600 times faster than does Hb in erythrocytes. Free hemoglobin is responsible for the acute constriction and hypertension associated with hemolytic anemia as in sickle cell anemia.

How much NO diffuses into the blood, into red blood cells and is consumed and swept away and how much NO diffuses into the vessel wall and is consumed by superoxide and how much is left to activated sGC and cause vasodilation is a delicate balance between NO production, hematocrit, blood velocity, redox state, lipid vs. aqueous partitioning, ATP level, O2 level, L-arginine levels, asymmetric dimethyl arginine levels, nitrite, R-SNO thiols, NO from the extravascular space and other things. We know that all of those things are important, none of them can be measured on the length and times scales that we know are important in vivo. Neurogenic or receptor mediated production of superoxide can acutely consume NO and cause acute constriction. Superoxide can also be dismutated into H2O2 which can also cause vasodilation (but that is usually at high metabolic rate, not at rest).

When hematocrit is acutely decreased (taking out blood and replacing it with cell-free fluid, plasma or starch solution) as in isovolemic anemia, exhaled NO levels increase.[3] As Hct was decreased by dilution with hydroxyethyl starch (30, 23, 17, 11 %), cardiac output rose (0.52, 0.60, 0.70, 0.76 L/min), and exhaled NO levels rose (30, 34, 38, 43 nL/min). This demonstrates that NO levels in exhaled air are coupled to hemoglobin concentration in blood. This actually makes sense because the hormone that determines when more red blood cells need to be made is erythropoietin (Epo) and Epo is regulated by HIF-1-alpha which is regulated by low O2 (hypoxia) and also high NO. Both low O2 and high NO are signals of "not enough hemoglobin". HIF-1-alpha also causes expression of VEGF (vascular endothelial growth factor) which is one of the major factors that triggers angiogenesis.

There is starting to be some appreciation that the anemia observed in many chronic diseases may be an adaptive response and not solely something pathological. Anemia increases NO levels. High hemoglobin levels will decrease NO because hemoglobin is the sink for NO. It is the product of NO concentration and hemoglobin concentration that fixes the NO destruction rate. That destruction rate equals the production rate because there is no accumulation. The NO concentration (which is what NO sensors react to) then goes inversely with hemoglobin concentration. In a number of disorders associated with anemia (especially end stage kidney failure), increasing hemoglobin levels to "normal" causes increased death rates over increasing it to somewhat less than normal. Not enough hemoglobin is bad, but not enough NO (because a high hematocrit is destroying it) is worse. Increased hematocrit had the largest adverse effect on vascular disorders. The increased death rates are not concentrated in one or a few categories, but spread out over many. I see this as evidence of how many physiological systems are dependant on proper levels of NO, and how closely coupled that level is to hemoglobin levels in blood. Another example is systemic sclerosis, the death rate is ~2x that of standardized death rates after subtracting out deaths due to systemic sclerosis, but the causes of death are spread out over multiple causes. Presumably what ever is causing the systemic sclerosis is also causing the increased death rates.

When more flow is needed, NO levels are increased.

When greater blood flow is needed acutely through a particular vessel, the velocity goes up, that shear then activates eNOS and NO is generated which causes the vessel to dilate. When tissue becomes hypoxic, NO is generated via reduction of nitrite by deoxyhemoglobin and by other enzymes. When the vessel cannot supply sufficient oxygenated hemoglobin via blood, the increased NO level becomes chronic. Increased NO level would then be the ideal signal to trigger generation of more blood vessels through angiogenesis. It turns out that increased NO does trigger angiogenesis, and blocking NOS does inhibit angiogenesis. Supplemental IP nitrite substantially accelerates compensatory angiogenesis around a blocked artery in mice. The positive effects of nitrite were observed over a very broad dose range, something like a factor of 400.

Increased NO mediates increased blood flow over time scales from seconds to weeks. Presumably these multiple mechanisms for regulating blood flow evolved from an archetypal blood flow regulation mechanism which involved NO.

Acute Regulation of blood flow by NO, not by O2

Under conditions of isovolemic anemia, blood flow increases. The "conventional wisdom" is that it is "hypoxia" that causes the increased blood flow; however that cannot be correct because there actually is no hypoxia. There is no reduction in the O2 level in either the arterial blood, or the venous return blood. With no reduction in O2 level, there is no hypoxia. With no hypoxia, hypoxia cannot be a signal for the body to use to regulate blood flow.

At rest, acute isovolemic anemia is well tolerated. A 2/3 reduction in hematocrit has minimal effect on venous return PvO2, indicating no reduction in either O2 tension or delivery throughout the entire body. At 50% reduction (from 140 to 70g Hb/L), the average PvO2 (over 32 subjects) declined from about 77% to about 74% (of saturation). The reduction in O2 capacity of the blood is compensated for by vasodilatation and tachycardia with the heart rate increasing from 63 to 85 bpm. That the compensation is effective is readily apparent. The mechanism is not. The “obvious” explanation is that “hypoxia” sensors detected “hypoxia” and compensated with vasodilatation and tachycardia. However, there was no “hypoxia” to detect. There was a slight decrease in blood lactate (a marker for anaerobic respiration) from 0.77 to 0.62 mM/L perhaps indicating less anaerobic respiration and less “hypoxia” (though lactate production occurs under oxic conditions). The 3% reduction in venous return PvO2 is the same level of “hypoxia” one would get by ascending 300 meters in altitude (which from personal experience does not produce tachycardia). With the O2 concentration in the venous return staying the same, and the O2 consumption staying the same, there is no place in the body where there is a reduction in O2 concentration. Compensation during isovolemic anemia cannot occur because of O2 sensing.

When red blood cells of dogs are replaced with red blood cells that have been fully oxidized to methemoglobin (and so cannot carry O2), compensation for reduced O2 carrying capacity of blood is greatly reduced.[4] While maintaining the same hematocrit Hct (43%) and substituting (0, 26, 47%) fully metHb erythrocytes, the cardiac output (CO) declined (178, 171, 156 mL/m/kg) while the arterial PaO2 (93, 87, 84 mmHg) and PvO2 (55, 46, 38) also declined. In contrast, when acute isovolemic anemia (Hct 40, 30, 22) was induced using plasma, compensation was much better, CO (155, 177, 187), PaO2 (87, 88, 91), and PvO2 (51, 47, 42). When anemia was induced using dextran solution (Hct 41, 25, 15) cardiac output (143, 195, 243), PaO2 (89, 92, 93), PvO2 (56, 56, 51) compensation was better still. As part of their experiments with the metHb tests, a final dilution was done with dextran to lower the Hct to 26% while still maintaining 47% metHb. Compensation was much improved with CO (263 mL/m/kg), PaO2 (86 mmHg), and PvO2 (41 mmHg) all were increased, despite lower Hct, greater O2, and less “hypoxia”. The compensatory mechanisms to deal with this “hypoxia” cannot be due to reduced O2 levels because the O2 levels were not reduced, in fact, the O2 levels were increased. MetHb does bind NO, not quite as well as does Fe(II)Hb, but the presence of metHb erythrocytes clearly adversely effects compensation. The authors attributed the increased cardiac output to reduced blood viscosity in the case of reduced cell concentration. However when viscosity is increased, blood flow does increase.

When blood viscosity is increased during acute anemia, NO levels increase, flow mediated vasodilation increases and flow increases.

The optimum hemoglobin concentration for O2 delivery is as low as 15%. For O2 delivery to the brain it is about 30%. Normal hemoglobin levels are ~44%.

In summary, when the O2 carrying capacity of blood is reduced by removing erythrocytes, there is essentially complete compensation over a wide range by increased blood flow such that reduced O2 levels never occur. When the O2 capacity of blood is reduced by oxidizing hemoglobin to methemoglobin, there is much less compensation and reduced O2 levels do occur. When viscosity of blood is increased, there is increased shear, increased NO production and increased flow.

Long Term Regulation of vascularization

If acute regulation of flow of blood in the vasculature is not regulated by O2 levels, but is regulated by NO levels, should we expect that physiology uses a control system operating over a different time scale utilizing a different control scheme for other regulation?

Using a different control system presents potential difficulties when transitioning from one control scheme to the other. For the control to be stable, there can't be control regimes where one system is calling for more and the other system is calling for less. The control needs to be monotonic when averaged over a period that is long compared to the response time. We know that the control system evolved. In virtually all cases evolution takes an existing pathway or structure and modifies it for improved or different functionality.

If we know that acute increases in blood flow are mediated through increased NO, and we know that some instances of angiogenesis are increased by increased NO, it is likely that increased NO is the generic control system used for regulating vascularization.

Regulation of vascularization is a critically important physiological effect, and it is regulated exquisitely well and exquisitely complexly. Some of the details are known, many (probably most) are not. The vasculature is "well formed", that is it is very closely matched to the physiological needs of the tissue compartment it is in. There is no great excess of vessels and no great deficiency either. Organisms grow from a single cell, and have a well formed vasculature at all sizes. Organs grow in size, organs also shrink.

For vascularization to be regulated over so many orders of magnitude in size in so many different tissue compartments as organs grown and regress, there must be at least two types of regulation. There must be a mechanism that senses when there is not enough perfusion in a tissue compartment and signals the generation of more vasculature. There must also be a mechanism that senses when there is excess vascularization in a tissue compartment and ablates that excess. We know that there must be at least those two mechanisms. No doubt there are others, but I will focus on those two. It may also be a single mechanism operating in different regimes. I think a single mechanism is the most likely, that mechanism being NO with high NO triggering angiogenesis and low NO triggering ablation of vessels.

When are those signals generated?

They must be generated "at rest". Most growth occurs "at rest". The vasculature of organisms remains well formed after long periods of rest. The blood flow through some tissue compartments doesn't change much between periods of activity and inactivity. In utero, the fetus is always "at rest", capillary spacing is and must be regulated equally well in utero. Actually it must be regulated better in utero than after birth. A fertilized egg increases in size by many orders of magnitude very rapidly. An infant increases in size only about 1 order of magnitude as it becomes an adult and over a much longer time period. At rest would be the ideal time to fix the basal capillary spacing. Metabolic demands are low and constant. The appropriate level of vascularization could be established with the proper excess safety factor. At rest is a good time to remodel important physiological systems because metabolic need for those systems is at a minimum. While running from a bear is a bad time to divert resources into remodeling active systems. There may be additional signals that occur at other times (such as during exercise), but I will focus on the one(s) "at rest" which presumably are involved in all tissue compartments and so is likely to be the archetypal signaling system.

We know that hypoxia is involved in regulation of vascularization via HIF-alpha. Cells not getting enough O2 could generate a signal to generate more vasculature to bring more oxygenated blood to that tissue compartment. How can excess vasculature be measured? It cannot be measured by O2 level. The O2 level in arterial blood is very close to the level in air. That is the level in tissues at rest. At rest, the O2 level is essentially independent of capillary density. O2 demand is low, there are no large gradients in O2 concentration. All arterial blood is at near the saturation level in air. Venous blood is also regulated to a fairly constant O2 level. Gradients in O2 concentration between blood and mitochondria (where O2 is consumed) are in the extravascular space, not in the vasculature.

It also needs to be remembered that when embryos regulate capillary density they do so with quite different O2 partial pressures than do ex-utero organisms.

Physiology needs to generate a signal that measures how diffusively close red blood cells are to cells that require O2 and other nutrients in blood, that is, is a particular cell close enough to red blood cells such that it can obtain enough O2. If there are enough red blood cells close to a cell, that cell can indicate when it has sufficient vascularization and when there is not enough.

I suggest that an important component of that signal is NO. NO has physical properties very close to that of O2, the diffusion of NO through tissues is virtually identical to that of O2. O2Hb is the sink of NO, so the vasculature has the lowest NO level in the body (neglecting formation of superoxide (which is generated in mitochondria and microsomes) for the moment). If there was a volume source of NO, the basal NO level would be higher the farther from a capillary that tissue compartment was.

In summary, there must be a signal by which insufficient vascularization triggers angiogenesis, and also a mechanism by which excess vascularization is ablated. NO can signal both instances due to O2Hb acting as the sink for NO and with the extravascular space acting as a volume source.

Hypoxia and NO activate HIF-alpha which causes the expression of VEGF which is important in angiogenesis. NO is known to be important in angiogenesis, expression of iNOS is important in angiogenesis surrounding vascular infarcts. Neurogenic release of NO is what causes vasodilation by activating sGC. If the neurogenic NO were not sufficiently swept away by enough O2Hb, it would be a good signal for angiogenesis.

Regulation Oxygen delivery, Oxygen extraction, Ischemia reperfusion injury

Physiology can only use what are called intensive properties, properties that are proportional to the concentration or chemical potential of a substance, and not extensive properties, properties that are dependant on the amount of substance available. O2 partial pressure is the same as O2 chemical potential. O2 partial pressure is not proportional to the O2 content of blood because hemoglobin has a non-linear O2 dissociation functionality. O2 partial pressure is proportional to O2 content of plasma because plasma does have a linear O2 dissociation functionality (actually it is simple solubility via Henry's Law).

There are numerous misconceptions about this regarding O2 delivery, O2 extraction and the blood. O2 only moves by passive diffusion down a gradient in chemical potential. In homogeneous media this is down a concentration gradient via Fick's law of diffusion. By homogenous media I mean media where the chemical potential is strictly proportional to the concentration. In non-homogenous media such as blood or mixtures of lipid and aqueous phases one has to be careful. The "concentration" of O2 in red blood cells (mL/L), is not the same as the "concentration" of O2 in plasma in equilibrium with those red blood cells. The chemical potential of O2 in red blood cells and in plasma in a sample of blood is the same (and is the same in all fluids in mutual equilibrium that is the definition of equilibrium). In hemoglobin there is a non-linear relationship between O2 partial pressure and O2 concentration. Physiology can't measure O2 concentration in blood, all it can measure is O2 partial pressure, or more precisely the O2 chemical potential of the O2 sensors in equilibrium with that blood. Lipids have ~10x higher solubility of O2 and NO than do aqueous fluids. This can affect rates of chemical reactions a lot, as can the lower dielectric constant inside lipids. Ions can't enter lipids. Highly polar compounds like water or H2O2 can diffuse through lipid, but not as quickly as something nonpolar like NO or O2.

Some of the physiology literature talks about "oxygen extraction" from blood as if that is a real parameter. It isn't. Oxygen only moves by diffusion. There is no active transport. Tissues don't "extract" O2 from blood, O2 diffuses out of blood if the tissue the blood is flowing through has a lower O2 chemical potential than the blood flowing through it. If tissues are at the same O2 partial pressure as the blood, then they do not extract O2. If the tissues are at a higher O2 partial pressure, O2 diffuses out of the tissues and into the blood. There are no barriers to O2 diffusion. There is nothing that can block O2 diffusion. The vasculature can regulate where blood flows, and bypass less important organs to divert blood to more important organs. Tissues can only regulate O2 consumption by regulating the affinity for O2 of enzymes that consume O2.

Mitochondria are the ultimate sinks of O2; cytochrome c oxidase is the enzyme that reduces one O2 to two H2O's. The binding coefficient (Km) of cytochrome c oxidase for O2 is a sensitive function of the NO level. NO binds to cytochrome c oxidase and inhibits the binding of O2. This is an extremely important regulatory system for mitochondria. It is by regulating the NO level that the affinity of mitochondria for O2 is regulated. High NO, low O2 affinity. Low NO, high O2 affinity. The generation of superoxide by mitochondria under conditions of hypoxia is then seen as a necessary regulatory function. When cells become hypoxic, their mitochondria generate superoxide, that superoxide (confined to the inner matrix!) pulls down the NO level, cytochrome c oxidase is disinhibited, binds O2 at a lower O2 partial pressure, O2 is consumed to a lower partial pressure, the partial pressure gradient between the blood vessel (where it is nearly constant) and the mitochondria (where it is consumed by mitochondria) increases and so the flux of O2 (moles O2 per second) diffusing to the space where the mitochondria is now increases, relieving the "hypoxia". The problem of insufficient "oxygen extraction" is too much NO on the mitochondria. But is that really a problem? Cells don't need "oxygen extraction", they need ATP. If cells have enough ATP, they don't need anything else. Mitochondria are not the only source of ATP. Cells can make ATP via glycolysis which does not consume O2.

It is the attempt to make ATP using O2 under conditions of very high NO during sepsis that causes the mitochondrial damage and the multiple organ failure.

Under conditions of hypoxia, mitochondria first generate superoxide, and pull the NO level down to extract as much O2 as possible. Once that O2 is exhausted, mitochondria have a different need, to prevent the production of a massive amount of superoxide if and when O2 levels are restored. Most of the damage that occurs during ischemia-reperfusion occurs during the reperfusion, not the ischemia. When all the enzymes in mitochondria are primed to make superoxide, it is a bad time to supply a large bolus of O2 because much of it can get turned into superoxide.

Preventing damage following reperfusion is I think where nitrite comes in. There are many different enzymes that reduce nitrite to NO in the cytosol, in mitochondria, in microsomes. The nitrite reductase activity of these enzymes is O2 level dependant. O2 inhibits the production of NO from nitrite by them. When the O2 level drops, they become active nitrite reductases and produce large quantities of NO. This NO binds to heme enzymes and blocks their take-up of O2. This NO blocks the formation of superoxide following reperfusion.

As mentioned earlier, damage due to peroxynitrite from NO and superoxide occurs only when both are generated at near equimolar fluxes. This occurs during the transitions from a state dominated by one to a state dominated by the other.

Regulation of nutritive blood flow

In fMRI BOLD testing, it has been observed that the quantity of blood that flows in the region activated by the neurogenic NO release exceeds the nutritive quantity needed to supply the metabolic activity of that activated region. If the normal mechanism produces blood flow in excess of nutritive needs, there is a "factor of safety" and other mechanisms regulating blood flow are not needed. This is an important point. Blood contains many factors that are needed at different levels, O2, glucose, albumin, insulin, various proteins, hormones, immune cells, cytokines, etc. The levels of many of these factors in blood change week to week, day to day, even minute to minute. The needs for each of them in a particular tissue compartment also change. How many of them can the local regulation of blood vessel tone be determined by? In principle many of them, however if there is more than one control parameter, the control system may become over specified and instabilities may occur. It is much easier for evolution to modify and elaborate on a primitive ancestral trait than to evolve one de novo. The shortest time scale need is for O2. The time scale for O2 need is seconds, the time scale for glucose is minutes, the time scale for angiogenesis is days. If NO is both the shortest time scale control parameter and also the longest time scale, it seems implausible that a different control parameter and/or control system would have evolved to handle an intermediate time scale.

If blood flow in the brain is not acutely regulated to provide for acute nutritive needs, how is it that nutritive needs are met long term? The signals that do regulate acute blood flow either have some dependence on nutritive needs, or those nutritive needs are met by always providing an excess, or the tissue remodels to reduce demand when there is not sufficient excess.

If blood flow to the brain is reduced, how will the brain remodel itself to accommodate? Presumably brain cells self-regulate and reduce their metabolic load until it matches the supply of substrates provided by the blood. Presumably this involves pruning of lesser used cells. Pruning of brain cells is observed following a stroke. As cells necrose, the inhibitory signals they produce are lost, down stream cells become disinhibited, cells become over excited and excitotoxic death occurs. Excitotoxic death strikes cells with compromised metabolic capacity. Compromised due to insufficient blood supply, compromised due to mitochondrial damage, compromised due to other damage.

Capillary rarefaction

I suggest that the capillary rarefaction observed in many disorders, systemic sclerosis, Raynaud's, hypertension, dilative cardiomyopathy is ultimately caused by normal vascular remodeling via the same mechanism that leads to reduced blood flow, that of low NO. Reduced blood flow is observed in many neurological disorders, many even before there are overt signs of neurodegeneration. If blood flow is regulated to be chronically low, presumably efficient regulation of vascularization would ablate the seemingly excess vasculature that is seemingly present, resulting in capillary rarefaction. It needs to be appreciated that this is the completely normal response to a reduced perfusion setpoint. Physiology can't "compensate" because it is the compensatory pathways that are actually doing it.

If there is capillary rarefaction in an organ such as the heart, how will it respond? There isn't sufficient blood supply to maintain normal cell density, the cells "too far" from a capillary become stressed, die, and are cleared. If they are replaced, the replacement cells are insufficiently supplied also. The space could become filled with non-metabolically active fibrotic tissue. The heart still needs to pump sufficient blood, so it gets bigger, but weaker as fibrotic tissue replaces muscle. I think this is what eventually leads to dilative cardiomyopathy.

If the liver doesn’t have enough mitochondria to dispose of reducing equivalents, what does it do with them? Usually it makes fat. I think that is the source of fatty liver from chronic alcohol consumption. Alcohol is metabolized by alcohol dehydrogenase which makes NADH which can only be disposed of in complex I in mitochondria, or by making lipid. Ectopic lipid is an end stage symptom of many degenerative diseases associated with vascular abnormalities, liver failure, kidney failure, dilative cardiomyopathy. Excess NADH makes superoxide, and that superoxide lowers NO levels. Acutely that is adaptive, in that it disinhibits cytochrome c oxidase and allows for more O2 reduction to dispose of the reducing equivalents. In the long term, insufficient NO reduces mitochondria biogenesis resulting in systemic excess reducing equivalents which can only be disposed of by generating lipid. I think this is how the extreme obesity of hundreds of kg occurs. Individuals without enough mitochondria generate ATP via glycolysis; this generates lactate which is disposed of by generating lipid.

Hypertension

Hypertension occurs via increased vascular tone, the stiffness of vessels increases requiring higher pressure to drive the same volume of blood through the vessel bed.

Hypertension is associated with capillary rarefaction, with capillaries getting farther apart then is considered "normal". Capillaries farther apart means fewer capillaries in a tissue compartment and so blood flow through the remaining capillaries must be increased if the same blood delivery is going to occur.

Flow through a capillary and pressure drop across that capillary can be regulated independently. There is no need for a higher pressure drop to drive more blood through a capillary, the capillary could increase in cross section and accomplish the same thing. However bulk flow of blood through a capillary is not the only requirement. As mentioned before, extravascular flow of plasma is equally important (but on a different time scale, minutes as opposed to seconds). Extravascular cells derive nutrients only from local flow of plasma through the extravascular space. This plasma "leaks" out of the capillaries (however it is not "leakage" per se, it is a required flow). That "leakage" is likely proportional to the surface area of capillaries in that tissue compartment and also to the pressure drop from the inside to the outside of the capillaries. As capillary rarefaction reduces the number of capillaries, the total cross section goes down, to supply the same flow of extravascular fluid the pressure would have to go up.

I suspect that increasing extravascular flow is the physiological reason that blood pressure increases. Increased pressure drop through capillaries increases flow through the extravascular space that bypasses those capillaries. I suspect that increased extravascular flow is required to compensate for capillary rarefaction, both to supply the same extravascular flow, but also to increase extravascular flow to compensate for fewer mitochondria and greater ATP from glycolysis (which requires 19x more glucose for the same ATP production). NO is what triggers mitochondria biogenesis, so low basal NO will result in fewer mitochondria and more ATP from glycolysis necessitating increased extravascular fluid through fewer capillaries requiring higher pressure.

Raynaud's Syndrome

Raynaud's occurs when exposure to cold causes acute constriction of blood vessels in the skin, leading to pain, and in some cases necrosis. It is sometimes one of the first symptoms of capillary disorders, and usually accompanies all the others.

Because the output of the heart is limited, control of blood flow to peripheral tissues requires the regulation of both the pressure drop through the specific tissue being perfused, but also the pressure drop in the rest of the vasculature. The skin is a large organ, and constituting the outside surface, the skin is the only place where heat can be dissipated. Heat is brought to the surface by the blood and is easily observed as flushing. The external few hundred microns of skin derive O2 from the external air, they receive all other nutrients from the extravascular flow of plasma.

Conserving heat by constricting blood vessels in the skin when the skin is too cold is an essential part of maintaining the proper internal body temperature. Constricting blood vessels is usually done by generating superoxide, destroying NO and causing a reverse of the vasodilation that NO is producing.

Tortuous vessels

It is the take up of NO by hemoglobin in blood cells that results in the particular morphology of low NO damaged vessels, what is called "tortuous" vessels. There are some cases of familial tortuosity. This tortuosity is produced by essentially the same mechanism that stream meander is produced by. At high velocity there is erosion of the stream bank, and deposition of material at regions of low velocity. The same characteristic mechanism occurs in vessels but via different mechanisms. The crucially important similarity is that the flow of fluid inside the vessel affects the morphology of the flow pattern. In a stream the flow is within the stream banks. In blood vessels the flow is within the vessel.

Red blood cells are denser than plasma, so when there is accelerated flow there is segregation to the outside of the curved flow. This reduces the thickness of the boundary layer along the endothelium and increases the removal of NO at the high velocity outside region by the hemoglobin in the red blood cells that are now closer to the wall (just like in a stream meander). Removal of NO reduces the NO level on the inside and outside of the vessel, and this causes regression of that tissue via apoptosis due to low NO. This regression at high velocity allows for a vessel with an isotropic high velocity to enlarge in diameter. The tissue outside the vessel must regress so as to allow space for the vessel to enlarge in diameter. A series of images that just scream "low NO induced apoptosis" is here.[5] The images are of brain sections at autopsy of an individual with white matter hyperintensities. The vessels show a characteristic "tortuous vessel inside a cavity". The vessel is highly tortuous and the surrounding tissue has regressed leaving a cavity. Since there is no scaring, and there are markers characteristic of apoptosis, presumably the vessel is either a source of a pro-apoptotic diffusible factor, or a sink of an anti-apoptotic factor. It turns out that vessels are sinks for NO, which is an anti-apoptotic signal. The tortuousness of the vessel relates to how the flow changes the local source-sink properties of the vessel.

These blood vessels are often tortuous and appear as a tortuous arteriole in a cavity. These images are quite striking. The vessel is quite corkscrew-like, very tortuous and is in an empty cavity, devoid of white matter. These are sites of apoptosis. Low NO causes the tissues outside the vessel to regress (via apoptosis) and so the vessel migrates in that direction. Tortuous vessels like this are easily seen in retinopathy (where they accompany WMH) where they are caused by the same mechanism. In retinopathy, often when vessels cross there is observed to be "nicking", that is, a reduction in the diameter of the vessels. This is due to the decreased NO at the site of crossing (my hypothesis) where there is more hemoglobin to act as a sink of NO. This migration and remodeling of blood vessels by local NO levels is part of the normal regulation of capillary spacing (my hypothesis).

As cardiovascular risk factors increase there is decreased vascular reactivity; [6] that is there is reduced responsiveness shear induced increased blood flow, of exercise induced hyperemia, and even of NO induced hyperemia. This is what would be expected if basal NO levels are reduced because it is generation of NO by the endothelium that activates sGC and generates cGMP which relaxes the vascular smooth muscle. With a lower background level of NO, it takes more neurogenic NO, more shear generated NO, or more NO donor to achieve the same cGMP level and the same level of vasodilation.

Reductions in NO mediated vasodilation are observed in aged rats.[7]

These observation of long term remodeling of vascular morphology suggest that the remodeling is coupled to NO physiology, and that the basal level of NO is important in that regulation.

White matter hyperintensities

The tortuous vessels and cavities apparent on autopsy around those tortuous vessels indicate loss of neuronal tissue. There is also generalized reduction in capillary density observed in white matter hyperintensities,[8] which can be considered to be capillary rarefaction in the brain.

WMH are also observed during seizures. On acute occlusion of cerebral arteries, WMH occur very rapidly, in 2.7 minutes in the rat. It has been suggested that edema is the cause of WMH, however edema does not occurs this quickly, but ATP depletion does.

There may be other changes that result in WMH too. WMH are associated with markers for hypoxia. In any case, the relevance of this diversion into WMH is only to connect WMH to the ATP status of the brain. The association of WMH with low brain ATP is pretty well established, even if the mechanism(s) for that association is not.

NO and superoxide from iNOS are protective against excitotoxic injury, and this protection can be induced via LPS treatment.

Subjects with WMH have reduced density of blood vessels in regions which show hyperintensity. The reduced blood vessel density is likely due to a remodeling of the vasculature due to chronic low NO. With O2Hb being the sink for NO, if the level of NO is lower, then less hemoglobin is needed to act as a sink. I think this remodeling of the vasculature is the mechanism behind the lower brain blood flow observed in all of the neurodegenerative disorders characterized by WMH. I think it is also the mechanism for capillary rarefaction in non-neuronal tissues observed in hypertension and other disorders.

Ischemic preconditioning in the Brain

Ischemic precondition is a lower ATP state, but more importantly is a lower ATP consumption state. Some aspects of ischemic preconditioning are the same as the fight or flight state. Not enough is known about both to know if they are identical. They might be in some tissue compartments and not in others. All mammalian cells are aerobic and require continuous supply of O2 and substrate for continued ATP generation by mitochondria (except for red blood cells). When cells are deprived of O2 and substrate, they undergo ischemia and become damaged and eventually will die depending on the severity and length of the ischemia. This is the source of the injury when a vessel is occluded in the brain or heart for example. Ischemic preconditioning occurs when a tissue compartment is exposed to brief periods of ischemia prior to a prolonged severe ischemia. In an ischemic preconditioned state, tissues can survive ischemia that would otherwise cause necrosis. It only takes a few brief instances of ischemia to trigger the ischemic preconditioned state, which then persists for variable lengths of time, but it can be as much as a day or longer. The standing down from the ischemic preconditioned state takes longer.

Ischemic preconditioning can be triggered in a few minutes, and persists for hours to days. In the ischemic preconditioned state cells use less ATP. Presumably if cells could survive/reproduce while in the ischemic preconditioned state they would have evolved to do so because it would then free up more ATP for reproduction. Cells did not, so there is something incompatible with long term survival/reproduction with being in the ischemic preconditioned state too long. Presumably the time period that is "too long" depends on the tissue compartment and is probably longer than the normal duration of the normal ischemic preconditioned state.

Migraine

Migraine is a characteristic episodic type of headache that is often localized to a portion of the head, is sometimes preceded by visual hallucinations called aura or pro droma, and is sometimes triggered by a number of different environmental and/or physiological circumstances. The details of the physiology behind migraine are not well understood.

There has been considerable work on migraine using nitroglycerine because nitroglycerine does reliably induce migraine in susceptible patients. It is unfortunate that the effects of nitroglycerine on migraine have been attributed to nitroglycerine being a "NO donor". Nitroglycerine is not a "NO donor" in the classic sense. The chemistry and physiology behind the effects of nitroglycerine are complex and are not well understood. It can be a source of NO and nitrite via chemistry which is not fully understood, and which is subject to significant changes in fairly brief periods of time (few hours). Nitroglycerine exhibits what is termed "nitrate tolerance", where the dose of nitroglycerine must be increased; other NO donors such as sodium nitroprusside or authentic NO does not cause nitrate tolerance.

Nitroglycerine does irreversibly inhibit aldehyde dehydrogenase which appears to be the main enzyme responsible for generation of NO. This irreversible inhibition is exacerbated by oxidative stress. Nitroglycerine does induce late ischemic preconditioning. Ischemic preconditioning is a state where ATP concentration and consumption is reduced; it is a state that is protective in the short term, but (my hypothesis) detrimental in the long term. Long term treatment with organic nitrates increases cardiac events in patients with healed myocardial infarctions. I suspect that the therapeutic mechanism of nitroglycerine may be to induce ischemic preconditioning pharmacologically. This may be useful at reducing pain, and in reducing acute injury, but may not be helpful in the long term. That may be why some groups see increased cardiac events in long term treatment with nitroglycerine.

Migraine induced by nitroglycerine is not associated with changes in brain perfusion. This article is quite interesting and goes against a lot of conventional thinking and assumptions. It is consistent with the idea that migraine is not caused by vasodilation associated with NO. They did observe the prompt vasodilation associated with acute infusion of nitroglycerine, however there was no vasodilation associated with migraine following the nitroglycerine.

Migraine is pretty reliably triggered by sildenafil (Viagra). Sildenafil inhibits the phosphodiesterase 5 that is the main esterase that removes the cGMP produced by sGC after it is activated by NO. This is the mechanism by which sildenafil potentiates the action of NO through the cGMP pathway. However because there is feedback inhibition of NO production through the cGMP pathway, potentiating the level of cGMP will reduce the level of NO that is produced. Sildenafil thus will reduce the effects of NO mediated through non-cGMP pathways. This is apparent in men with obstructive sleep apnea, where a single dose of sildenafil significantly increases desaturation events. One of the triggers for breathing is S-nitrosothiols and is not mediated through cGMP.

When migraine is visually triggered, there is increased O2 levels by fMRI BOLD. This has been generally interpreted as being due to vasodilation, however the nitroglycerine study shows no vasodilation. I think it is more likely that the increase in O2 levels may be due to reduced O2 consumption due to triggering of ischemic preconditioning. Reduced O2 consumption is also consistent with reduced metabolism. The dynamic range of O2 level is substantially reduced, that is the level between activated and deactivated brain regions.

Migraine has been hypothesized to be associated with spreading depression. Spreading depression is a depolarization of neuronal tissue that propagates at up to a few mm/minute. It is not propagated by axons, but by some other type of signaling. It has many characteristics one would expect of ischemic preconditioning, and likely is related. This review article is interesting because it discusses both spreading depression (SD), and also hypoxia spreading depression like depolarization (HSD). I like the discussion of how they are different and how they are the same and the adherence to precision in naming and discussing the phenomena.

I suspect that they are even more similar than the author suggests, and perhaps are even indistinguishable. The major difference, that SD occurs in normal O2 environments and HSD occurs in low O2 environments simply means that O2 is not the causal factor. I think they are both simply ischemic preconditioning that has been turned on abruptly and hard. Under normal O2 levels, ischemic preconditioning turns off some pathways of ATP consumption, which reduces O2 consumption, so O2 levels go up. Under hypoxic conditions ATP production is reduced, so ATP consumption gets turned off. If the hypoxia is too severe, ATP cannot be produced and cells eventually die during HSD. SD can be tolerated many times with (apparently) little or no damage. I suspect that there is damage, that there is a "pruning" of a few neurons during each instance of SD to reduce the metabolic load and so bring it into better balance with what can be supplied by the vasculature. This "pruning" occurs during any type of seizure or excitotoxic damage. Cells that are firing too much and exceed their metabolic capacity are the ones that succumb to excitotoxic death. The cells that are the "weakest link" in the neural network of the brain. This pruning may not have apparent consequences with each episode, simply due to the redundancy and reserve of neurons present. When that reserve is exhausted, increased dysfunction will occur with each occurrence.

There are reports that people with migraine are at higher risk for lesions of various types visible on MRI. The increased risk due to migraines is additive to other risk factors and is somewhat higher in migraine with aura. Males who experience migraine are at slightly higher risk for cardiovascular disease. Women with migraine are at somewhat greater risk. I see the association of migraine with cardiovascular disease as both being due to and exacerbated by low NO.

Patients with migraine show subtle reductions in grey matter diffusivity compared to controls via high field MRI. Diffusivity relates to ATP levels as discussed above. These same patients also had reductions in grey matter density. The grey matter is where the cell bodies of neurons are, where protein synthesis and mitochondria biogenesis occurs.
The confinement of SD to the gray matter may be the attempt by physiology to spare the cell bodies of neurons. The white matter is mostly axons, which in principle can be replaced if the cell body of that axon remains in tact.

A number of conditions that are associated with mast cell activation are also associated with migraine including allergies, asthma, and irritable bowel syndrome. Elevated levels of histamine are sometimes associated with migraine, suggesting that mast cells in tissues associated with neurons in the brain may be involved in migraine. Agents that sensitize and activate mast cells also increase the sensitivity of intracranial meningeal pain receptors. These are thought to be the source of much of the pain felt during migraine.

When mast cells degranulate they release histamine as well as other agents that cause the production of ROS. ROS destroys NO, and this increases the sensitivity of mast cells to degranulation. Mast cells are responsible for release of ROS that is the inflammatory response to hypoxia.

There is another type of headache that is associated with excessive numbers of immune cells in the CSF, termed pseudomigraine lymphocytic pleocytosis. I see this as the production of superoxide by larger numbers of lymphocytes in the CSF, this superoxide reduces NO levels and triggers ischemic preconditioning and the low NO state.

Migraine is observed more frequently in people with other capillary/connective tissue disorders such as Sjögren's syndrome, Raynaud's and other rheumatic disorders. I think this relates to low NO being the final common pathway in all of these.

In conclusion, migraine is the triggering of ischemic preconditioning in the brain.

Reductions in Brain Blood flow associated with neurodegenerative diseases

Essentially all of the neurodegenerative diseases are characterized by reductions in blood flow, reductions in metabolism, accumulation of damaged proteins, and atrophy and shrinkage of the brain. These changes are not acute, but are progressive sometimes over many years and involve the whole brain.

I see this characteristic decline as the inevitable consequence of low basal NO. Once the NO level gets low enough that basal blood flow is affected, then basal blood flow is reduced, and tissues remodel themselves to accommodate to the now reduced nutritive blood flow. The reduced metabolic demands then set up another round of ablation of excess vasculature, reduced basal blood flow and still more remodeling. This progressive atrophy of tissue due to low NO is what I term the "low NO death spiral". The fundamental problem is the shifted setpoint brought about by reduced basal NO levels. Physiology is still regulating vascularization appropriately, it is simply to the wrong setpoint. The only way to fix the vascular remodeling is by restoring the correct setpoint. The only way to restore the correct setpoint is by restoring the appropriate basal NO level. This level is local to the tissue under consideration, and cannot be measured in vivo. It is on the order of nM/L.

In some ways the "low NO death spiral" is similar to the cell death that occurs during seizures or spreading depression. Those are acute episodes where cells are "tested" and the weakest cells ablated. We know there must be mechanisms for ablating cells because organs can and do shrink. Acute infarcts cause necrosis and scarring, less acute infarcts cause apoptosis and cell removal without scarring.

Diabetic vasculopathy

Vascular abnormalities leading to tissue damage are a common outcome in diabetes type 1 and diabetes type 2. I prefer the term metabolic syndrome over diabetes type 2 because there is a lot more going on than simply high blood sugar, and it is fundamentally different than diabetes type 1. One can have both diabetes type 1 and the metabolic syndrome simultaneously. I won't go into a lot of detail because there is quite an extensive literature on it. Diabetic vasculopathy is a chronic condition, it is not caused acutely. A serious complication is the very slow healing of even minor wounds which then become infected and if not treated adequately that healing occurs, amputation is not infrequently necessary.

There is a lot of thought that it is simply the high blood sugar that causes the damage. This is not strictly correct, but it is certainly related. There have been two recent large trials on standard blood glucose regulation vs. intensive glucose regulation, and with conflicting results. In one trial increased regulation of blood sugar doesn't reduced the death rate, it actually increases it. My interpretation is that in some cases trying to prevent hyperglycemia can be counterproductive. There is another study with a different conclusion, that intensive blood glucose control is beneficial. However in this study the incidence of hypoglycemic events requiring assistance and medical assistance was much higher in the intensive control group.

A recent (1999) "review points out that there is no compartment of glucose in the body at which all glucose is at the same concentration, and that models of glucose metabolism, including effects of insulin on glucose metabolism based on assumptions of concentration homogeneity, cannot be entirely correct." I would be more blunt; such models are wrong.

All cells in all tissue compartments need sufficient glucose. The only place where glucose can easily be measured is in bulk blood which is well mixed and essentially uniform in composition. Most cells derive glucose not from blood, but from plasma in the extravascular space. This plasma has a lower glucose level than bulk blood because cells have removed glucose from it before it reaches the sampling point. Physiology can't regulate extravascular glucose independently of blood glucose because it is plasma from the blood that makes up that extravascular plasma.

Preventing hyperglycemia will be counterproductive if it causes pathologically low glucose levels in the extravascular space (where it cannot be measured). Too much glucose is bad, but not enough glucose is worse. Not enough glucose in the extravascular space can occur even when there is pathologically high glucose in the blood stream. I suspect that to some extent that is the reason that physiology causes hyperglycemia in the first place (in the case of the metabolic syndrome, not diabetes type 1). NO is the signal for mitochondria biogenesis. With low NO, there ends up being not enough mitochondria. This shifts ATP production more to glycolysis, which takes 19 times more glucose per ATP molecule. If 5% of ATP production is shifted from mitochondria to glycolysis, that cell needs twice as much glucose to accommodate it. How can the vasculature deliver twice as much glucose? Only by increasing glucose concentrations in blood. If blood levels of glucose are not allowed to go up, then cells too far from a capillary become starved for glucose.

I suspect that if the groups were stratified for on the basis of capillary density that intensive glucose control would be beneficial for those with high capillary densities and the adverse events occur more in the group with low capillary densities, but it is probably more complicated than that.

In the intensive trial that was stopped, patients averaged 4 years younger and started out ~15 kg heavier and some exhibited larger weight gain since baseline (27.8% gained 10 kg or more compared to 14.1% in the standard group) (the averages are not provided). The starting weight in that trial was 93.5 and 93.6 kg. In the other trial, the starting weights were 78.2 and 78.0 and weight change was smaller, the ending weights were 78.1 and 77.0 kg. The standard treatment leg actually lost weight.

I suspect that weight and weight gain is a marker for degree of ATP production from glycolysis. When ATP is produced by glycolysis, lactate is produced and that lactate must be disposed of. Without enough mitochondria in the liver to recycle lactate into glucose via the Cori cycle, I think the excess lactate gets disposed of as fat. Since mitochondria biogenesis is triggered by NO, low NO will cause fewer mitochondria.

Diabetic vasculopathy is somewhat more complicated than just hyperglycemia. Low NO is a major final common pathway, but the cause is somewhat different. Acute hyperglycemia causes acute production of superoxide which reduces NO mediated regulation of vascular tone. What is interesting in this paper is that a transient elevation of glucose caused a sustained reduction in NO mediated vasodilation. This makes sense from a physiological control sense. When does blood glucose go up? When the body calls for more glucose to deal with an acute event such as running from a bear. The glucose is needed not in the bulk blood, but in the peripheral tissues, in the extravascular space. The only way that pulse of glucose can get to the extravascular space is to increase the pressure drop through the capillary bed and so transiently increase the extravascular flow and the flow velocity in the extravascular tissue compartment.

In obese Zucker rats, flow induced remodeling is characterized by low NO. Treatments that reduce NO decrease vasodilation due to shear, treatments that decrease superoxide (and so increase NO) increase vasodilation.

There have been suggestions that individuals with recurrent diabetic wounds have increased blood NO. This is incorrect. A paper which purports to have found this didn't actually measure NO, they measured the sum of nitrate plus nitrite. This is a common and fundamental error. NO has a very short lifetime in blood (less than 1 second) and is present at only nM/L levels. It is converted into nitrite and nitrate by oxyhemoglobin. Nitrite and nitrate are present at tens of microM/L. NO is extremely difficult to measure, nitrite and nitrate are easy to measure. Nitrite and nitrate are the terminal metabolites of NO, so there is some relationship between NO and nitrite and nitrate levels. Precisely what that relationship is remains largely unknown (and is likely very different in different tissue compartments). NOx levels in blood are more related to NO production rate than to NO concentration. The effects of NO as a signaling molecule are local and are related to the local NO concentration, not the NO production rate averaged over long times and multiple tissue compartments.

NO is one of the cytokines that has major regulatory effects on the immune system. NO attracts immune cells to the site of infection and regulates their function once they are there. This regulation is complex, and is affected by such things as temperature (NO being increased by fever range temperatures). NO causes vasodilation, bringing increased flow of blood. NO inhibits biofilm formation by Pseudomonas and Nitrite inhibited the formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis, and caused dissociation of biofilms already formed. Biofilm formation is a major virulence factor in infection. Suppression of virulence factor production renders even infectious strains of bacteria non-infectious. This is a point that is not always appreciated. Bacterial strains are infectious only because they produce toxins, proteases, and other virulence factors. Bacteria that do not produce virulence factors are non-virulent. Expression of virulence factors is regulated by bacteria, and until their expression is triggered, bacteria are non-virulent. Raising NO levels locally and systemically will improve the healing of diabetic wounds. Improving vascularization by increasing NO will prevent them from happening in the first place.

Summary

NO and NOx physiology is intimately connected with the regulation of vascularization. Capillary spacing is regulated not by gradients of O2, but by gradients of NO. Low NO causes physiology to decrease capillary spacing because low NO mimics the local signal of oxyhemoglobin being diffusively close. Physiology can't compensate because it is the compensatory pathways that are affected.

Reference:

1 Pechánová O, Simko F. The role of nitric oxide in the maintenance of vasoactive balance. Physiol Res. 2007;56 Suppl 2:S7-S16. Epub 2007 Sep 5. Review.

2 Espey MG, Thomas DD, Miranda KM, Wink DA. Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide. Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11127-32. Epub 2002 Aug 12.

3 Deem, Steven, Richard G. Hedges, Steven McKinney, Nayak L. Polissar, Michael K. Alberts, and Erik R.
Swenson. Mechanisms of improvement in pulmonary gas exchange during isovolemic hemodilution. J. Appl. Physiol. 87(1): 132–141, 1999.

4 MURRAY,JOHN F., AND EDGARDO ESCOBAR. Circulatory effects of blood viscosity: comparison
of methemoglobinemia and anemia. JOURNAL OF APPLIED PHYSIOLOGY Vol. 25, No. 5, 594-599
November 1968.

5 Brown WR, Moody DM, Challa VR, Thore CR, Anstrom JA. Venous collagenosis and arteriolar tortuosity in leukoaraiosis. J Neurol Sci. 2002 Nov 15;203-204:159-63.

6 Silber HA, Lima JA, Bluemke DA, Astor BC, Gupta SN, Foo TK, Ouyang P. Arterial reactivity in lower extremities is progressively reduced as cardiovascular risk factors increase: comparison with upper extremities using magnetic resonance imaging. J Am Coll Cardiol. 2007 Mar 6;49(9):939-45. Epub 2007 Feb 16.

7 Sun D, Huang A, Yan EH, Wu Z, Yan C, Kaminski PM, Oury TD, Wolin MS, Kaley G. Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats. Am J Physiol Heart Circ Physiol. 2004 Jun;286(6):H2249-56. Epub 2004 Jan 29.
8 Brown WR, Moody DM, Thore CR, Challa VR, Anstrom JA. Vascular dementia in leukoaraiosis may be a consequence of capillary loss not only in the lesions, but in normal-appearing white matter and cortex as well. J Neurol Sci. 2007 Jun 15;257(1-2):62-6. Epub 2007 Feb

What does it mean to be old?

I was talking with someone I just met in person, and she remarked that I seemed much younger than my actual age. She is not the first person to say that, at the Gordon Conference someone less than half my age said I seemed like I was his age. Someone at work thought I was his age, late 30's.

I do nothing to try and appear younger. I don't color my hair, I don't use make-up, I don't use any cosmetics at all, no lotions, no moisturizers, nothing except the very occasional soap and water and my nitric oxide bacteria culture. I am in pretty good shape, nothing remarkable, nothing that I would consider "youthful". High NO levels do retard aging, and do facilitate healing and repair of injured tissues.

I think the youthful appearance is more due to communication, and to a lack of being identifiable with a certain age and cultural group.

In the art field, once art has reached a certain age, it becomes trivial to place it in the age that it is from. An example people might be familiar with is in the movie 2001. Most of it was pretty non-era specific, except the bee-hive hairdo in one of the scenes simply screams 1968. There have been art frauds that were undetected when they were painted which became obvious years later because they could then be identified by the style of the era they were done in.

I think this relates to how language is generated by humans. When children acquire language, they acquire a language with a well-formed grammar and syntax. If the language the adults are speaking doesn't have a well-formed grammar and syntax, the children will synthesize a new language that does, they will create a Creole that combines aspects of what the adults are communicating with, but with a well-formed grammar and syntax.

When that happens, the language becomes "fixed", that is the mapping of sounds to mental concepts becomes fixed. At a fundamental level, communication is only the transfer of data from one individual to another which invokes a certain mental concept in the recipient. The only thing that can be communicated is mental states, ideas that are mapped into the neurological hardware that the person receiving the communication has.

I was looking through lyrics and came across the lyrics to Never Never Land.

You'll have a treasure if you stay there
More precious far than gold
For once you have found your way there
You can never, never grow old

And that's my home where dreams are born
And time is never planned
Just think of lovely things
And your heart will fly on wings
Forever in Never Never Land

The usual conceptualization of Never Never land is perpetual childhood and a perpetual lack of responsibility. It is not a lack of responsibility that is characteristic of youth, it is flexibility and ability to consider and learn new things. It is the old dogs inability to learn new tricks that makes the dog "old", not irresponsibility. It is the old dogs rigidity of neurological hardware that makes the old dog unable to learn new tricks. If your brain can't adapt to learn new ideas, then you have become old no matter what your chronological age is. If your thinking is old, you are old.

That is the essence of Conservatism, the essence of old and rigid thinking. Sarah Palin may be younger than I am, but her ideas are as old as the hills, from well before the 19th century. Teach Creationism and not teach sex education? Ban books? Open sports centers and close museums? That will prepare the next generations for the 16th century, not the 22th century.

Not just time when "dancing was a sin and beer came in buckets", but a time when thinking was a sin. Their next step will be to make thinking a crime. Think those times won't come again?

Mechanism for mitochondria failure during immune system activation

The roots of autism: How immune system activation can cause regression with/without autism and with/without mitochondrial failure. Non-immune system mediated mitochondrial failure. The connection of mitochondria to autism and regression.

This has gotten longer and more complicated than I intended, but I felt it was necessary to include a fair amount on how low NO causes the acute behavioral effects associated with immune system activation and how those effects can become self-sustaining.

The first third of this is devoted to neural connectivity, how that changes, and how those changes matter. Neural connectivity is the fundamental "cause" of all properties of neural networks. That includes all neural activity and all behaviors. This is true in the same sense that the meaning of a book is tied up in how the letters are arranged. That arrangement of letters determines the words, the phrases, the sentences, the paragraphs, the pages, the chapters, and the sections, everything that is "important" about the book. The devil is in the detail, and there are a lot of details. Virtually all of those details (more than 99.999%) remain unknown. This is not a measure of how little we know; rather it is a measure of how complicated physiology and the brain actually are. Most researchers do not appreciate how complicated physiology is.

There has been some discussion about vaccines causing mitochondria failure. There is nothing special about vaccines that cause mitochondria failure; any sufficiently severe immune system activation can/will cause mitochondria failure even in completely healthy individuals even with completely healthy mitochondria. The turning off of mitochondria under conditions of extreme immune system activation is a completely normal, important, and necessary regulatory feature of mitochondria. It can occur with or without neuropathy or regression. In no case is this mitochondria failure due to "toxins" or toxic effects of exogenous materials on mitochondria. This is the cause of death in sepsis, failure of mitochondria leading to multiple organ failure. If too many mitochondria fail, there is nothing that can be done to prevent death. Any successful treatment can only be to prevent too many mitochondria from failing.

This turn-off has nothing to do with any toxic components in vaccines. It is easy to see that the mitochondrial failure following vaccination has nothing to do with "toxic" components in vaccines. If it did, we would expect to see a dose-response effect; that is that mitochondria subjected to the highest dose of "toxins" would be expected to be killed the fastest and the most. If "toxins" in a vaccination cause mitochondrial failure, that failure should be greatest at the site of injection (which is never intravenous, it is either IM or IP) where the dose is many orders of magnitude higher than in a remote site such as the brain. We would expect to see acute necrosis at the site of injection. Acute necrosis at the site of injection is virtually never observed, therefore there is not acute mitochondrial toxicity from "toxins" in vaccines. In other words, every person injected with a vaccine has their mitochondria at the site of injection exposed to many thousands of times higher levels of "toxins" than is the brain of anyone who has ever developed mitochondrial neuropathy following vaccination. If the vast majority of vaccinated individuals don't have toxic effects at the site of injection it is virtually impossible for mitochondrial neuropathy effects observed in some individuals to be due to any kind of "toxicity". I discuss this more later.

Regression (with or without neuropathy) can occur as a consequence of an immune system activation, where transient high NO due to iNOS reprograms the basal NO level slightly lower by feedback inhibition of nNOS and eNOS expression, which then decreases the range of the NO release during neuronal activation (the cause of the BOLD fMRI signal), which lowers the functional connectivity of the brain which then drops below the percolation threshold and the functionality of the neural network drops exponentially especially in regions using NO to mediate social behaviors. Many social behaviors are mediated through neurotransmitters utilizing NO effects, including oxytocin, and steroids. There is positive feedback, where increased social isolation programs the brain to be less social and more on the ASD spectrum. There will be a future blog about that.

The root cause of autism: decreased actual and functional connectivity in neural structures mediating communication

Some of the mechanism(s) by which regression occurs is discussed in the blog on how there is acute resolution of autism symptoms during fever in the section on the "low NO ratchet". The regression of autism is called regression because children so affected lose behaviors mediated through the brain that they previously had. In that sense, any loss of any neuronally mediated function could be called "regression". The regression of autism is similar (my hypothesis) to the "regression" of neurosyphilis (which isn't called that, it is called paresis or paralysis), but depending on which part of the brain is affected the symptoms of paresis would mimic "regression". It is similar to the "regression" of Alzheimer's, which can be resolved acutely by the injection in the spine of agents which block TNF (and so acutely stop neuroinflammation). I see these different types of "regression" as being due to neuroinflammation which raises superoxide levels and so lowers NO levels. It is the low NO that accompanies neuroinflammation that causes the characteristic "regression". With low NO, the range of the neurogenic NO produced to regulate neuronal function is reduced. It is neurogenic NO that produces the vasodilation observed in the fMRI BOLD signal. Low NO in the brain, reduces the range of those NO signals, reduces the functional connectivity, reduces the ability of that brain to engage larger volumes of brain to achieve complex computations. Social computations are the first affected because many of the neural structures involved in social computations have effects mediated through NO, they are also extremely complex high-level responses requiring large computation volumes, so low NO is going to affect them a lot.

The brain is actively rewired to modify functionality. This is observed following stroke and other trauma to the brain. It must happen during development and learning. This remodeling occurs over the entire life span. The many details of how that wiring and rewiring occurs over the lifespan are virtually all unknown. If the brain is able to accomplish a computational task, it must have the neural structures to accomplish that task. Once the task is accomplished, those neural structures may not be needed any more. An example I give later is the ability of children to learn or synthesize new languages. Once the language is incorporated into hardware (i.e. the neural networks comprising those parts of the brain mediating communication), the ability to synthesize a new language isn't needed any more, and that ability is lost. Adults have "regressed" in that they have lost the ability they had as children to learn and synthesize new languages. It is not called "regression" and is not appreciated as "regression" because it happens to virtually all humans, and the lost abilities are not missed, and had no significant utility in evolutionary time (which is why evolution configured development to lose those abilities). Humans didn't need to acquire multiple new languages as adults 100,000 years ago. Once the language synthesizing scaffold generates the neural hardware to use language, the scaffold isn't needed any more and can be taken down and those resources (brain volume, neurons, blood supply, glucose, etc.) can be used for more important things, what ever those things might be.

The meta-programming of the brain occurs via modulation of functional connectivity. The details of how this happens are mostly unknown. It is known that it does happen, so there must be mechanisms that make it happen. These mechanisms cause functional connections to occur between parts of the brain such that activity in one part can then stimulate activity in another part which can then stimulate activity in another part.

I think a large part of that meta-programming of functional connectivity occurs through NO, through the same NO that causes the vasodilation observed by fMRI BOLD imaging. That NO is coupled to NO from each and every other source. That coupling is what modulates brain activity in sync with the physiology of the rest of the body. When the body experiences metabolic stress, NO is lowered, and this modulates the activity of the brain accordingly. There is nothing "abnormal" about this. Usually it takes extreme changes in physiology to produce extreme changes in brain activity. How "extreme" is a matter of degree. Low NO is going to change what counts as "extreme". Superoxide from inflammation or from metabolic stress is going to lower NO levels and reduce the functional connectivity.

I am necessarily describing a schematic cartoonish simplification. The details to go beyond a cartoon are not known. We know that there are lots of details we just don't know what they are yet. There are many details that I am leaving out of what I have written here. There is lots of stuff that connects what I am saying together. The "big picture" is very big, very complex and hard to get even a simplified cartoon of it to fit in something that anyone has even a small chance of reading, much less understanding ;)

Types of autism: Chronic via neuroanatomy vs. Acute via functional connectivity

As I see it, there are fundamentally two types of autism. The distinctions that I make are not precisely the same ones that other people make and there is considerable overlap. One can have either, both, or neither and to very different degrees. Both result from low NO, and are consequences of the fundamental programming of the brain to adapt to the environment the person is being born into, or is living in. The main difference is when the low NO happens, and for how long and does it come back up again and where one is in their development, in utero, infancy, childhood, puberty, early adult, middle age, or elderly.

First, there is autism due to neuronal development in utero. This results in the characteristic brain structures that are visible on MRI. The most notable characteristic is smaller and more numerous minicolumns, increased brain size and increased asymmetries. This fundamentally programs the brain for Asperger's and autism. Depending which particular part of the brain is affected by this neurodevelopment determines the spectrum of positive and negative mental abilities. There is a fundamental trade-off in brain function. The two main characteristic traits of humans are tool use and communication via language. Both of these require large brains. Since the size of the brain at birth is limited by the mother's pelvis, evolution has forced a compromise, an optimization of brain function, depending on the environment the fetus expects to be born into.

Social isolation in early life does cause low NO in the brain in later life. This occurs due to fewer NO producing neurons. This makes sense because if the brain doesn't need pathways to do social-type stuff (because there are no other individuals around to be social with), it is better to divert those resources to non-social-type stuff. This is the fundamental tradeoff along the autism spectrum.

As I see it, autism that develops in utero is more complicated because it depends on the idiosyncratic neurodevelopmental pathway of each individual. Which brain regions are affected and in what ways will determine the spectrum of positive and negative mental abilities and attributes. Autism that develops later in life is more simple because the underlying neuroanatomy is already fixed and isn't as malleable (it can still be extremely malleable, but the time scale is longer). The prompt behavioral effects discussed in the blog on fevers are due to resolution of the prompt and acute aspect of autism (lower functional connectivity). There is plasticity in that the brain can remodel itself even in adults but remodeling takes time, time that was not available during the acute fevers. My own experience is that increased NO has shifted my position on the autism spectrum making me more social. I still have Asperger's, but I am more aware of thing social, less anxious and aware that I am missing a lot that I was unaware of before. Those changes have taken years and are still ongoing. I am quite sure that this has involved neuronal remodeling. I will blog about this in more detail later.

The breakdown of ability to communicate that occurs later than in utero mimics some aspects of the autism which develops in utero but is fundamentally different and more mutable (to some extent). It is when the parts of the brain that deal with communication of certain types are forming (and remodeling) during early childhood that the communication aspects of autism occur. They can only occur when the brain is developing those structures.

It is wrong to think of "autism" as something extra that is added on or taken away from someone who is NT. To some extent, an individual's location on the autism spectrum will be shifted by their NO/NOx status. They can never be "cured". "Cure" is the wrong term to apply to autism. Autism is a range of neurodevelopment, the same way that muscle strength occurs in humans in a range (but neurodevelopment is much more complex). One can move in the muscle strength spectrum, but only to an extent. Autism is like the trade-off between fast-twitch and slow-twitch muscle. Most people have a mix, which lets them do a mix of different things. If you had only fast twitch, you would be a good sprinter but terrible at long distance. If you had only slow-twitch you would be good at long distance but terrible at sprinting. The trade-off on the autism spectrum is similar, a trade off of social skills useful for understanding and manipulating people for skill at understanding and manipulating non-social concepts. "Curing" a sprinter so they can no longer sprint is a wrong concept.

Autism is a complete spectrum in many dimensions. The most visible decreased ability is in social interactions, the most visible increased abilities are in the savant abilities. Usually savant abilities are different in different individuals. These are different dimensions of the autism spectrum. Everyone has some ability at calendar. People with savant calendar are just really good at it. NTs have communication abilities with other NTs that are "savant" compared to the communication abilities of people on the autism spectrum.

The fundamental problem of autism is that some people who are NT are unable to "connect" with people who have autism and that inability to "connect" is intolerable to those NT individuals. This is not a problem of people with autism; it is a problem of some people without autism. This is not to minimize the real difficulties that people with autism have, but those problems are greatly amplified by being treated badly by NTs. The distress that NTs feel when around people with autism is about the NTs, not the people with autism. A fundamental problem that many NTs have is a tendency to project human attributes on non-human things, as for example in Uta Frith's work on the emotional states and motivations of triangles. Communication between individuals requires those individuals to transmit information (i.e. language) allowing each of them to construct (to some fidelity) a representation of the mental state of the other. Projecting one's own mental state onto another is common. When two people are able to map their mental states onto each other, they are able to "connect".

Mapping a mental state from one individual to another individual requires neuroanatomy that can accomplish this. If the mental state cannot be mapped from one individual to another, those two individuals cannot communicate that mental state and cannot "connect" on that level. I think this is a reason for some of the fundamental difficulties in communication that NTs and ASDs have with each other. There are dissimilarities in neuroanatomy that make mapping of mental states between them difficult.

I think (and this is perhaps the most controversial idea in this blog entry) that all NTs have very complex neural net "hardware" that allows NTs to communicate easily and effectively with other NTs. This NT neural net hardware is what NTs use to think their NT thoughts. ASDs lack that neural net "hardware", but can emulate a facsimile of it (to some extent) in the neural net "hardware" that ASDs use for thinking their ASD thoughts. The emulation that ASDs can do is not as high fidelity as the hardware that NTs have and this bothers NTs a lot. Most NTs can't emulate ASDs at all and this doesn't bother those NTs at all, or rather any discomfort those NTs feel is attributed to and projected onto the ASDs and the ASDs are blamed for it (and bullied) as a consequence.

It is the NT neural network that gives NTs the "savant" ability to communicate with other NTs. They don't know how they do it, they can't imagine not being able to do it, they can't imagine anyone else not being able to do it. That is the problem.

Genetic abnormalities: What is the final common pathway? Low NO phenotype (my hypothesis)

There have been many single gene mutations and some more complex mutations associated with autism. I think that most of the single mutations and deletions that are associated with autism "cause" autistic symptoms by invoking metabolic stress and shifting metabolism to a low NO state where the low NO phenotype is invoked and which has more autism-like symptoms. It is the low NO state which triggers the epigenetic development (controlled by many genes), not the simple genetic abnormality per se. If the low NO state can be reversed, I think it is likely that many of the autism-like symptoms can be reversed also. I think this is what happens in Rett Syndrome. The MeCP2 single gene defect puts metabolism under substantial stress by greatly interfering with methylation signaling. Metabolism responds by invoking low NO (my hypothesis), the low NO skews ongoing function and continuing neurodevelopment onto the low NO autism path. In the MeCP2 mouse model, restoring MeCP2 function restores normal physiology, removes the stress, allows NO levels to return to the non-stressed state and restores the non-Rett neurological phenotype. This demonstrates that the "autism" of Rett Syndrome in the mouse model is fundamentally not an issue of neurodevelopment. The neurological phenotype of mature adult mice is restored by restoration of MeCP2. In humans it is more complex because lots of neurodevelopment has occurred under conditions of low NO produced by the MeCP2 defect. People with RS don't have the larger brains that people who develop autism in utero typically do.

The autism phenotype is caused by low NO (my hypothesis). How that low NO occurs doesn't matter. NO can diffuse everywhere, there are no barriers to NO, and each NO sensor only senses the sum of NO from all NO sources. The NO sensor sums and integrates NO from all sources and then produces an output which evolution has determined is likely to be the most suitable. Sometimes it isn't. That is why anaphylaxis sometimes kills people and sometimes saves their life. Physiology has to respond before it "knows" exactly how strong a response is necessary.

There are multiple ways for low NO to occur. The main focus of this article is on the generation of superoxide by mitochondria under high NO levels causing mitochondria depletion which causes high superoxide due to higher mitochondrial potential. Inflammation also generates superoxide from activated immune cells. Metabolism of normal and xenobiotic chemicals by the cytochrome P450 enzyme system also makes superoxide. Xanthine oxidoreductase also makes superoxide.

I think it is better and more correct to call autism-like behavioral symptoms associated with metabolic stress due to genetic abnormalities as "autism-like" syndromes. The "degree" of autism is purely an arbitrary definition. I agree with Michelle Dawson that terms such as "high functioning" and "low functioning" are not useful and should be discouraged. The behaviors characterized as affected in autism are multi-factorial, diverse with a great deal of variability within and between individuals. "Functionality" doesn't correlate well with any traits that can be easily characterized. The largest factor in an individual's ability to cope is likely the other people in that individual's environment. Are they helpful and supportive or hurtful and non-supportive. I think that much of the variability observed in individuals relates to their acute NO status. During fevers the NO level is raised acutely by expression of iNOS and I think that increased NO is the mechanism for the resolution of autism symptoms during fever. Any mechanism to raise NO levels will have similar effects. Raising NO levels during childhood puts the child back on the high NO development paradigm which leads to a more NT phenotype. Maintaining a low NO level causes development on the ASD developmental paradigm and results in the ASD phenotype. There is some degree of movement on the ASD spectrum at any age.

Smaller and more numerous minicolumns are also observed in the brains of distinguished scientists. I suspect they were exposed to low NO in utero and early childhood, developed the characteristic neuroanatomy, and then had high NO levels restored later which allowed for more normal social and communication abilities. My expectation is that if the appropriate NO levels are restored sufficiently early during neurodevelopment in childhood this is how people with autism in utero will turn out.

The diagnostic criteria for autism are all behavioral. The behaviors are mediated through neuronal structures that develop over time. Until those neural structures have or have not developed, a diagnosis as to how they behave once they do develop cannot be made.

What are Mitochondria?

Mitochondria are major energy sources of cells. Each cells has many mitochondria, some have many thousands, some have many more. All the mitochondria are essentially identical. There are some differences in different tissue compartments but the basic mitochondrial DNA in an individual is all identical and inherited 100% from your mother. They produce ATP by oxidizing substrates (the only site in the body that does so). They also produce superoxide where usually a few percent of O2 consumed ends up as superoxide. This superoxide production is a normal, necessary and irreplaceable part of mitochondria regulation. Mitochondria cannot function properly unless they produce superoxide. That superoxide is an important part of very important signaling pathways. Superoxide is confined to the mitochondrial matrix where it is dismutated into H2O2 which can diffuse through the two mitochondrial membranes. Mitochondria are also important in a number of chemical synthesis steps. Urea is produced from ammonia to detoxify it. All urea synthesis occurs in mitochondria in the liver. Heme is made (in part) in mitochondria as are the iron-sulfur clusters which are also used in oxidation enzymes. Hemoglobin is made using mitochondria (to synthesize the very large quantities of heme) and then the mitochondria are removed from red blood cells during their maturation.

Nitric oxide regulates mitochondria at multiple levels, by multiple mechanisms, at multiple time scales, for multiple different reasons in multiple different tissue compartments at multiple different sites on multiple different enzymes in the respiration chain and elsewhere. None of this simple, or is even close to being well understood. Much of this regulation is in synergy with production of superoxide. Mitochondria generate superoxide which is confined to the inner matrix, the local NO level communicates the effects of that superoxide to neighboring mitochondria (and elsewhere) so all the mitochondria can operate "in sync" and so that all of physiology can work "in sync" too. This is an extremely important regulatory function of NO diffusion, keeping mitochondria "in sync" in each cell, in each tissue compartment, in each organ, and in the entire organism. Mitochondria not being "in sync" is a source of reduced efficiency and can lead to dysfunction or even death.

Acute mitochondria failure

In a nutshell, mitochondria failure during immune system activation results from mitochondria being pushed to a high potential (where they generate a high superoxide flux) under conditions of high NO. This generates a high peroxynitrite flux in the inner matrix which eventually deactivates MnSOD causing superoxide levels to increase further which accelerates the production of peroxynitrite. This causes respiration chain inhibition and eventually mitochondria turn-off. Some early steps in this inhibition by this regulation are reversible; eventually a point is reached where the inhibition depletes ATP production in the cell so severely that the inhibition is irreversible. That is, the cells become so depleted in ATP that they do not have enough ATP to repair the damage and recover.

This is not a "disorder" per se; rather it is the normal regulation of mitochondria causing a bad outcome, sort of like the way anaphylaxis can cause a bad outcome. Is anaphylaxis a "disorder"? No, if you had bacteria in your blood stream you want an enormously powerful immune system response because in "the wild", that is the only thing that has a possibility (however remote) of saving your life. Evolution has configured the immune system to minimize the sum of deaths from too strong an immune response (death from anaphylaxis) and from too weak an immune response (death from infection). In an attempt to avoid death from infection, some risk of death from anaphylaxis is acceptable.

Destruction of too many mitochondria under the extreme NO production that occurs during sepsis occurs for the same (ultimate) reason that anaphylaxis occurs. Evolution has configured mitochondria to be turned off under certain conditions so as to minimizes the sum of deaths from mitochondria not turning off enough and from mitochondria turning off too much. It is a balance between two extremes, the way that most things in physiology are. This is not to suggest that there is only one "switch" that determines mitochondrial fate, no doubt there are many. NO happens to be an important one in the context of immune system activation.

This effect is related to the resolution of autism symptoms during fever which I discussed before. It is related in that both effects are caused by high NO, but the level of NO is considerably higher for mitochondria destruction compared to the resolution of autism symptoms. These levels are still quite low (and difficult to measure). Normally the basal NO level is less than the order of a nanomole per liter. That is less than 30 parts per trillion by weight. If the NO level gets up as high as 300 parts per trillion, guanylyl cyclase is about 50% activated and there is massive vasodilation. This does happen in septic shock. It doesn't happen in a simple fever except perhaps locally where there is a local infection, local inflammation and local vasodilation. Locally this is a healing mechanism to bring more blood flow, more immune cells which then generate more NO and more H2O2 to fight the local infection. When the infection becomes systemic, then there is a life-threatening crisis. In the "wild", such a life threatening infection was likely to be fatal and extreme desperate measures are called for by physiology. This is the extreme desperate measure of septic shock.

This paper has a cartoon that shows the mitochondria respiration chain and its regulation by NO and NOx. The point of the cartoon is that there are many different parts of mitochondria that are known to be regulated by NO and NOx, and that regulation is complex in time, space, and under diverse metabolic conditions only some of which are known. The "details" of none of these mechanisms are well understood and are the topics of intense and ongoing research (which is quite challenging). The regulation of mitochondria by nitration occurs in seconds to minutes under conditions of hypoxia, and then is reversed (to some extent) also in a few minutes. Isolating mitochondria so they can be analyzed but without perturbing what is going on (and then demonstrating there has been no perturbation) is very challenging. Mitochondria turn-over; that is they have a finite lifetime and some are replaced each day, usually at night when humans are least active. So when mitochondria are isolated from experimental animals, the entire mitochondria population is isolated, which includes mitochondria of all different life spans (that is time since created).

Mitochondria in neurons

It is loss of ATP from mitochondria in neurons that causes neuron death and neuropathy. Mitochondria in neurons are simpler than in the rest of the body because they don't oxidize lipids. Neurons also don't generate ATP via glycolysis, it is pretty much only generated by mitochondria through oxidation of substrates, lactate, ketone bodies, small acids such as acetate, aspartate. Mitochondria are small organelles that have a few thousand proteins, only 13 of which are coded for by mitochondrial DNA, all the others are coded by nuclear DNA. Mitochondria have 2 lipid membranes, inside the inner one is where the DNA is and the protein manufacture stuff (the mitochondria matrix). Mitochondria work by generating an electrical potential and a pH gradient across that inner membrane. The different respiration complexes take electrons and protons from chemical compounds and extract energy from the chemical reactions as those electrons and protons are moved across the membrane and store that energy in the electrical and pH gradient. That energy gradient is then used to make ATP. Eventually those electrons are added to protons and added to O2 making water. Four electrons and four protons are added simultaneously to make two molecules of water. Doing a four electron reaction is very tricky.

The 13 proteins are all parts of the respiration chain, usually the part containing the active site. All animals (except for a few invertebrates) have these same 13 proteins coded in their mitochondria. Plants have a few extra. These proteins are all large and quite hydrophobic. Why these (and only these) proteins are coded in mitochondria is not understood. I think it has to do with the necessary regulation of mitochondria, some of which has to be local to each mitochondrion and sometimes that regulation means turning off part of the respiration chain and then turning it back on which means making the protein again. In most cells mitochondria are close to the nucleus so that proteins can be made from DNA in the nucleus and then transported to mitochondria (in principle, whether this happens or not is unknown). In neurons that can't happen because the distance between the cell body (where the nuclear DNA and the protein synthesis capacity is) and mitochondria can be inches or even a meter in motor neurons. There simply isn't time for a signal to propagate from mitochondria to the cell body, trigger protein synthesis and then transport proteins out to mitochondria in need of them. If protein synthesis is needed for control of mitochondria, that synthesis must occur locally using locally available DNA. There is pretty good evidence of local regulation of mitochondria activity by the de novo synthesis of proteins in mitochondria. The ability of mitochondria to synthesize the active site of respiration chain enzymes allows irreversible inhibition of the active site to be an acceptable control scheme. If the active site is inactivated it can be replaced in situ by de novo synthesis from mtDNA. The active site is the parts of the respiration chain most exposed to oxidative damage. No other proteins can be replaced in situ. Mitochondria do have the capacity to degrade individual proteins with ATP powered proteases. Amino acids can be degraded and fed into the citrate cycle and oxidized to CO2. Use of highly reactive agents (superoxide derived peroxynitrite) to regulate the active sites of the respiration chain, allows the more distant non-active regulatory sites to be spared contact with the inhibiting agent (because it reacts before it reaches them). The sparing of regulatory components from oxidative damage increases their lifetime and so prolongs the time that mitochondria can survive solely using mtDNA. It think this is also part of why neuronal mitochondria are more simple, fewer proteins to carry means the lifetime of functional mitochondria can be longer while remote from the cell body.

Motor neurons are long, up to a meter in length. Mitochondria can only be made in the cell body, which is in the spine, because that is where the nucleus is and the only place that 99% of the proteins in the mitochondria can be synthesized. Once made, mitochondria are carried out via ATP powered motors to the tippy end of the axon, and when they get "tired", they are carried back for reprocessing via autophagy. In the rat CNS, mitochondria have a half life of about a month. That is in rats, it is likely somewhat longer in humans, but probably only a few months, likely not years.

As the largest cells in the body, neurons are unique. Virtually all of the metabolic load is in the axon far from the cell body. Because the metabolic load depends on the axon length, and the axon length can vary from less than a mm to a meter, the metabolic load (and hence the number of mitochondria must also vary by more than 3 orders of magnitude. A very important question is how does the cell regulate the mitochondria number in neurons over multiple orders of magnitude? The answer is: extremely well. The only type of regulation that would be able to work with such fidelity is feedback control. I suspect that some of the peculiarities of neurons as cells (such as absence of glycolysis and lipid oxidation) reflect physiological constraints imposed by the need for this regulation.

The time constant of that mitochondria feedback control has to reflect the time constant of the lifetime of the mitochondria in the neuron. The neuron needs to control both the number of neurons and also the age distribution of those mitochondria. This is an important point. If the age distribution gets too far out of whack, then at some point too many mitochondria will get old simultaneously and ATP production drops. If ATP demand exceeds the ATP that the remaining mitochondria can supply, the neuron becomes ATP depleted and either sheds metabolic load or dies. I suspect that maintaining the age distribution of mitochondria is one reason why regrowth of nerves is so slow.

Normally cytochrome c oxidase (the enzyme that consumes O2) is tonally inhibited by NO, which blocks O2 from binding and is the major regulatory pathway by which mitochondria regulate their O2 consumption. The only reason that mitochondria can regulate their O2 consumption is because NO "poisons" cytochrome c oxidase and inhibits O2 consumption. Remove that inhibition and mitochondria consume O2 to very low partial pressure, an order of magnitude below what is the "normal" basal O2 level at the location of the mitochondria. At "rest", the O2 flux to a mitochondria in the heart is 1. The O2 consumption by that mitochondrion can increase by 10x. The flux of O2 from the blood vessel to the mitochondrion is purely passive down a concentration gradient. For the flux to go up 10x, either the gradient has to go up 10x, or the distance has to go down by 10x because the concentration at the blood vessel stays the same. For the gradient to go up by 10x, the concentration at the mitochondrion has to go down, and go down a lot, by a factor of 10x. It has to go down while the mitochondrion is increasing its O2 consumption by 10x. The specific O2 consumption by that mitochondrion, moles O2/mg protein/Torr O2 has to go up by a factor of ~100. This is only achieved by removing the "poisoning" of cytochrome c oxidase by NO. This removal is accomplished by the generation of superoxide. The ATP production of neuronal mitochondria probably doesn't change by an order of magnitude. The mitochondria in heart muscle can.

The capacity of mitochondria to generate superoxide is limited only by the supply of O2 and reducing equivalents. The same substrates that mitochondria use to generate ATP.
Superoxide is generated vectorally into the inner matrix. It is charged, so it can't pass through lipid membranes except through anion channels. There is also a pretty high potential, ~140 mV across the inner mitochondrial membrane that tends to keep anions inside. Superoxide is dismutated to H2O2 which is uncharged and so can diffuse through lipid membranes. Normally a few percent of O2 consumed is converted into superoxide (O2-). Superoxide is generated when the mitochondria potential gets high; it is also generated if the respiration chain becomes too reduced, such as when cytochrome c oxidase is blocked. When cytochrome c oxidase is blocked by NO, that blocking is reversed when superoxide is generated (this destroys NO resulting in disinhibition).

What happens when there is insufficient cytochrome c oxidase activity? The respiration chain becomes reduced and superoxide is generated, but the NO level can only go to zero where there is no more inhibition of cytochrome c oxidase. Before that happens other parts of the respiration chain start to be inhibited in many cases also by NO and NO metabolites; including complex I, which introduces reducing equivalents into the respiration chain from NADH, and also complex III which takes reducing equivalents from succinate (from the citrate cycle). There are a couple of different pathways by which that inhibition occurs, some critical thiols become S-nitrosated, and some tyrosines become nitrated. The S-nitrosation is pretty much reversible, some of the nitration is reversible too. The details of this regulation are not well understood and involve NO, superoxide, glutathione, CO2 and no doubt other things. There are over a thousand different proteins in mitochondria. Which ones are regulated by NO and by what mechanisms, in what order and for what purposes under what conditions are mostly unknown. It is obvious that all of those proteins are regulated to work together and "in sync".

Cells can't allow the regulation of mitochondria to break down.

What happens if that regulation were to break down? If the regulation of the mitochondria were to somehow fail such that production of superoxide was not limited? Making superoxide from O2 requires only a single electron, reducing that O2 to two H2O requires 4 electrons. Mitochondria have the theoretical capacity to make at least 4 times more superoxide than they do to consume O2 to make ATP. Mitochondria can increase their metabolic rate many times over the basal rate, some as much as 10x. A few "bad" mitochondria could consume O2 and substrate and produce high levels of superoxide and/or H2O2. Cells cannot afford to have even a few mitochondria running out of control. They could easily kill the cell. Mitochondria generating superoxide at maximum rate could consume the O2 that 30 or 40 times more mitochondria could use at rest. To stop a few bad mitochondria from killing the cell, there must be a "fail-safe" mechanism that reliably turns mitochondria off.

Turning off mitochondria when they produce too much superoxide is easy to observe. and there are multiple mechanisms to decrease superoxide levels when there is too much, including inhibition of the respiration chain, and also expression of uncoupling protein which short circuits the membrane potential dissipating it as heat. Uncoupling protein is from nuclear DNA, so it can't be used in neurons. Mitochondrial uncoupling is a major factor in the heat production during malignant hyperthermia. The problem of malignant hyperthermia isn't just temperature; it is the consumption of substrate and the turn-off of mitochondria which causes a profound reduction in ATP levels and in ATP production capacity. If ATP levels in cells drop enough, the cell will die. Cell death due to ATP depletion in irreversible. There is no way to supply ATP from outside cells. Either a cell has the metabolic machinery to generate sufficient ATP and survives or it does not survive.

Superoxide, a necessary evil.

Because much of the regulation of mitochondria depends on the interplay between NO and superoxide, what happens when mitochondria don't make enough superoxide? Because the regulation requires superoxide, there need to be mechanisms to increase superoxide when the level falls too low. These have not been as well described in the literature. I think largely because there are no good experimental techniques that are well recognized for reducing the superoxide production in part because the physiological pathways are so well regulated. There is a technique which I think does this, but which isn't well appreciated as such, that is the use of near infrared light to photodissociate NO from cytochrome c oxidase. I mention this technique in my blog on the magic light helmet for Alzheimer's.

What does chronic lack of mitochondria biogenesis look like?

I suspect the symptoms will mimic the delayed symptoms of mercury poisoning such as the dimethylmercury poisoning experienced by a woman heavy metals researcher where she had a lethal body burden of dimethyl mercury following acute exposure (estimated at 1,344,000 micrograms at exposure) with no symptoms for 5 months. At 5 months (time of diagnosis) she had a measured blood level of 20,000 nM/L, and a body burden of 336,000 micrograms. This has nothing to do with the non-existent mercury poisoning that the quacks and frauds attribute autism to (which they assert occurs promptly (days) following vaccination with a trivial quantity of mercury (~15 micrograms). These delayed symptoms are for real mercury poisoning, which occurs at levels that are unmistakably diagnosed via testing of any specimen, blood, urine or hair. The level she was exposed to was roughly 100,000 times the level in vaccines.

The very long symptom free period (5 months) demonstrates that even these extremely high mercury levels are not acutely toxic to mitochondria. If they were acutely toxic, she would have died much sooner. Nerve cells can only function for a few seconds without mitochondria. Cells that can do glycolysis can function longer, perhaps minutes. There are essentially no cells that can function indefinitely only on glycolysis. Red blood cells can, but they have a finite lifetime. The major important tissues, muscle, liver, kidney, gut, skin, etc. all require mitochondria. Mitochondria are required to make heme and also to make the iron sulfur complex that is the active site of many proteins. Since the exposure was through essentially a point contact, a spill of pure material on her gloved hand, the local dose to those skin cells was absolutely gigantic. Essentially pure dimethylmercury ended up on her skin. There was no report of acute necrosis of the skin, presumably it didn't happen. If it had happened, perhaps her exposure would have been recognized and she would have been treated. That treatment might have saved her life. I think the five month delay was due to the normal turnover of mitochondria without replacement due to a blockage of mitochondria biogenesis due to the extremely high levels of mercury. I think this relates to the interference with the recycling of mitochondria during autophagy, and specifically in the blunting of the NO/NOx signal that occurs during autophagy. What is interesting is that the organ that failed was the brain, not the other organs. My explanation of this is that these levels of mercury disrupted the normal feedback regulation of mitochondria biogenesis, but only in neuronal tissue. The mechanisms that regulates mitochondria turnover in neuronal tissue (or in any tissue) have not been identified. That there must be such mechanism(s) is certain. With zero mitochondria biogenesis in neurons I would expect the onset of symptoms of failure of the CNS to occur pretty abruptly as observed in the dimethyl mercury poisoning. There is significant redundancy and fewer mitochondria running at high potential can produce the same ATP as many running at low potential. I would expect the abruptness of the transition from essentially no symptoms to death to be pretty rapid (as observed). The abruptness relates to the average of the mitochondria and the number that fail at any one time. The higher the metabolic load each mitochondria experience, the faster it will age and ultimately fail. The longest nerves are the ones affected first, as experienced by peripheral numbness. This is typically the same pattern observed in other neurodegenerative diseases such as amyotrophic lateral sclerosis. The peripheral nerves are (typically) the ones that go first. Mitochondria biogenesis likely doesn't go to zero in ALS the way it likely did in the mercury poisoned woman.

The fundamental control paradigm of mitochondria is for them to produce more superoxide when they require producing ATP at a higher rate. Mitochondria biogenesis can only occur when the superoxide level is low. If the number of mitochondria drops below the level where they can produce sufficient ATP while maintaining a superoxide level sufficiently low for mitochondria biogenesis to happen, then mitochondria biogenesis will stop and cannot be resumed. This is the point of no return beyond which the cell it occurs in is doomed.
The first symptoms this woman noticed were neurological. The CNS has the longest lived mitochondria. For mitochondria depletion to be observed in the CNS first, it must have been essentially non-existent in other tissue compartments. That the disruption is only in neuronal tissue puts quite severe constraints on what the mercury could be doing. It is likely not due to inhibition of key mitochondrial enzymes (that would lead to acute mitochondrial inhibition in many tissues and prompt death) or even inhibition of enzymes that make key mitochondrial enzymes (that would lead to reduced mitochondria biogenesis in all tissues and multiple organ failure on the time scale of mitochondrial turnover for that organ). Mitochondria in neurons are the simplest mitochondria. They don't oxidize lipid or transaminate most amino acids so they likely have only a subset of the enzymes that all other mitochondria have. They should be the most resistant to toxicity because they have fewer enzymes to be disrupted. In some cases of mitochondrial toxicity, the first mitochondria to be damaged are those in the liver with mitochondria in muscle and brain being spared, as for example in Reye's Syndrome. Salicylate increases superoxide production in liver mitochondria and this is what causes Reye's syndrome. Reye's syndrome is characterized by fatty liver and encephalopathy. It would make sense for liver mitochondria to be the most susceptible to toxicity. The liver has the greatest capacity for detoxification, the liver can regenerate itself, a lot of the toxicity of xenobiotics is actually due to the xenobiotic metabolites, not the parent compound.

Since the mitochondria in neurons are the simplest, mitochondria in other tissue compartments have more proteins and enzymes to do more complicated things. The disruption of mitochondria biogenesis in neurons is likely not due to disruptions in transcription because transcription of mitochondrial proteins in other tissue compartments is continuing. Since those mitochondria are more complex than neuronal mitochondria, the loss of neuronal mitochondria biogenesis is likely not due to blocking transcription.

That leaves the signaling upstream of transcription. This relates to a poster I presented at the NO conference 2 years ago, where I hypothesized that the long term regulation of mitochondria number in neurons was mediated through NO/NOx generation during autophagy of dead or dying mitochondria from nitrated proteins. The concentration of nitrated proteins in recycled mitochondria is dependent on the level of metabolic stress that mitochondrion experienced over its lifetime. In other words, the degree of metabolic stress a mitochondrion experiences regulates its membrane potential which regulates its superoxide production which regulates its peroxynitrite production which regulates how many proteins get nitrated by how much. Autophagy reads back that signal and generates the appropriate number of new mitochondria to meet the need. I will discuss the details in a later blog. Autophagy is the only way by which mitochondria are recycled, and recycling of mitochondria is "tricky". Mitochondria are quite dangerous. They contain Fenton active metals, Fe, Cu, and Mn, which can produce hydroxyl radical from H2O2. Hydroxyl radical is extremely reactive. It is so reactive that antioxidants are ineffective against it. Virtually any organic molecule is reactive enough toward hydroxyl that the first molecule hydroxyl hits is damaged.

Inhibition of mitochondria by NO/NOx a critical regulatory feature

There must be a fail safe mechanism that turns off dysfunctional mitochondria to prevent the useless (and dangerous) consumption of O2 and substrates. I suggest that mechanism occurs by the simultaneous generation of too much superoxide in an environment of too much nitric oxide. Normally this mechanism protects cells from a few aberrant mitochondria, the loss of which is of no serious consequence. Under conditions of very high immune system activation many mitochondria can be turned off such that normal metabolism of the cell becomes impossible and the cell dies. When many cells in an organ die, this leads to organ failure, and eventually to multiple organ failure.

The normal regulation of O2 consumption of cytochrome c oxidase is via the destruction of NO by superoxide by a reduced respiration chain. One of the most important targets for regulation by NOx is MnSOD, manganese superoxide dismutase. This enzyme is only found in the mitochondrial matrix, but it is coded for in nuclear DNA, not mtDNA. This means that the total amount of MnSOD a particular mitochondria has is finite and can't change over that mitochondria's lifetime except by going down as it is either inhibited or degraded. MnSOD dismutates superoxide into H2O2 at near diffusion limited kinetics. Those kinetics are close to the rate that superoxide reacts with NO. Superoxide is vectorally generated in the mitochondrial matrix, where there is competition between reaction with MnSOD and with NO. When NO reacts with superoxide it forms peroxynitrite (ONO2-). What happens then depends in part on the CO2 level but we will ignore that complexity.

Peroxynitrite can nitrate proteins, it can also decompose generating NO2 which can also nitrate proteins. The amino acid most susceptible to nitration is tyrosine forming nitrotyrosine. Human MnSOD is inhibited by nitration of a single tyrosine. Bacterial FeSOD (which is highly homologous with MnSOD) is not inhibited when 8 of 9 tyrosines are nitrated. That the two enzymes are homologous demonstrates that they derive from a common ancestor. That the bacterial FeSOD is virtually totally resistant to inhibition due to nitration demonstrates that inhibition by nitration is not an intrinsic property of SOD enzymes. If bacteria evolved SOD enzymes that are highly resistant to inhibition due to nitration, then organisms with mitochondria could too. They haven't, implying that inhibition due to nitration is a "feature" that has been positively selected for by evolution. I think it is, and the very important feature that that nitration accomplishes is the feature of turning off mitochondria when they become damaged, a function that is not necessary for bacterial SOD enzymes. I think this nitration is also important in transducing and integrating the degree of metabolic stress that each mitochondria has experienced over its lifetime, so that during autophagy that signal can be read out and the appropriate number of mitochondria generated to match the load. I think this occurs by the nitrated tyrosine being converted to NO/NOx by conditions during autophagy.

When mitochondria generate ATP, it is always very important that all the mitochondria work "in sync" that is that all the mitochondria generate ATP in concert. If there were differences in how the load of ATP generation were distributed among the mitochondria, then the ones generating more would be overloaded (relatively) and those generating less would be underloaded (relatively). That represents inefficient allocation of resources. Fewer mitochondria efficiently loaded could generate more ATP and at a lower metabolic cost than more mitochondria inefficiently loaded. Over evolutionary time organisms with efficient mitochondria loading will out reproduce organisms with inefficient mitochondrial loading. I suspect this may be one of the primary reasons that all mitochondrial inheritance is only through the maternal line. It is extremely important that all mitochondria in a cell be as identical as possible so they are controlled in sync. If the mitochondria were not identical, then the regulatory mechanisms to share the load could not affect them uniformly. If mitochondria were not reset to all identical in each oocyte, then over multiple generations there would end up being considerable variation in mitochondria. A variation that would preclude precise regulation of all the mitochondria in a cell in sync. I think this is especially important in organs such as the brain where NO regulation of mitochondria is required to be extremely precise and synchronous in both time and space for good function.

It needs to be remembered that there are mitochondria of different ages in each cell. Mitochondria have a finite life time, the longest is about 30 days in rat CNS. Each mitochondrion is "born" in the cell body, loaded with proteins coded in nDNA and then transported out the axon by ATP powered motors.

When a cell needs more ATP, the ATP level drops and the mitochondria "turn on". The details of that process are not important for our discussions. One of the things that regulates the NO level is sGC which is also controlled by ATP. The sensitivity of sGC to NO is modulated by ATP, with low ATP causing greater sensitivity. This is the mechanism by which cells control their ATP level, and via NO how they communicate that ATP level inside the cell and between cells. Communication of ATP levels between cells is very important so that entire organs (and the entire organism) can be regulated "in sync". In the heart for example, different muscle cells need to be equally loaded, to maintain efficient allocation of the resources needed by the heart, glucose, insulin, lipid, O2, hormones, etc.

Normally, the basal NO level is modest, ~1 nM/L, and the mitochondria work together in sync to consume O2, generate and consume NO to regulate cytochrome c oxidase and generate ATP. With all the mitochondria working together, they all experience about the same ATP, O2, and NO levels, and the proportionality of ATP and NO is maintained via sGC. In the brain, this synchronicity is important because NO is a neurotransmitter, one of the very few that passes through cell membranes without requiring a receptor. NO signaling could (conceivably) even occur in the white matter where NO could generate "cross talk" between axons.

Under such circumstances if one mitochondrion begins producing superoxide at a higher rate than all the others, it generates more superoxide, pulls the NO level down locally to itself. This reduces the local ATP level via sGC which accelerates mitochondria ATP production. A period of positive feedback ensues where the mitochondrion generates more superoxide and more peroxynitrite than its neighbors and the mitochondrion begins to down regulate different parts of the respiration chain. Either the mitochondria achieves a new stable operating point where the consumption of O2, NO and generation of superoxide and ATP matches that of its neighbors, or it becomes irreversibly inhibited.

The critical parameters for this regulation are the ATP production rate by the mitochondria and the NO concentration. High ATP production requires a high mitochondria membrane potential and so generates a high superoxide flux. That superoxide flux pulls down the NO level and is dismutated to H2O2. If ATP production or superoxide production is high in the presence of high NO, then there is inhibition of the respiration chain.

If all the mitochondria in a cell are operating at the same level, then the NO and superoxide levels go up and down in sync. The ATP demand is shared between the mitochondria. If one mitochondria gets overloaded, then that mitochondria has a superoxide level that is out of sync with the NO level that all the other mitochondria are experiencing. That overloaded mitochondria makes more peroxynitrite which down regulates that mitochondria. Reversibly at first and then irreversibly. If this happens to one or a few mitochondria, there are plenty left to support the ATP demand of the cell.

With this understanding of mitochondria regulation, the life cycle of mitochondria becomes clear. Mitochondria are made in the cell body, they migrate out the axon carried by ATP powered motors. When mitochondria have a high potential, they move out away from the cell body. When they have a low potential they move back toward the cell body. The sorting of mitochondria by potential, keeps active mitochondria out in the axons and returns dead, dying and dysfunctional mitochondria to the cell body for reprocessing.

In neurons essentially all the metabolic load is out in the axons, and the axons can be different in length by 3 or 4 orders of magnitude. This means the mitochondria number must also be variable by 3 or 4 orders of magnitude. This variability occurs in each cell. When a cell first divides, it is small and then grows larger. The number of mitochondria must be matched to the cell's metabolic demand at every stage in that cell's lifetime, which for a human is the entire lifespan (because many CNS neurons do not divide).

In the cell body mitochondria are reprocessed by autophagy. Cytoplasm including mitochondria is engulfed in a vacuole, protease and other lyase enzyme precursors are ported in, and a pH gradient is set up by the ATP powered proton pump VH-ATPase. The pH gradient is then used to power the transport of other things too. There are a pretty large number of proteases, the cathepsins and they catalyze the breaking of peptides into smaller ones some of which are sorted out and recycled.

The details of autophagy remain mostly unknown. It is the only mechanism by which organelles can be recycled. It is something that all eukaryotes do. It is the only way to recycle mitochondria.

The recycling of mitochondria occurs regularly. In rats, it occurs during the period of low activity, during the day. This makes perfect sense. Mitochondria biogenesis requires a high NO level, and also first requires the destruction of the mitochondria being recycled. This temporarily reduces the ATP production capacity of the cell, so it is not something that the cell can allow to happen if there isn't enough ATP to start with, or to complete the process. The period of lowest ATP demand is during sleep, when activity is lowest. With the highest NO level during sleep, the highest ATP level would be during sleep also.

This is a very important point. The number of mitochondria in a neuron is adjusted every day. Some are disposed of through autophagy, and new ones are made. Disposing of mitochondria takes ATP, as does making new mitochondria. During times of low ATP, this is put off until later. Even crappy dysfunctional mitochondria make ATP. Completely dead mitochondria don't consume ATP until they are reprocessed. If there isn't enough ATP, it is better to put those things off until later when more ATP is available.

How the mitochondria number changes over time demonstrates the mechanism for mitochondrial dysfunction.

If there is mitochondrial "toxicity", the number of mitochondria changes acutely, in a single day. That produces an acute effect on physiology. If that is a severe effect, the consequence is immediate death. This is what causes death from sepsis, hyperpyrexia, malignant neuroleptic syndrome, cyanide poisoning, CO poisoning and a few others.

If there is a change in physiology that is not abrupt, it cannot be due to mitochondrial toxicity. If there is slow mitochondria depletion, that is a problem with mitochondria biogenesis, with the ongoing replacement of mitochondria. If that replacement goes to zero, the result is death with the time scale depending on the tissue compartment. The longest living mitochondria are in the CNS, lack of replacement of mitochondria there would follow the clinical course of the woman with dimethyl mercury poisoning (discussed earlier), essentially no symptoms until the mitochondria depletion reaches a certain level then very rapid decline and death.

If someone has survived for a year, they are replacing mitochondria in their CNS. They might not have "enough" mitochondria, but not enough mitochondria is due to a disruption of the regulation of mitochondria number, not due to blocking mitochondria biogenesis. Mitochondria biogenesis is triggered by NO. Low NO is going to skew the mitochondrial number to a lower value. This is one fundamental cause of insufficient mitochondria, too low a basal NO level. This is what causes physical detraining and also chronic fatigue (which is just an extreme form). If the background NO level is too low, exercise may not be able to raise it enough to trigger sufficient mitochondria biogenesis. In that case there isn't a way to increase mitochondria levels.

That clinical course, no symptoms and then rapid decline and death could occur in any organ. When it happens in the liver it is called fulminate liver failure.

So what happens during sepsis?

During sepsis the level of NO can become very high. The mechanism for the NO increase is that activation of NFkB causes the expression of iNOS, which generates NO via open loop control, that is, the NO generated is not regulated other than by the amount of iNOS produced and the availability of substrates and the presence of inhibitors. This NO inhibits NFkB and prevents the expression of more iNOS. Thus the level of NO before NFkB activation determines in part the amount of NO after NFkB activation. However it is an inverse regulation. The lower the initial NO level, the higher the iNOS expression and the higher the ultimate NO level. This high NO level then reduces the expression of eNOS and nNOS, lowering the basal NO level when the iNOS is degraded in a day or so.

The high NO in acute sepsis from expression of iNOS leads to high ATP concentration. This is not generally appreciated. During acute sepsis, ATP levels are actually higher (if the patient survives) than normal controls. It is my interpretation that the authors of this last report don't appreciate what their own data clearly shows. They show higher ATP levels in skeletal muscle during sepsis than in uninfected controls (p =0.05). ATP is higher because NO is higher. High NO blocks cytochrome c oxidase, so mitochondrial ATP generation is shut down (mostly). This is why septic shock causes cachexia. The body is generating ATP via glycolysis. The mitochondria are shut off by the high ATP, so the body needs to make glucose without using ATP, so it does so by turning the muscles into alanine which the liver can turn into glucose without consuming ATP. All of this glycolysis generates a lot of lactate, which can't be turned back into glucose because the mitochondria are shut down. So the body turns it into fat. That is what septic shock does, it turns muscle into fat. Turning protein into fat and carbohydrate liberates a lot of ammonia. If that is sweated out to the skin, a resident biofilm can turn it into NO/NOx while conserve NOS substrates arginine, NADPH and O2.

Note this ATP measurement during sepsis was in muscle, however the ATP levels of all the cells in the body have to go up and down in sync for physiology to be regulated in a stable way. There has to be a "signal" that communicates the ATP level in cells, so that level can be regulated up and down in sync. This "signal" has to be uncharged to penetrate lipid membranes, and rapidly diffusible to communicate the signal quickly. There are hundreds of different cell types; it is implausible that they would use different signals. It is pretty clear that the signal has to be NO. The coupling of NO and ATP via sGC makes perfect sense in this light. NO is the diffusible signal that causes all cell to regulate their ATP levels up and down "in sync". This is especially important in the brain, where everything really does need to operate "in sync" for the brain to function properly.

When basal NO is low, any immune system activation raises NO levels higher (due to less inhibition of NFkB) than if NO was higher before immune system activation. I hypothesize that this can lead to what I call the low NO ratchet, where activation of the immune system under conditions of low basal NO causes basal NO levels to ratchet lower each time the immune system is activated. When NFkB is activated, more iNOS is expressed under conditions of low basal NO, leading to higher NO levels following immune system activation. That high NO level then causes the feedback inhibition of the expression of eNOS and nNOS, which add to the normal basal NO level. When the iNOS is degraded, the basal NO level falls to below the level where it was before the immune system activation.

I discuss some effects of NO/NOx on bacteria earlier. NO is used as a quorum sensing agent by bacteria, low NO is the trigger for bacteria to form a biofilm. As bad as bacteria floating around in your blood stream is, those bacteria coming out and forming a biofilm is much worse. Much much worse. I think this is the reason that the body cranks the NO level up so high, the attempt is to suppress bacterial quorum sensing for a day or so, so the immune system can knock out the bacteria and prevent them from forming a biofilm which makes them much much harder to get rid of. Preventing a biofilm from forming is so important that it is worth a significant risk of death from the preventative response.

Regressive autism and chronic fatigue syndrome

I think high NO induced switching of physiology (the low NO ratchet) is one of the fundamental causes of regressive autism, and also of low basal NO in adults as characterized in chronic fatigue syndrome (CFS). Many people with CFS can identify when they acquired it, and it corresponded with an acute serious infection other causes include trauma of surgery or accidents. Similarly, many parents anecdotally identify the immune reaction of a vaccination as a precipitating event leading to regression. However the large scale epidemiology shows no change in incidence of autism with changes in vaccination. My hypothesis is that in susceptible individuals, any immune system activation is sufficient to activate the "low NO ratchet", a vaccination, or one of the zillions of infections of childhood. It is the low NO ratchet that (I hypothesize) causes Gulf War Syndrome. Receiving multiple immune system activations (vaccinations) during a high stress period (being deployed to a war zone) causes basal NO to ratchet lower with each immune system activation until it saturates and produces chronic fatigue. This takes a few weeks, while the mitochondria turn-over and are not replaced (due to the low NO level from the chronic stress). Once mitochondria numbers are low, the low NO state is perpetuated due to superoxide from too few mitochondria being pushed to higher potentials to supply the same ATP. With continually low NO, mitochondria biogenesis can't occur enough to get back to the level that is "normal".

Simple oxidative stress alone can cause low NO, and if that low NO persists for long enough, then chronic fatigue will be induced by insufficient replacement of mitochondria.

Mitochondria depletion need not be so severe as to cause neuropathy for "regression" or chronic fatigue to occur. All that is necessary is for mitochondria depletion to exceed a threshold such that they achieve a new operating point with fewer mitochondria working at a higher potential. The higher potential generates more superoxide which lowers NO levels and if not enough mitochondria biogenesis occurs, that state can be perpetuated.

Only rarely is regressive autism or even any type of autism characterized by neuropathy as in the case of Hannah Poling. I think it is more appropriate to call such cases "neuropathy with autism-like symptoms". Normal "autism" is not characterized by neuropathy. Sufficient neuropathy will cause symptoms of the lack of communication. Lack of communication is also exhibited by some people with autism. Neuropathy is neuronal damage. Autism can occur with zero neuronal damage. I consider it fundamentally wrong to call any disorder characterized by neuropathy "autism". People with autism can experience neuropathy unrelated to their autism and again that is fundamentally wrong to connect that neuropathy to autism.

Whether mitochondria depletion progresses to neuropathy depends on how severe the mitochondria depletion is. Perfectly healthy and normal mitochondria can be turned off by this mechanism, which can result in failure of any organ where too many mitochondria are turned off, or in death, or anything in between. The critical factors are how much ATP mitochondria are called on to produce during the high NO state of sepsis (and so how much superoxide they produce), and how high the NO is level during that time.

This irreversible turn-off of mitochondria has been demonstrated in rats by injection of lipopolysaccharide, a component of Gram-negative bacteria which causes an extremely robust immune system response. This is also known as LPS, and endotoxin. This material can cause anaphylaxis, and it is thought that LPS from bacterial contamination in vaccines in 1928 (before thimerosal was used) that killed 12 of 21 children inoculated from a vial that (obviously) became contaminated a few days after 21 children were vaccinated from the same vial without ill effects.

The acute turn-off of mitochondria by LPS was accompanied by damage to mtDNA; that is a reduction in copy number of mtDNA and also the presence of deletions. Later the mtDNA copy number was restored and the presence of deletions greatly diminished. This decline and then increase in mtDNA copy number reflects the number of mitochondria present in the cells. As the number of mitochondria go down, so does the DNA they contain. As mitochondria biogenesis restores mitochondria the number goes back up, and the new mitochondria have intact mtDNA. This reflects the intact DNA required for mitochondria biogenesis. A cell can tolerate some damaged mitochondria, provided sufficient mitochondria remain to maintain the cell while it makes more mitochondria. If they are all damaged, the cell is going to die and be cleared.

This turn off of mitochondria during sepsis occurs in multiple organs including heart, liver, diaphragm and others. Because the cells in an organ communicate (via NO), they tend to fail in sync. If sufficient mitochondria remain viable to support the organ, the organism doesn't die and can recover from the sepsis. How likely mitochondria are to fail depends on the ATP load they are called upon to produce and how many mitochondria there are to share that load. Reducing the ATP demand by being immobile is the primary reason that people feel so crappy and lethargic during illness. That feeling of weakness is to prevent consumption of ATP which turns on mitochondria and can cause them to fail. This is also why putting people on a respirator helps. It reduces the load on their diaphragm muscles which improves the survival of the mitochondria and the survival of the muscle and the survival of the organism. This is also why masking symptoms of fatigue and weakness during immune system activation can be dangerous. Those symptoms of weakness and fatigue are important warning signals that ATP supplies are low, even dangerously low. There are times when overriding those danger signals are useful and lifesaving, such as when running from a bear. There are few instances in modern life where overriding weakness and fatigue are lifesaving. It may be convenient and more comfortable to block pain signals, but it always needs to be remembered that usually pain signals indicate overload, and continued overload will eventually lead to damage, eventually irreversible damage, and eventually death.

What turns off mitochondria is a superoxide level that is too high for the NO level that is present. Very high superoxide can do it, even if the NO level is not that high. That is what causes mitochondria failure during malignant hyperthermia, malignant neuroleptic syndrome or due to a hypermetabolic state from other causes such as surgical trauma. The details of how this happens are not understood. NO levels are low at those times to disinhibit the mitochondria. But the NO level can only go to zero. If at zero NO level the activity of cytochrome c oxidase isn't high enough to fully oxidize the electrons being put into the respiration chain, superoxide will be generated.

Excitotoxicity, seizure induced neuropathy, and delayed neuropathy following stroke

Following ischemic stroke, there are complex responses of the brain to the acute ischemia due to the stoppage of blood flow. First there is the acute death of the neuronal tissue where the blood supply was stopped. This is followed by release of glutamate which leads to excitotoxic death of the affected neurons. There is also death of neurons that have lost the "upstream" neurons that produce the signals for them to process.

It is not clear how much of this ongoing neuronal death can strictly be called "pathological", it is pathological in the sense that it causes greater long term dysfunction, but it may be non-pathological in the sense that it is a programmed physiological function which has understandable benefits in the shorter term. It may be thought of as pathological in the same sense that anaphylaxis is pathological. The neuronal "wiring" of the brain is extremely complex and mostly not understood. Fortunately it occurs and is regulated spontaneously. Occasionally there are disorders such as epilepsy, where seizure activity initiates in one area and propagates to other areas causing disruption of normal brain activity. During a seizure, the seizing part of the brain is disabled and bodily functions depending on that part cannot be performed properly. If that occurred in motor areas while "running from a bear", it is easy to understand how a seizure could prevent escape from the bear and result in death. If inhibitory pathways in the brain are damaged, such that a seizure threshold is reduced, ablating the pathways that may cause seizure later would be a near term benefit, even if there was significant loss of function in the long term. The "gain in function" of a lower seizure threshold can be so life-threatening (under some circumstances) that the immediate benefit of ablating those pathways would be worth the long term reduction in neuronal function. The precise balance of ablation vs. preservation over what period of time is obviously an extremely complicated instance of neuronal remodeling. Presumably the balance depends in part on the organisms' perception of the immediacy of the need to avoid near term seizures vs. preservation of long term cognitive function.

Once mitochondria depletion has occurred, neurons affected are more susceptible to excitotoxic injury. They have reduced metabolic resources to draw on and those most susceptible neurons will be the ones to be "pruned" first in the event of excitotoxic injury.

Neuroinflammation

Many cases of autism are characterized by neuroinflammation. This is discussed in the blog on how acute fevers can temporarily resolve the symptoms of autism. That autism symptoms can be acutely resolved during fever conclusively demonstrates that those symptoms are not caused by damage or by other permanent alterations to neuroanatomy, but rather are caused by the acute regulation of brain function. We know that people with ASDs do have alterations in their neuroanatomy. The alterations that are observed, minicolumn morphology, increased asymmetries occur during early brain development in utero. There isn't time during a fever for that anatomy to remodel. If neuroanatomy did remodel during the fever, it wouldn't change back when the fever resolves. I conclude that any changes observed during a fever must be from changed regulation, not changed in anatomy.

Regulation of active tissue

The brain is "active" tissue in that it can sustain self-perpetuating activation. All self-activating systems have the potential for positive feedback and collapse from a state of meta stable dynamic equilibrium to one extreme state or another. A seizure is one extreme state where all nerves are activated, a state of zero activation is the other extreme state. Neither of these brain states is functional, proper neuronal function requires very delicate control of the balance between activation and deactivation. When this balance is perturbed by the loss of nerves that produce either activation or deactivation, the balance needs to be restored ASAP. Restoration of the balance and immediate function is likely to have been more selected for because the brain is such a critical system that cannot be "offline" for even short periods of time. If the loss of inhibitory or excitatory neurons causes an imbalance that balance can be restored most rapidly by "pruning" which ever type of neuron is in excess. Speed of restoration of balance is probably more important than ultimate restoration of maximum function.

Following a stroke, death of neurons continues for a considerable period of time, even after normal brain vascular dynamics have been restored. Much of this neuronal death is due to excitotoxicity injury. Mitochondria depletion makes neurons particularly sensitive to excitotoxicity induced cell death and there is considerable thought that cell death is due to ATP depletion and not due to oxidative stress. Neuropathy due to acute mitochondrial depletion occurs quickly. That is what causes the neuropathy of stroke, the acute ischemia causes the death of neurons due to ATP depletion, and due to mitochondria depletion.

The brain doesn't have the metabolic capacity for all nerves to fire simultaneously and continuously. That would quickly lead to exhaustion of the supplies of glucose and O2, and likely exceed heat dissipation capacity. Preventing run-away metabolic overload is an absolutely necessary control function.

The brain matches its metabolic requirements with the metabolic resources supplied by the blood stream exquisitely well. That regulation can only occur via feedback and active control. That necessarily includes regulation in both directions, angiogenesis when there is insufficient blood supply and ablation of blood vessels when there is too much.

The same goes for regulation of cognitive functions. When more cognition is needed in a certain area, the brain increases neural connections in that area, recruits more connections in that area and so allocates a greater volume of neuronal resources to whatever computation function is required.

Total brain volume is fixed by the size of the skull. If local brain volume is going to be increased in one region, it must be decreased in another. This is a necessary trade-off. Precisely how this happens is unknown, that it happens is virtually certain. It is well known that brain size does decrease during normal aging, and that this decrease is accelerated in many types of neuronal atrophy.

All individuals "regress" to some extent. Infants and children have the ability to learn any human language. If a group of children is raised without a "well formed" human language (as in societies formed by mixes of immigrants speaking only pidgin versions of multiple languages), the children will synthesize a new language, a Creole, with its own well-formed syntax and grammar. Adults cannot do this. Adults can learn new languages, but it is difficult and they can only (for the most part) learn languages that are already well-formed, or pidgin languages. Adults cannot synthesize a new Creole language. Adults have lost this ability, they have in effect "regressed". My presumption is that this "regression" produced during normal neurodevelopment is to free up brain volume for other purposes that are more important, such as being a parent.

Implications

There are several implications from this analysis of mitochondrial dysfunction during immune system activation. Any activation will do it, a vaccine, a cold, an infection, a vaccine preventable disease. It is not possible to produce vaccines that do not produce an immune response. The immune response is the reason for the vaccine in the first place. It is the immune response itself that causes the mitochondria turn-off.

An important factor is the magnitude of the immune response and that depends on the NO/NOx status of the individual before the immune system stimulation.

A high NO/NOx status before immune system stimulation has several protective effects. Probably the most important one is the increased mitochondria number before any immune stimulation happens. NO is what triggers mitochondria biogenesis, with a greater basal NO level you will have a greater basal mitochondria level. That leaves more mitochondria to share the ATP production load, increasing ATP then requires less superoxide production, and there are more mitochondria available in case some get deactivated by this mechanism.

A high NO/NOx level will also reduce immune system activation by reducing the activation of NFkB. So far there are no generally approved methods for raising NO/NOx levels. What about unapproved methods? Meditation will work, but infants don't know how to meditate. Consuming nitrate, as in lettuce (lettuce is ~2000 ppm nitrate) has been shown to increase plasma nitrate, nitrate is concentrated ~10x in saliva and nitrate is reduced to nitrite on the tongue in adults. I have been told that children don't develop the characteristic bacteria on the tongue that do this until they are ~1 year old. I haven't seen any published data on this.

The method I am working on is a topical biofilm of ammonia oxidizing bacteria. I think this is how people lived in "the wild", before the modern era of frequent bathing. Before modern indoor plumbing, humans couldn't bathe every day. In Africa people probably never bathed in their entire lives. It was too dangerous to go into parasite and predator infested natural bodies of water.

More on the magic light helmet for Alzheimer's

I blogged earlier about the magic light helmet which has been reported to help with symptoms of Alzheimer's.

I have been working on my post on the mechanisms of how immune system activation causes mitochondria turn-off. This is a "normal" property of the immune system and of mitochondria. There is nothing special about vaccines, any immune system activation (if sufficiently severe) can do it (as can some other things). It has nothing to do with any toxins of any sort.

Some of the stuff on mitochondria turn-off is related to the magic light helmet so I thought I would put that up first. There is a section here that talks about the woman who was poisoned by dimethyl mercury, who received a lethal dose and then had no symptoms for 5 months despite carrying a lethal body burden of mercury. Her experience at a gigantic dose puts quite severe constraints on what possible effects mercury can have at the microscopic doses in vaccines.

I had a chance to go to the library and look up some more background on the effects of NIR (near infra red) on physiology. There are plenty of real effects that are well known and well described. NIR is also called IRA (the way that the UV spectrum is divided up into A, B and C). IRA is from visible to about 1400 nM (700 to 1400 nM). The energy of these photons is 1.77 to 0.89 eV/photon. For comparison, the mitochondria membrane potential is about 150 mV, so these photons have much higher energy than the electrons that mitochondria are gathering energy from. The energy is considerably lower than the energy of UV photons, UVA, UVB, UVC (400 -320 nM, 320-280 nM, or less than 280) having energies of 3.1-3.87, 3.87-4.43 eV. For comparison, the usual germicidal wavelength from mercury vapor lamps is 254 nM or 4.88 eV. This is a high enough energy to break chemical bonds including those in DNA.

Melanin absorbs strongly in the visible, but is essentially transparent beyond 800 nM so skin color doesn't affect IR transmission through the skin much. The major absorption of skin in that region is due to water. The major reflectance of skin in that region is due to scattering due to the difference in index of refraction of the different tissue compartments, water, lipid, protein. Reflectance due to scattering may not be particularly relevant in the Magic Light Helmet because the inside of the helmet might be reflective and light scattered out would be reflected back in. The tissue being treated, the brain, can receive scattered light from any of the light sources, or via reflection after being scattered out of the skin.

There are at least 3 relevant parameters when thinking about light interactions, wavelength, total dose, and dose rate. All natural sources of NIR such as the Sun, fire, hot objects are continuous wave that is they are on continuously and the average dose rate and the instantaneous dose rate are the same. They are also "black body", that is they have a continuous spectrum not a single wavelength. This is not true for some artificial sources of NIR such as are used in the Magic Light Helmet. They use pulsed solid state sources so the instantaneous dose rate may be many times (or even many orders of magnitude) higher. Doses of anything that may be safe at one rate may be completely unsafe at another. For example, the daily RDA of sodium (as salt) is less than 6 grams. In other words, 6 grams a day is ok, but if you got a year's worth of salt in a day (about 5 pounds) it would kill you.

Similarly an NIR dose rate that is ok indefinitely, lets say 10 Watts on your head from the Sun might cause problems if it is delivered as 10 megaWatts in a microsecond from a pulsed monochromatic source. At high dose rates non-linear things start to happen with light, there are multiple photon absorption events.

These non-linear effects increase dramatically as the dose rate increases. Two photon absorption goes as the intensity squared. Double the dose rate and there are 4 times more two photon events. My presumption is that the people developing the magic light helmet noticed that as they increased the dose rate by using pulsed NIR sources it worked "better". That is a sign of non-linear effects going on.

If multiple NIR photons are absorbed, the cumulative energy might be enough to cleave bonds the same way that UV can. Tissues are mostly opaque to UV, they are not opaque to NIR. I don't know at what dose rate NIR starts to cause problems, but neither do the people pushing the magic light helmet (or if they do they have not reported either what levels they are using or what safe levels are by what theory of safety). There are a great many potential sites of damage, none of which have been characterized.

If there is damage from pulsed NIR, that damage can accumulate over the lifetime of the things being damaged. Neurons don't divide. The DNA in neocortical neurons is not replaced over their lifetime and neocortical neurons are only generated in the perinatal period. If the NIR exposure did damage neuronal DNA in addition to what ever positive effects, that damage would accumulate and at some point would cause problems. Without knowing what dose is required to cause problems, the dose at which problems do not occur can't be known.

There are mechanisms where IR can reduce the UV damage to skin cells. IR is also known to deactivate activated chemical species in a non-damaging way. This is the photostimulated emission of visible light that is sometimes used as a detector of IR. This is explained as the reason why people burn worse on a cloudy day. The clouds don't block the UV which causes the damage (they scatter it) but do block the IR from the Sun which deactivates the excited bonds in a non-damaging pathway before they can decay via a damaging pathway. Broad spectrum IR as from the Sun or thermal sources is probably more effective than is monochromatic IR from diodes. The photon energy has to couple to the activated bond to deactivate it. The energy usually needs to be in a narrow window for that to happen. With a broad spectrum there are lots of photons of different energy available.

NIR does cause the photodissociation of things like cyanide from cytochrome c oxidase. It would also cause the photodissociation of NO. This has the effect of increasing consumption of O2 by accelerating the removal of electrons from the respiration chain onto O2 and causing the respiration chain to become more oxidized. In other words, with the activity of cytochrome c oxidase accelerated by the photodissociation of NO, there are fewer electrons hanging out on complex I and complex III which are the major sites of superoxide production, so superoxide levels go down. I think this acute reduction in superoxide is the likely mechanism for the perceived beneficial effects of the magic light helmet. With less superoxide produced, NO levels go up, and there is an improvement in the ATP level due to sGC. That is there is an improvement observed in Alzheimer's until physiology adapts. This improvement is transient and illusory (in my opinion) and will very likely be followed by rebound. In other words, the transient increase in ATP levels (above the regulatory setpoint) will cause the regulatory setpoint for ATP to be moved still lower. The magic light helmet might provide a temporary boost to ATP levels, but will likely accelerate the long term decline.

When mitochondria are irradiated by NIR, there is a transient reduction in membrane potential, and also a release of cytochrome c. After 18 hours the membrane potential is substantially restored. My interpretation is that the photodissociation of NO from cytochrome c oxidase increased electron flow to O2, this reduced the membrane potential which reduces superoxide production, resulting in a loss of superoxide production. This loss of a necessary part of mitochondria regulation is intolerable, so the mitochondria respond by releasing cytochrome c, to interrupt the respiration chain after complex III but before cytochrome c oxidase. This restores the production of superoxide.

This restoration of mitochondria potential and superoxide production comes at a cost, the loss of cytochrome c. Cytochrome c is a small soluble protein that is in the space between the inner membrane and the outer membrane. It ferries electrons from complex III to cytochrome c oxidase. Cytochrome c is coded for in nuclear DNA, and so is only capable of being produced in the cell body of a neuron. Once mitochondria in a neuron lose cytochrome c, that cytochrome c is gone for good. Could the cytochrome c be recaptured by the mitochondria that lost it? No, all proteins in mitochondria that are coded for in the nucleus are targeted to mitochondria by the addition of a special hydrophobic targeting sequence attached to the protein. That targeting sequence pulls the protein through a special pore. Heme is only attached to proteins inside mitochondria. Cytochrome c is produced as the apo enzyme, is transported to the mitochondria outer membrane where it binds, and then a special enzyme, cytochrome c lyase attaches heme to the apo enzyme making cytochrome c. Cytochrome c is the only heme containing enzyme where heme is covalently bound to the protein. In all other heme containing enzymes the heme is not chemically bound but is only held by the conformation of the protein.

Could there be a mechanism by which intact cytochrome c was transported back to mitochondria? Maybe, it seems doubtful. Better regulation of cytochrome c loss in the first place would be a better evolutionary target. Cytochrome c loss is a critical event in apoptosis. Apoptosis is not something cells want to do partially. The damage to a cell very rapidly becomes irreversible. Cytochrome c level inside mitochondria has to be something that each mitochondrion regulates by itself independently of its surroundings (and the level of cytochrome c in those surroundings which is usually zero). Usually there is no excess cytochrome c in the cytoplasm a mitochondrion is in, so there would be no way that a mechanism to import it would have any utility. The time scale that mitochondria require for regulation of cytochrome c levels doesn't allow transcription of cytochrome c DNA into RNA, generation of cytochrome c protein (necessarily heme free) and then transport to the mitochondria where heme could be added. There simply isn't time, and in neurons it simply can't happen because mitochondria may be inches away from the cell nucleus.

This progressive loss of cytochrome c is (in my opinion) an effect of the NIR irradiation of mitochondria that will make the side effects of the magic light helmet unacceptable. I expect that there will be serious and irreversible neurodegeneration with prolonged use of the magic light helmet, even in previously normal individuals. I suspect that this irreversible neurodegeneration may creep up and not be noticed until it reaches a threshold beyond which recovery is not possible.

The fundamental control paradigm of mitochondria is for them to produce more superoxide when they require producing ATP at a higher rate. Mitochondria biogenesis can only occur when the superoxide level is low. If the number of mitochondria drops below the level where they can produce sufficient ATP while maintaining a superoxide level sufficiently low for mitochondria biogenesis to happen, then mitochondria biogenesis will stop and cannot be resumed. This is the point of no return beyond which the cell it occurs in is doomed.

Mitochondria are necessarily producers of superoxide. That superoxide production cannot be blocked without disrupting normal mitochondrial function. Mitochondria won't allow their superoxide level to fall to zero, they will compensate by interrupting the respiration chain by releasing cytochrome c into the cytosol. That can also be a trigger for apoptosis, but I will leave a discussion of that for another time.

What does chronic lack of mitochondria biogenesis look like?

I suspect the symptoms will mimic the delayed symptoms of mercury poisoning such as the dimethylmercury poisoning experienced by a woman heavy metals researcher where she had a lethal body burden of dimethyl mercury following acute exposure (estimated at 1,344,000 micrograms at exposure) with no symptoms for 5 months. At 5 months (time of diagnosis) she had a measured blood level of 20,000 nM/L, and a body burden of 336,000 micrograms. This has nothing to do with the non-existent mercury poisoning that the quacks and frauds attribute autism to (which they assert occurs promptly (days) following vaccination with a trivial quantity of mercury (~15 micrograms). These delayed symptoms are for real mercury poisoning, which occurs at levels that are unmistakably diagnosed via testing of any specimen, blood, urine or hair. The level she was exposed to was roughly 100,000 times the level in vaccines.

The very long symptom free period demonstrates that even these extremely high mercury levels are not acutely toxic to mitochondria. If they were acutely toxic, she would have died much sooner. Nerve cells can only function for a few seconds without mitochondria. Cells that can do glycolysis can function longer, perhaps minutes. There are essentially no cells that can function indefinitely only on glycolysis. Red blood cells can, but they have a finite lifetime. The major important tissues, muscle, liver, kidney, gut, skin, etc. all require mitochondria.

Since the exposure was through essentially a point contact, a spill of pure material on her skin, the local dose to those skin cells was absolutely gigantic. Essentially pure dimethylmercury ended up on her skin. There was no report of acute necrosis of the skin, presumably it didn't happen. If it had happened, perhaps her exposure would have been recognized and she would have been treated. That treatment might have saved her life.

I think the delay is due to the normal turnover of mitochondria without replacement due to a blockage of mitochondria biogenesis due to the extremely high levels of mercury. I think this relates to the interference with the recycling of mitochondria during autophagy, and specifically in the blunting of the NO/NOx signal that occurs during autophagy. What is interesting is that the organ that failed was the brain, not the other organs. My explanation of this is that these levels of mercury disrupted the normal feedback regulation of mitochondria biogenesis, but only in neuronal tissue.

With zero mitochondria biogenesis I would expect the onset of symptoms of failure of the CNS to occur pretty abruptly as observed in the dimethyl mercury poisoning. There is significant redundancy and fewer mitochondria running at high potential can produce the same ATP as many running at low potential. I would expect the abruptness of the transition from essentially no symptoms to death to be pretty rapid. The abruptness relates to the average of the mitochondria and the number that fail at any one time. The higher the metabolic load each mitochondria experience, the faster it will age and ultimately fail. The longest nerves are the ones affected first, as experienced by peripheral numbness. This is typically the same pattern observed in other neurodegenerative diseases such as amyotrophic lateral sclerosis. The peripheral nerves are (typically) the ones that go first. Mitochondria biogenesis likely doesn't go to zero in ALS the way it likely did in the mercury poisoned woman.

That the disruption is only in neuronal tissue puts quite severe constraints on what the mercury could be doing. It is likely not due to inhibition of key mitochondrial enzymes (that would lead to acute mitochondrial inhibition in many tissues and prompt death) or even inhibition of enzymes that make key mitochondrial enzymes (that would lead to reduced mitochondria biogenesis in all tissues and multiple organ failure). Mitochondria in neurons are the simplest mitochondria. They don't oxidize lipid so they likely have only a subset of the enzymes that all other mitochondria have. They should be the most resistant to toxicity because they have fewer enzymes to be disrupted.

In short, I see the magic light helmet as potentially quite dangerous, even if it works. Non-physiological treatment (subjecting the brain to NIR fluxes many orders of magnitude above normal) can't have effects via normal physiological mechanisms. There is no reason to suppose that a non-physiological mechanism is benign or has no side effects.

I will discuss mitochondria depletion due to immune system activation in a future post.