passionless Droning about autism

Posts Tagged ‘Microglia

Hello friends –

The concept of glial priming (and implicit double multi hits) is the nexus of developmental programming, low penetrant effects, and an altered microglial responsiveness, a blueprint for a change in function in the tightly entangled neuroimmune environment; sort of an all time greats theory mashup for this blog.  The basic idea is that microglia can become sensitized to insults and subsequently respond to similar insults with greater robustness and/or for increased timespans later in life.  Here is a snippet from Microglia in the developing brain: A potential target with lifetime effects on the primed glial phenotype:

There is a significant amount of evidence regarding what is often termed ‘‘priming’’ and ‘‘preconditioning’’ events that serve to either exacerbate or provide neuroprotection from a secondary insult, respectively. In these states, the constitutive level of proinflammatory mediators would not be altered; however, upon subsequent challenge, an exaggerated response would be induced. The phenomena of priming represent a phenotypic shift of the cells toward a more sensitized state. Thus, primed microglia will respond to a secondary ‘‘triggering’’ stimulus more rapidly and to a greater degree than would be expected if non-primed.

Glial priming may be the fulcrum on which much of the underlying early immune activation research balances, the machinery that drives environmental influences during development leading to irregular neuroimmune functionality through the lifespan.  Even though this type of finding is not really unexpected when considered within the prism of programming effects in other systems and the perturbed immune milieu in many (all?) neurological disorders, it is still pretty cool.

The first paper that I read that specifically mentioned glial priming was Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulation, (Somera-Molina KC , 2007) which totally kicked ass.  They brought a lot of heat at design time of the study; (very powerful) seizures were induced /saline given in animals at postnatal day 15 and 45; at day 55 animals were analyzed and showed distinct increases in microglial activation, neurologic injury, and future susceptibility to seizures in the ‘two hit’ group (i.e., animals that got seizure inducing kainic acid instead of saline on both day 15 and 45).  Even better, it was shown that a CNS available inhibitor of inflammatory cytokine production rescued the effect of the seizure.  In other words, it didn’t matter if the animals had a seizure, what mattered was the presence or absence of an unmitigated inflammatory response associated with the seizure.

Treatment with Minozac, a small molecule inhibitor of proinflammatory cytokine upregulation, following early-life seizures prevented both the long-term increase in activated glia and the associated behavioral impairment.

That is an important step in understanding the participation of inflammation in seizure pathology.  There were also observable effects (worse) in animals that got seizures just once, if they got induced on day 15 versus 45, and even worse symptoms for the “double hit” animals.  That was pretty fancy stuff in 2007.  The similarity in terms of seizure susceptibility really reminded me of another paper, Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats, which also showed altered susceptibility to seizures in animals subjected to seizures in early life, with the effect mediated through inflammation related cytokines.   Here, however, the same effect observed, but with the addition of clinical evidence of chronically perturbed microglia phenotype in the treatment group.  Nice!

The same group followed up with Enhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model (full version), with more of the same.  Here is part of the Discussion:

First, in response to a second KA ‘hit’ in adulthood, there is an enhancement of both the upregulation of proinflammatory cytokines, microglial activation, and expression of the chemokine CCL2 in adult animals who had previously experienced early-life seizuresConsistent with the exaggerated proinflammatory cytokine and microglial activation responses after the second hit, these animals also show greater susceptibility to seizures and greater neuronal injury. Second, administration of Mzc to suppress of the upregulation of proinflammatory cytokines produced by early-life seizures prevents the exaggerated cytokine and microglial responses to the second KA hit in adulthood. Importantly, regulating the cytokine response to early-life seizures also prevents the enhanced neuronal injury, behavioral impairment, and increased susceptibility to seizures associated with the second KA insult. These results implicate microglial activation in the mechanisms by which early-life seizures lead to increased susceptibility to seizures and enhanced neurologic injury with a second hit in adulthood.

Not only that, but the authors speculated on the possibility of a rescue effect through neuroimmune modulation!

Our data support a role for activated glia responses in the mechanisms by which early-life seizures produce greater susceptibility to a second neurologic insult. The improved outcomes with Mzc administration in multiple acute or chronic injury models where proinflammatory cytokine upregulation contributes to neurologic injury (Hu et al., 2007; Somera-Molina et al., 2007; Karpus et al., 2008; Lloyd et al., 2008) suggest that disease-specific interventions may be more effective if combined with therapies that modulate glial responses.  These results are additional evidence that glial activation may be a common pathophysiologic mechanism and therapeutic target in diverse forms of neurologic injury (Akiyama et al., 2000; Craft et al., 2005; Emsley et al., 2005; Hu et al., 2005; Perry et al., 2007). Therapies, which selectively target glial activation following acute brain injury in childhood, may serve to prevent neurologic disorders in adulthood. These findings raise the possibility that interventions after early-life seizures with therapies that modulate the acute microglial activation and proinflammatory cytokine response may reduce the long-term neurologic sequelae and increased vulnerability to seizures in adulthood.

(Please note, the agent used in the above studies, kainic acid, is powerful stuff, and the seizures induced were status epileptcus, a big deal and a lot different than febrile seizures.  That doesn’t mean that febrile seizures are without effect, I don’t think we are nearly clever enough to understand that question with the level of detail that is needed, but they are qualitatively different and not to be confused.)

The idea of modulating glial function as a preventative measure seems especially salient to the autism community alongside the recent (totally great) bone marrow studies observing benefits to a Rett model and an early life immune activation model of neurodevelopment.

A lot of kids with autism go on to develop epilepsy in adolescence, with some studies finding prevalence in the range of 30%, which terrifies the shit out of me.  Is a primed microglial phenotype, a sensitization and increased susceptibility to seizures one of the mechanisms that drive this finding?

After Somera-Molina, I started noticing a growing mention of glial priming as a possible explanation for altered neuroimmune mechanics in a lot of places.  Much of the early life immune literature has sections on glial priming, Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (full / highly recommended / Staci Bilbo!) is a nice review of 2010 data that includes this:

However, there is increasing support for the concept of “glial priming”, in which cells can become sensitized by an insult, challenge, or injury, such that subsequent responses to a challenge are exaggerated (Perry et al., 2003). For instance, a systemic inflammatory challenge in an animal with a chronic neurodegenerative disease leads to exaggerated brain inflammation compared to a control animal (Combrinck et al., 2002). The morphology of primed glial cells is similar to that of “activated” cells (e.g. amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. Upon challenge, however, such as infection or injury in the periphery, these primed cells will over-produce cytokines within the brain compared to cells that were not previously primed or sensitized (Perry et al., 2002)This overproduction may then lead to cognitive and/or other impairments (Cunningham et al., 2005; Frank et al., 2006; Godbout et al., 2005).

Other studies included increased effects of pesticide exposure following immune challenge, Inflammatory priming of the substantia nigra influences the impact of later paraquat exposure: Neuroimmune sensitization of neurodegeneration, which includes, “These data suggest that inflammatory priming may influence DA neuronal sensitivity to subsequent environmental toxins by modulating the state of glial and immune factors, and these findings may be important for neurodegenerative conditions, such as Parkinson’s disease (PD).”  Stress was also found to serve as a priming agent in Glucocorticoids mediate stress-induced priming of microglial pro-inflammatory responses, which studied the effect of stress mediated chemicals on inflammatory challenges; the authors get bonus points for using glucocorticoid receptor agonists and surgical procedures to eliminate glucocorticoid creation to observe a priming effect of stress on neuroimmune response.

Here is a terrifying but increasingly unsurprising study on how neonatal experience modifies the physical experience of pain in adulthood, recently published in BrainPriming of adult pain responses by neonatal pain experience: maintenance by central neuroimmune activity

Adult brain connectivity is shaped by the balance of sensory inputs in early life. In the case of pain pathways, it is less clear whether nociceptive inputs in infancy can have a lasting influence upon central pain processing and adult pain sensitivity. Here, we show that adult pain responses in the rat are ‘primed’ by tissue injury in the neonatal period. Rats that experience hind-paw incision injury at 3 days of age, display an increased magnitude and duration of hyperalgesia following incision in adulthood when compared with those with no early life pain experience. This priming of spinal reflex sensitivity was measured by both reductions in behavioural withdrawal thresholds and increased flexor muscle electromyographic responses to graded suprathreshold hind-paw stimuli in the 4 weeks following adult incision. Prior neonatal injury also ‘primed’ the spinal microglial response to adult injury, resulting in an increased intensity, spatial distribution and duration of ionized calcium-binding adaptor molecule-1-positive microglial reactivity in the dorsal hornIntrathecal minocycline at the time of adult injury selectively prevented both the hyperalgesia and early microglial reactivity associated with prior neonatal injury. The enhanced neuroimmune response seen in neonatally primed animals could also be demonstrated in the absence of peripheral tissue injury by direct electrical stimulation of tibial nerve fibres, confirming that centrally mediated mechanisms contribute to these long-term effects. These data suggest that early life injury may predispose individuals to enhanced sensitivity to painful events.

One of the primal drivers of behavior in any animal, paincan be persistently modified at a molecular level!  Have you ever known someone that seemed to have a higher pain tolerance than you?  Maybe they did, and the training of their microglia (or yours) in early life might be why.  The most basic physiologic responses can be organized through the crucible of early life events sensitizing microglia to the future environment.  Multi hit wow!

The effect that befalls us all, getting older, has a ton of studies on the effect of aging on glial priming, with greatest, err, ‘hits’ including Immune and behavioral consequences of microglial reactivity in the aged brain,  Aging, microglial cell priming, and the discordant central inflammatory response to signals from the peripheral immune system (full),Immune and behavioral consequences of microglial reactivity in the aged brain (full), and the autism implication heavy Microglia of the Aged Brain: Primed to be Activated and Resistant to Regulation,  and others.  Broadly, these studies spoke of the same pattern, a primed neuroimmune response, except in this instance, the “hits” that predisposed towards altered microglial reactivity weren’t a vigorous insult during development, but just the hum drum activity of growing older.  It wasn’t a hit so much, more like a then gentle force of a relentless tide, but the functional effect on microglia response was largely similar, responses to stimuli were changed and programming was observed.  I do not believe that the underlying instrument of change in age related priming is understood, but the thought occurs to me that it could simply be an exhaustion effect; a lifetime of exposure to inflammatory cytokines gradually changes the microglial phenotype.

So what about autism?

First and foremost, it provides us a line of insight into the likelyhood of a causal relationship between an altered neuroimmune milieu and autism (or nearly any other neurological disorder); that is, the question of whether or not our continued and repeated findings of altered neuroimmune parameters in the autism population represent a participating force in autism, as opposed to an artifact, a function of something else, which is also causing autism, or perhaps a result of having autism.  While these are still possible explanations, the findings of glial priming provide additional detail on available mechanisms to affect brain activity and behavior through neuroimmune modifications alone.

If nothing else, we now know that we need not rely on models with no underlying substrate except the lamentations of ‘correlation does not equal causation’ and the brash faith of another, as of yet undefined, explanation.  These models tell us that immune mediated pathologies can be created (and removed!) in very well established animal models of behavioral disturbances with corollaries to autism findings.

For more direct links to autism, we can look at the autism immune biomarker data set and find evidence of primed peripheral (i.e., outside the CNS) programming, literal examples where the autism population responds with a different pattern than the control group including an increased response to some pathogen type agonists, increased immune response following exposure to pollutants, of even dietary proteins.

The pattern we see of an altered microglial phenotype in the autism population, a state of chronic activity, is certainly consistent with disturbed developmental programming; it does not seem unlikely to me that a priming effect is also present, the initial prime seems to be responsible for the programming.   As far as I know, there are no studies that have directly attempted to evaluate for a primed phenotype in the microglia of the autism population; I’d be happy to be corrected on this point.

Thinking about the possibility of increased microglial responsiveness and possible cognitive effects of a sustained neuroimmune toggling got me wondering if this is one of the mechanisms of a change in behavior following sickness?  Or, alternatively, for some of us, “Is This Why My Child Goes Goddamn Insane And Stims Like Crazy For A Week After He Gets Sick?

If we look to a lot of the studies that have shown a priming effect, they share a common causative pathway as some cases of autism, an early life immune insult.  For some examples, the interested reader could check out Neonatal programming of the rat neuroimmune response: stimulus specifc changes elicited by bacterial and viral mimetics (full paper), Modulation of immune cell function by an early life experience, or the often mentioned Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper).  If there are some cases of autism that have an early life immune insult as a participating input, it is very likely a primed microglia phenotype is also present.

The studies on aging are bothering me, not only am I getting older, but the findings suggest that a priming need not necessarily mandate a distinct ‘hit’, it can be more like a persistent nudge.   Our fetuses and infants develop in an environment with an unprecedented number of different nudges in the past few decades as we have replaced infection with inflammation.   Acknowledging this reality, however, raises the troubling thought that our embrace of lifestyles associated with increased inflammation has reached a tipping point that we are literally training the microglia of our children to act and react differently; we aren’t waiting a lifetime to expose our fetuses and infants to environments of increased inflammation, we are getting started from the get go.

Even with all of that, however, there is a genuinely microscopic Google footprint if you search for “autism ‘glial priming’”.  So, either I’m seeing phantoms (very possible), or the rest of the autism research community hasn’t caught on yet, at least in such a way that Google is notified.

Even if I am chasing phantoms, there is evidence of a widespread lack of understanding of the depth of the neuroimmune/behavioral crosstalk literature, even by the people who should be paying the most attention.  This was brought to my attention by a post at Paul Patterson’s blog, where Tom Insel was quoted as finding the recent Patterson and Derecki findings ‘unexpected’.

A bone marrow transplant, which replaces the immune system, corrected both the immune response and the behavior. This finding, which was unexpected, is surprisingly similar to another recent paper reporting disappearance of the symptoms of Rett syndrome in mice following a bone marrow transplant. 

Keep in mind, this is from the guy who is the head of the IACC!  I can tell you one thing; while the studies were impressive, I don’t think that the findings were especially unexpected.  The researchers took the time to give mice bone marrow transplants, and in Wild-type microglia arrest pathology in a mouse model of Rett syndrome, the authors utilized a variety of knockout mice and even partial body irradiation to illuminate the question of neuroimmune participation in disorder.   This work was not initiated in a vacuum, they did not throw a dart at a barn door sized diagram of study methodologies and land on ‘bone marrow transplants with subsequent analysis of microglia population properties and behaviors, accounting for different exposure timeframes, radiation techniques, and genotypes’.   These were efforts that had a lot of supporting literature in place to justify the expense and researcher time.  [I really want to find time to blog both of those papers in detail, but for the record, I did feel the rescue effects are particularly nice touches.]

So given that the head of the IAAC was surprised to find that immune system replacement having an effect on behavior was ‘surprising’, I’m not all together shocked at the relative lack of links on ‘glial priming’ and autism, but I don’t think it will stay that way for too much longer.  As more experiments demonstrating a primed phenotype start stacking up, we are going to have to find a way to understand if generation autism exhibits a primed glial phenotype.  I don’t think we are going to like the answer to that question very much, and the questions that come afterwards are going to get very, very inconvenient.

Spelling it out a bit more, with bonus speculation, we should remember our recent findings of the critical role microglia are playing in shaping the neural network; our microglia are supposed to be helping form the physical contours of the brain, a once in a lifetime optimization of synaptic structures that has heavy investment from fetushood to toddlerhood. Unfortunately, it appears that microglia perform this maintenance while in a resting state, i.e., not when they have been alerted of an immune response and taken on a morphology consistent with an ‘activated state’.  An altered microglia morphology can be instigated during infection, or perceived infection and consequent immune response.  For examples of peripheral immune challenges changing microglial morphology, the neuroimmune environment and behavior some examples include:  Peripheral innate immune challenge exaggerated microglia activation, increased the number of inflammatory CNS macrophages, and prolonged social withdrawal in socially defeated mice, Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system, or Long-term changes of spine dynamics and microglia after transient peripheral immune response triggered by LPS in vivo.

But what if we have a susceptible population, a population sensitized such that the effects of an immune challenge would result in an exaggerated and extended microglial response, effectively increasing the length of time the microglia would be ‘not resting’.  What might be the changes in this population in response to a series of ‘hits’?

It does not seem to be a large logical leap to assume that if some of the altered brain physiology in autism is due to abnormal microglia function during the period of robust synaptic pruning, triggering the microglia to leave their resting state for an extended period in response could be a reasonable participant.  Think of it as an exaggerated loss of opportunity effect, essentially a longer timeframe during which the microglia are not performing synaptic upkeep when compared to the microglia in an individual that is not sensitized.   While our brains do show a lot of ability to ‘heal’, that does not mean that all things or times are created equally; there are some very distinct examples of time and spatially dependent neurochemical environments during early synapse development, environments that change as time goes on; i.e., Dynamic gene and protein expression patterns of the autism-associated met receptor tyrosine kinase in the developing mouse forebrain (full paper), or A new synaptic player leading to autism risk: Met receptor tyrosine kinase.   In other words, recovering from a delay in microglial participation in synaptic pruning during development may not be as simple as ‘catching up’;  if the right chemical environment isn’t available when the microglia get done responding, you might not be able to restart like a game of solitaire.  The Met levels might be different, the neurexin levels might be different, a thousand other chemical rally points could be set that much of a nudge differently; in a system dependent on so many moving variables being just so, an opportunity missed is an opportunity lost.  For good.

While the effects of a series of challenges and consequent obstructions of synaptic maintenance might not be acutely clear, I am becoming less and less convinced of the ‘safety’ of an observed lack of immediately obvious effects.   I think that an intellectually honest evaluation of our recent ‘discoveries’ in many areas of early life disturbances (i.e., antibiotics and IDB risk, C-section and obesity risk, birth weight and cardiovascular risk) tell us that subtle changes are still changes, and many rise to the level of a low penetrant, environmentally induced effect once we get clever enough to ask the right question.  And boy are we a bunch of dummies.

Taking all of this into consideration, all I can think is thank goodness we haven’t been artificially triggering the immune system of our infants for the past two decades while we were blissfully unaware of the realities of microglial maintenance of the brain and glial priming!  What a relief that we did not rely on an assumption of lack of effect as a primary reason not to study the effect of an immune challenge.   If we had done those things, we might start kicking ourselves when we realized out that our actions could be affecting susceptible subsets of children who were predisposed to reacting in difficult to measure but real ways that could literally affect the physical structure of their brains.

Oops.

–          pD

Hello friends –

I have a confession to make.  The fact that a lot of very smart people have ignored or flat out laughed at my arguments bothers me sometimes.  I have applied non-trivial, not to be rebated time and effort to put forth what I considered to be logical views, scientifically defendable and important ideas; and yet people I knew were otherwise rational, and in some cases, very intelligent, just hadn’t seemed to get what I was saying.  Often this was within the context of a discussion argument of vaccination, but my larger concern, that of a non-imaginary, non-trivial increase in children with autism in the past decades, also usually falls on deaf ears.  If “environmental changes” incorporate the chemical milieu of our mother’s wombs, the microbial world our infants are born into, or the ocean of synthetic chemicals we all swim through every day, we have no rational conclusion but that our environment has changed a lot in the past few decades.  Considered within the context of the reality based model where the events of early life can be disproportionally amplified through the lifetime of an organism, clinging to the idea that there has been a stable incidence of autism seems dangerously naïve, at most charitable.

And yet, for the most part, many or most of the people who are alarmed are crackpots.   There were times I questioned myself.  Am I missing something?  Am I chasing phantoms?  Why aren’t any of these other smart people as worried as I am?

A while ago I got a copy of Microglia in the developing brain: A potential target with lifetime effects (Harry et all), a paper that tells me that if nothing else, I have some good company in pondering the potential for disturbances in early life to uniquely affect developmental outcome, in this instance through alterations to the neuroimmune system.  If I am incorrect about the validity of a developmental programming model with lifetime effects, lots of prolific researchers are wrong about the same thing in the same way.  Harry is a very thorough (and terrifying) review of the relevant literature.  Here is the abstract:

Microglia are a heterogenous group of monocyte-derived cells serving multiple roles within the brain, many of which are associated with immune and macrophage like properties. These cells are known to serve a critical role during brain injury and to maintain homeostasis; yet, their defined roles during development have yet to be elucidated. Microglial actions appear to influence events associated with neuronal proliferation and differentiation during development, as well as, contribute to processes associated with the removal of dying neurons or cellular debris and management of synaptic connections. These long-lived cells display changes during injury and with aging that are critical to the maintenance of the neuronal environment over the lifespan of the organism. These processes may be altered by changes in the colonization of the brain or by inflammatory events during development. This review addresses the role of microglia during brain development, both structurally and functionally, as well as the inherent vulnerability of the developing nervous system. A framework is presented considering microglia as a critical nervous system-specific cell that can influence multiple aspects of brain development (e.g., vascularization, synaptogenesis, and myelination) and have a long term impact on the functional vulnerability of the nervous system to a subsequent insult, whether environmental, physical, age-related, or disease-related.

Hell yeah!

The body of Microglia in the developing brain: A potential target with lifetime effects has tons of great stuff.  From the Introduction

The evidence of microglia activation in the developing brain of patients with  neurodevelopmental disorders(e.g., autism) and linkage to human disease processes that have a developmental basis (schizophrenia) have raised questions as to whether developmental  neuroinflammation actively contributes to the disease process. While much of the available data represent associative rather than causative factors, it raises interesting questions regarding the role of these ‘‘immune-type’’ cells during normal brain development and changes that may occur with developmental disorders. Within the area of developmental neurotoxicology, the potential for environmental factors or pharmacological agents to directly alter microglia function presents a new set of questions regarding the impact on brain development.

There is a short section on what is known about the colonization of the brain by microglia, it is a busy, busy environment, and while we are just scratching the surface, microglia seem to be involved in scads of uber-critical operations, many of which pop up in the autism literature.   It is just being confirmed that microglia constitute a distinct developmental path that diverges as an embryo, two papers from 2007 and 2010 are referenced as reasons we now believe microglia are a population of cells that migrate into the CNS before birth and are not replaced from the periphery in adulthood. From there, the beautiful complexity is in full effect; as the microglia develop and populate the brain there are specific spatial and morphological conditions, microglia are first evident at thirteen weeks after conception, and do not reach a stable pattern until after birth.   In fact, it appears that microglia aren’t done finishing their distribution in the CNS until the postnatal period, “With birth, and during the first few postpartum weeks, microglia disseminate throughout all parts of the brain, occupying defined spatial territories without significant overlap (Rezaie and Male, 2003) suggesting a defined area of surveillance for each cell.”

It occurred to me to wonder if there are differences in microglia settlement patterns in males and females in human infants, as has been observed in other models?  Could a spatially or temporally different number of micoglia, or different developmental profiles of microglia based on sex be a participant in the most consistent finding in the autism world, a rigid 4:1 male/female ratio?

Speaking towards the extremely low replacement rates for microglia in adulthood, the authors wonder aloud on the possible effects of perturbations of the process of microglial colonization.

The slow turn-over rate for mature microglia raises an issue related to changes that may occur in this critical neural cell population. While this has not been a primary issue of investigation there is limited data suggesting that microglia maintain a history of previous events. Thus, if this history alters the appropriate functioning of microglia then the effects could be long lasting. Additionally, a simple change in the number of microglia colonizing the brain during development, either too many or too few, could have a significant impact not on only the establishment of the nervous system network but also on critical  cell specific processes later in life.

(Emphasis mine)

Perhaps coincidentally (*cough*), we have abundant evidence of an altered microglial state and population in the autism population; while we do not know that these findings are the result of a disturbance during development, it is an increasingly biologically plausible mechanism, and thus far, I’ve yet to see other mechanisms given much thought, excepting the chance of an ongoing, undetected infection.

There is a brief section concerning the changes found in adult microglial populations in terms of density, form, and gene expression in different areas of the brain, “With further investigation into the heterogeneity of microglia one would assume that a significant number of factors, both cell membrane and secreted, will be found to be differentially expressed across the various subpopulations.”  Nice.

There is a section of the paper on microglial phenotypes, there are a lot of unknowns and the transformation microglia undergo between functional states is even more nebulously understood during  brain development.  “It is now becoming evident that in the developing brain, many of the standards for microglia morphology/activation may require readdressing.”  We haven’t even figured out what they’re doing in the adult brain!

There is a really cool reference for a study that shows altered microglial function dependent on the age of the organism.

In the adult rodent, ischemia can induce microglia to display either a more ramified and bushy appearance or an amoeboid morphology depending on the level of damage and distance from the infarct site(s). In the immature rodent, ischemia-induced changes in capillary flow or, presumably, altered CNS vascularization can retain the microglia in an amoeboid phenotype for longer and delay the normal ramification process (Masuda et al., 2011).

One way of looking at this would be to say that we should exercise extreme caution in trying to translate our nascent understanding of how mature microglia react when speculating on how immature microglia will act.  To follow up on just how little we know, there is a long discussion about the shortcomings of a the term ‘activated’ microglia with some details on chemical profiles of broadly generalized ‘classically inflammatory, ‘alternatively activated’, ‘anti-inflammatory’, and ‘tissue repair’ phenotypes.

Next up is a dizzyingly list of brain development functions that microglia are known, or suspected to participate in.  Without getting too deep in the weeds, of particular interest to the autism realm, that list includes neurogenesis and differentiation in the cortex [related: Courchesne, me], cell maturation via cytokine generation, axon survival and proliferation [related: Wolff, me],  programmed cell death of Purkinje cells, clearance of ‘early postnatal hippocampul neurons’, and the ‘significant contribution to synaptic stripping or remodeling events’, i.e., pruning (Paolicelli / fractaltine), and even experience dependent microglia / neuron interactions.  Taking all of this (and more) into consideration, the authors conclude “Thus, one can propose that alterations in microglia functioning during synapse formation and maturation of the brain can have significant long-term effects on the final established neural circuitry. “  Ouch.

Next up is a summary of many of the animal studies on microglial participation in brain formation, there is a lot there.  Interestingly (and particularly inconvenient) is the finding that a lot of the functional actions of microglia during development appear to operate after birth.  “Overall, the data suggest that microglial actions may be most critical during postnatal brain maturation rather than during embryonic stages of development.” Doh!

Early life STRESS gets some attention, and for once there is some good news if you look at it the right way.  There is something about a very cool study from Schwarz (et all / Staci Bilbo!) involving drug challenge that peered deep into the underlying mechanisms of an environmental enrichment model; animals given a preferential handling treatment were found by two metrics to have differential microglia response in adulthood with (biologically plausible) observations, increased mRNA levels for IL-10 production, and decreased  DNA methylation; i.e., less restriction on the gene that produces IL-10, and more messenger RNA around to pass off the production orders [totally beautiful!].  There is more including thyroid disruption (though in a way that I found surprising), and the observations of time dependent effects on immue disturbances.  (super inconvenient)

There is so much data that keeps piling on that the authors end up with “Overall, the existing data suggest a critical regulatory role for microglia in brain development that is much expanded from initial considerations of microglia in the context of their standard, immune mediated responses.”

A terrifying concept that I haven’t found time to dedicate a post towards is microglia priming, which gets some attention in Harry.

There is a significant amount of evidence regarding what is often termed ‘‘priming’’ and ‘‘preconditioning’’ events that serve to either exacerbate or provide neuroprotection from a secondary insult, respectively. In these states, the constitutive level of proinflammatory mediators would not be altered; however, upon subsequent challenge, an exaggerated response would be induced. The phenomena of priming represent a phenotypic shift of the cells toward a more sensitized state. . . Exactly how long this primed state will last has not been determined; however, data from microglia suggest that it can extend over an expanded period of time. Preconditioning can also represent changes that would occur not only over the short term but may be long lasting.”

I happen to think that microglia priming is going to be a very important cog in the machinery for this journey when all is said and done; the evidence to support a preconditioning system is strong, and in parallel, the things we see different in autism (and elsewhere) is consistent with a different set of operations of microglia, AND we also have evidence the disturbances that would invoke microglial change are subtle but real risk factors for autism.

What comes next is a type of greatest hits mashup of very cool papers on developmental programming in the CNS.

Galic et al.(2008) examined age related vulnerabilities to LPS in rats to determine critical age periods. Postnatal injection of LPS did not induce permanent changes in microglia or hippocampal levels of IL-1b or TNFa; however, when LPS was given during the critical postnatal periods, PND 7 and 14, an increased sensitivity to drug induced seizures was observed in 8-week-old rats. This was accompanied by elevated cytokine release and enhanced neuronal degeneration within the hippocampus after limbic seizures. This persistent increase in seizure susceptibility occurred only with LPS injection at postnatal day 7 or 14 and not with injections during the first day of life or at PND 20. Similar long-lasting effects were observed for pentylenetetrazol-induced seizures when PND 11 or 16 rat pups were subjected to LPS and hyperthermic seizures (Auvin et al., 2009). These results again highlight this early postnatal period as a ‘‘critical window’’ of development vulnerable to long-lasting modification of microglia function by specific stimuli. Work by Bilbo and co-workers demonstrated LPS-induced deficits in fear conditioning and a water maze task following infection of PND 4 rats with Escherichia coli. In the young adult, an injection of LPS induced an exaggerated IL-1b response and memory deficits in rats neonatally exposed to infection (Bilbo et al., 2005). Consistent with the earlier work by Galic et al. (2008), an age dependency for vulnerability was detected with E. coli-induced infection at PND 30 not showing an increased sensitivity to LPS in later life (Bilbo et al., 2006).

In particular, Galic 2008, or Postnatal Inflammation Increases Seizure Susceptibility in Adult Rats (full paper) was a very formative paper for me; it was elegant in design and showed alarming differences in outcome from a single immune challenge experience, if it occurred during a critical developmental timeframe.  If you haven’t read it, you should.

This paper has a nice way of distilling the complexity of the literature in a readable way.

One hypothesis for developmental sensitivity is the heterogeneous roles for inflammatory factors and pro-inflammatory cytokines during development, including their timing-, region and situation-specific neurotrophic properties. Many of the proinflammatory cytokines are lower at birth with a subsequent rapid elevation occurring during the first few weeks of life. In an examination of the developing mouse cortex between PND 5 and 11, mRNA levels for TNFa, IL-1b, and TNFp75 receptor remained relatively constant while a significant increase in mRNA levels of CR3, macrophage-1 antigen (MAC-1), IL-1a, IL-1 receptor 1 (IL- )R1, TNFp55 receptor (TNFp55R), IL-6, and gp130 occurred (Fig. 2). This data suggests that an upregulation of interleukins and cytokine receptors may contribute to enhanced cytokine signaling during normal cortical development.

One hypothesis put forward using a model reliant on postnatal exposure to LPS suggests that these types of exposure may ‘‘reprogram’’ neuroimmune responses such that adult stress results in hyperactivation of the hypothalamic pituitary adrenal (HPA) axis (Mouihate et al., 2010) and corticosterone  changes (Bilbo and Schwarz, 2009).While limited, the available data suggest that events occurring during development, especially postnatal development, have the  potential to cause long term alterations in the phenotype of microglia and that this can be done in a region specific manner.

[extremely inconvenient]

In what could, conceivably, be a coincidence, our available information on the autism brain also shows region specific changes in microglia populations, microglial activation profiles, and oxidative stress.   I do not believe the findings reviewed in Microglia in the developing brain: A potential target with lifetime effects will be meaningless artifacts; the likelihood that our observations of an altered neuroimmune state in autism are not, at least, participatory has become vanishingly small.

Can these findings inform us on the incidence question?  I was lurking on a thread on Respectful Insolence a while ago, and someone gave what I thought was a very succinct way of thinking about the changes that our species has encountered the past few decades; it went something like “we have replaced infection with inflammation”.  That’s a pretty neat way of looking at how things have gotten different for humanity, at least lots of us, and especially those of us in the first world.  We used to get sick and die early; now we live longer, but oftentimes alongside chronic disorders that share a common underlying biological tether point, inflammation.

Any dispassionate analysis of the available data can tell us that we have, indeed, replaced infection with inflammation; we suffer from less death and misery from infection, but more metabolic disorder, more diabetes, more hypertension, more asthma and autoimmune conditions than previous generations.   We have largely replaced good fatty acids with poor ones in our diet.  All of these conditions are characterized by altered immune biomarkers, including an increase in proinflammatory cytokines.   Those are the facts that no one can deny; we have replaced infection with inflammation.

But when we look to the findings of Microglia in the developing brain: A potential target with lifetime effects, it becomes clear that our newfound knowledge of microglial function and crosstalk with the immune system raises some very troubling possibilities.

Lately it has been quite in vogue among a lot of the online posting about autism to at least mention environmental factors which could participate in developmental trajectory leading to autism; that’s a big step, an important and long overdue acknowledgement.  If you pay close attention, you will notice that 99% of these admissions are handcuffed to the word “prenatal”.  This is likely an attempt to deflect precise questions about the robustness of our evaluation of the vaccine schedule, but the big question, the incidence question, still hinges on fulcrum of the genetic versus environmental ratio ; that is a problem for the purveyors of the fairytale because the prenatal environment of our fetuses, the chemical milieu of their development, is qualitatively different compared to generations past.  That chemical soup is their environment; and that environment has unquestionably changed in the past decades as we have replaced infection with inflammation.

Our previous analysis tells us that invoking inflammation outside the brain modifies microglial function inside the wall of the blood brain barrier; good or bad, no honest evaluation of the literature can argue against a lack of effect.  What happens outside the brain affects what happens inside the brain.  If, however, microglia are active participants in brain formation, as a swath of recent research indicates, can this fact give us insight into the incidence question?

Is a state of increased inflammation the pathway between maternal asthma, depression, stress, and obesity being associated with increased risk of autistic offspring?  Have we replaced infection with inflammation plus?

What could be more lethal to the fairytale of a static tale of autism than a positive relationship between a lifestyle characterized by increased inflammation and the chances of having a baby with autism?

Are we totally fucked?

We cannot know the answers unless we have the courage to ask the difficult questions with methods powerful enough to provide good data, and it won’t be easy.  The static rate of autism fairytale is a comforting notion; it expunges responsibility for the coronal mass ejection sized change to our fetuses developing environment, and while hiding behind the utterly frail findings of social soft scientists, we can happily place tin foil hats and accusations of scientific illiteracy on anyone who might be worried that our abilities have outstripped our wisdom.  That is a terrible, cowardly way to approach the incidence question, what we should be doing is exactly the opposite, ridiculing the epidemic sized error bars in prevalence studies and demanding more answers from the hard scientists.  Eventually we will get there and it will be a critical mass of information from studies like Harry that will propel decision makers to abandon the fairytale for a course regulated by dispassionate analysis.

–          pD

Hello friends –

A study with a beautifully terse title, Microglia in the Cerebral Cortex in Autism landed in my inbox the other day.  It adds to the growing literature showing perturbations in neuroimmune system in the autism population, this time by measuring the number of microglia in different parts of the brain.  Here is the abstract:

We immunocytochemically identified microglia in fronto-insular (FI) and visual cortex (VC) in autopsy brains of well-phenotyped subjects with autism and matched  controls, and stereologically quantified the microglial densities. Densities were determined blind to phenotype using an optical fractionator probe. In FI, individuals with autism had significantly more microglia compared to controls (p = 0.02). One such subject had a microglial density in FI within the control range and was also an outlier behaviorally with respect to other subjects with autism. In VC, microglial densities were also significantly greater in individuals with autism versus controls (p = 0.0002). Since we observed increased densities of microglia in two functionally and anatomically disparate cortical areas, we suggest that these immune cells are probably denser throughout cerebral cortex in brains of people with autism.

[Note: You don’t see p-values of .0002 too often!]  This paper is at a high level largely similar to another recent paper, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism (discussed on this blog, here).  The authors were clever here, they intentionally used two very anatomically different, and spatially separated parts of the brain to evaluate for microglia population differences, a sort of bonus slice to learn more about the population of microglia in the brain.

The specific measurement technique in use, staining for specific antibodies, does not give us information regarding the activated/non activated state of the microglia, a determination which must be made with evaluations of morphology, though several other studies have measured this directly, and many more provide indirect evidence of a chronic state of activation of microglia.   Not only did the author s report an increase in population density in the autism group, the number of microglia was also positively correlated between sites; i.e., a patient with more microglia in the visual cortex was also more likely to have more microglia in the fronto-insular.

These findings demonstrate that, at the time of death, there were significantly higher microglial densities in the subjects with autism compared to the control subjects, and that this change in microglial density is widespread throughout the cerebral cortex in autism. The microglial  densities in FI and VC in the same subject were significantly correlated (both measures were available in 10 controls and 8 autistic subjects for a total of 18 subjects) with Pearson’s r2 = 0.4285, p = 0.0024 (Fig. 6). This indicates that the elevation in density is consistent between these areas, and probably throughout the cortex, in both subjects with autism and controls.

Also of interest, in the control group microglia densities tended to decrease with age, but this change was not seen in the autism population.

There is some discussion about a big problem in the autism research world, a very real and meaningful dearth of available tissue samples, this study shared five patients with Morgan, and one from Vargas.  [Note: Sign up to help.  Morbid but necessary.]

The authors went on to ask the exact same question I had, “How and when does the increased density of autistic microglial arrays arise, and how is it maintained?”  Unfortunately, while there aren’t any good answers, I was still a little disappointed with the analysis.  There is a quick rundown of a variety of neuroimmune and peripheral immune findings in autism, and some thoughts on ‘sickness behavior’ with the implicit interconnectedness of the immune state and behaviors, and some discussion on some of the many animal models of maternal immune activation in autism.

In an stroke of amazing serendipity, the authors wonder aloud towards the possibility of a type of distracted worker effect of microglia on neural networks, sort of a bank shot on the autism paradox I struggled with in my previous post when I said,

Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain?  Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?

The authors of Microglia in the Cerebral Cortex in Autism state

In contrast, microglia can also phagocytize synapses and whole neurons, thus disrupting neural circuits. For example,when the axons of motor neurons are cut, the microglia strip them of their synapses (Blinzinger and Kreutzberg 1968; Cullheim and Thams 2007; Graeber et al. 1993). Another example of the disruption of circuitry arises from the direct phagocytosis of neurons. Neurons communicate with microglia by emitting fractalkine*, which appears to inhibit their phagocytosis by microglia. Deleting the gene for the microglial fractalkine receptor (Cx3cr1) in a mouse model of Alzheimer’s disease has the effect of preventing the microglial destruction and phagocytosis of layer 3 neurons that was observed in these mice in vivo with 2-photon microscopy (Furhmann* et al. 2010). In particular, Cx3cr1 knockout mice have greater numbers of dendritic spines in CA1 neurons, have decreased frequency sEPSCs and had seizure patterns which indicate that deficient fractalkine signaling* reduces microglia-mediated synaptic  pruning, leading to abnormal brain development, immature connectivity, and a delay in brain circuitry in the hippocampus (Paolicelli* et al. 2011). In summary, the increased density of microglia in people with autism could be protective against other aspects of this condition, and that a possible side-effect of this protective response might involve alterations in neuronal circuitry.

Oh hell yeah.  (* concepts and papers discussed on this blog, here)

Going back to the big dollar question, How and when does the increased density of autistic microglial arrays arise, and how is it maintained?”, the possibility of an ongoing infection was raised as a one option, “The increase of microglial densities in individuals with autism could be a function of neuroprotection in response to harmful microorganisms.”  Vargas had a dedicated section towards a failure to find agents of the peripheral immune system that are consistent with infiltration from the peripheral immune system commonly observed during acute infection, I do not think other papers have looked for that per se, but will cede to someone with better data.  (?)   There was a very weird paper from Italy that pointed to a possible polyomavirus transmission from the father in the autism group, though this study was not referenced in Microglia in the Cerebral Cortex in Autism. [Note:  I showed my wife this paper, and she told me, “Good job with the autism gametes.”  Nice.]  Could a virus cause autism, is a nice discussion on this that includes blog and personal favorites, Fatemi, Patterson, and Persico discussing the possibilities and limitations of the study.  Great stuff!

While I must admit the possibility that the chronically activated microglia in autism are working on purpose, the irony gods mandate that I wonder aloud if certain segments of the autism Some-Jerk-On-The-Internet population will cling to the possibility that autism is caused by a disease in order to disavow a causative role for neuroinflammation?  Those are some tough choices.

There is a discussion on the myriad of ways that microglia could directly participate in autism pathogenesis, starting the discussion off right to the point, “By contrast, there are diseases that arise from intrinsic defects in the microglia themselves which can cause stereotypic behavioral dysfunctions.”  There is a short discussion of Nasu-Hakola disease, something I’d never heard of, which has evidence of an increase in cytokines as a result of genetically driven microglial deficiencies, and shows striking behavioral manifestations.

The possibility of some areas of the brain being more susceptible to alterations than others is there too, “Thus, while changes in microglial density appear to be widespread in brains of autistic individuals, some areas may be more vulnerable than others to its effects.”   Considering this idea alongside the extremely heterogeneous set of symptoms assigned to autism, a curious question to ponder becomes; if neuroinflammation is a participatory process in the behavioral manifestation of autism, could some of the variability in autistic behaviors be explained by spatially specific gradients of microglial activity?  Going further, considering the still largely mysterious migration of microglia into the brain during development, could the temporal origin of microglial activation in autism be a determinant in the eventual behavioral manifestations?  These are tricky questions, and I don’t think that our current methodological capacities are sufficient to start thinking about forming a model for analysis.

One concept I was surprised to not receive attention was a developmental programming model, where animal studies tell us that if something happens during critical developmental timeframes, the effect can propagate into adulthood.    In fact, one study, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all)  exposed four day old animals to e-coli, which found, among other things, “significantly more microglia in the adult DG of early-infected rats”, something seemingly of considerable salience to the current findings, especially considering the known risk factors of early infections as autism risk factors.  In Bland, no external agent other than an infection during early life was necessary; this is the essence of the developmental programming model, even after the infection was long since cleared, patterns of physiology were imprinted, the animals recovered from e-coli but were changed from the experience.  This my biggest issue with the possibility of an as of yet undefined, and continued evidence free pathogen or process that is causing the immune abnormalities we see in autism, it mandates we ignore existing biologically plausible models that fit well within known risk factors for autism.  Why?

Another area this paper was curiously silent on is the data regarding differences in males and females in the timeframes of microglial migration into the brain, something I’d like to learn much more about soon.  As an example, Sex differences in microglial colonization of the developing rat brain [yet another by blog favorite, Staci Bilbo] reported “the number and morphology of microglia throughout development is dependent upon the sex and age of the individual, as well as the brain region of interest” among other findings broadly consistent with a beautiful complexity.  This is interesting fodder for a discussion concerning possibly the most persistent finding in autism, a very high male to female ratio that has a series of possible explanations [somewhat discussed on this blog, here].

So we know more, but still have only increased our knowledge incrementally.  It is increasingly likely that an increased number of microglia in many areas of the brain is characteristic of autism, but the whys, hows, whens, wheres, and whoms still hold many mysteries.  The more things change, the more they stay the same.

–          pD

Hello friends –

I’ve had a couple of interesting papers land in my pubmed feed the past few weeks that seem to be tangentially touching on something that has been at the back of my mind for a long time; namely, the repeated findings of a state of an ongoing immune response in the CNS of the autism population, coupled with a behavioral state that is either static, or in many cases, showing gradual improvement over time.  [Discussions of ongoing immune response in the brain in autism, here, here, or here].  This is exactly the opposite of what I expected.  Most of the conditions I had generally associated with a state of neuroinflammation, i.e., Alzheimer’s or Parkinson’s show a behavioral profile opposite to autism over time, i.e., a deterioration of skills and cognitive abilities.   The diagnosis for these conditions is never a straight line or a gradual curve upwards, but a dispassionately reliable trajectory of a downward spiral.

This is something that has been really bugging me a lot as a riddle, I’ve mentioned it here in comments, and other places on the Internet.  While outright signs of neuroinflammation are clearly associated with conditions you would rather not have, as opposed to have, we must admit that the available evidence tells us that  we cannot just wave our hands, say ‘neuroinflammation!’, and know much more than the broad strokes.  [Note: In my early days of my AutismNet life, my view was somewhat less nuanced.]  I think that part of what was bothering me is the result of an oversimplified model in my mind’s eye, but I’d formed that model on top of a set of measurements that had empirical precision but underpowered understandings, alongside a more fundamental lack of knowledge.

We know a little more now.

The first paper that really got me thinking along these lines was Synaptic pruning by microglia is necessary for normal brain development, (discussed on this blog, here), which provided evidence of microglial involvement in the ‘pruning’ of synapses, an important step in brain development thought to streamline neural communication by optimizing neuron structure.  This was the first paper I’d read that hinted at microglia participation in ‘normal’ brain function; it was only very recently that microglia were considered to have any role in non pathological states.  Another paper, Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease, also implicated microglia in synaptic pruning.

Then I got myself a copy of The role of microglia at synapses in the healthy CNS: novel insights from recent imaging studies. It is a review of several recent studies on the non-excited life of microglia.

In the healthy brain, quiescent microglia continuously remodel their shape by extending and retracting highly motile processes. Despite a seemingly random sampling of their environment, microglial processes specifically interact with subsets of synaptic structures, as shown by recent imaging studies leading to proposed reciprocal interactions between microglia and synapses under non-pathological conditions. These studies revealed that various modalities of microglial dynamic behavior including their interactions with synaptic elements are regulated by manipulations of neurotransmission, neuronal activity and sensory experience. Conversely, these observations implied an unexpected role for quiescent microglia in the elimination of synaptic structures by specialized mechanisms that include the phagocytosis of axon terminals and dendritic spines. In light of these recent discoveries, microglia are now emerging as important effectors of neuronal circuit reorganization.

This review by Tremblay was published in 2012, evidence of the nascent nature of our available data on microglial involvement in the normal brain environment; Tremblay states that part of the reason this type of finding is so recent is the relative difficulty of measuring microglia in non excited states.  They were the electrons of brain measurements; our previous attempts to measure them were capable of causing them to change morphology.

The roles of ‘resting’ or immunologically quiescent microglia have remained relatively unknown (also see Tremblay et al., 2011). This is largely due to the difficulties of studying microglia in their non-activated state. Microglia respond promptly to any changes occurring in their environment, and therefore experimental ex vivo and in vitro preparations inevitably result in transformation of their normally prevailing behavior.

Nice.

Anyway, some new whizbang technologies (i.e., in vivo two-photon laser scanning microscopy)[?] are allowing researchers to peer into the ho-hum everyday activities of ‘non activated’ microglia, and what they are finding is that the term ‘activated microglia’ might be a bit of a misnomer, microglia have been participating in brain function all along, it is just that our filters were insignificantly powered to detect some of their actions until very recently.   Several studies have shown that so called ‘resting’ microglia are constantly evaluating their environment with protusions that seemed to operate rather quickly in relationship to other types of neurons.

This unexpected behavior suggested that resting or surveillant microglia may continuously survey the brain parenchyma as part of their immune function, which would justify the substantial expenditure of energy required to continuously maintain microglial dynamics in the normal brain, without excluding the possibility of an additional, distinct contribution to normal brain physiology

Several papers are reviewed that utilized a couple of highly technical methods, including double roll your own transgenic mouse models to visualize the interactions of microglia in a non excited state and synapses.  Specific areas of the brain were measured in different studies, microglia were observed transiently engaging with neurons and seemed to target some dendrites for removal.  The authors speculate that this could be a mechanism by which neuronal network maintenance, plasticity, could be affected.

In the mature healthy CNS, neuronal networks are continuously remodeled through the formation, modification and elimination of synaptic structures (see Fortin et al. (2011) for molecular mechanisms of structural plasticity) in relation with behavioral and sensory experience.

And

To determine a possible role of surveillant microglia in the structural remodeling of synaptic structures under normal physiological conditions, Tremblay et al. (2010b) also examined the size changes of spines and terminals before, during and after microglial contacts. Spines contacted by microglial processes during imaging (30–120 min sessions) were found to be smaller initially than those which remained non-contacted. Spines, but not terminals, also underwent transient increases in size during microglial contact, with smaller spines showing the most pronounced changes. Surprisingly, chronic imaging over 2 days further revealed a statistically significant difference in the elimination rate of microglia-contacted spines: spines contacted by microglia were more frequently eliminated than non-contacted spines (24 versus 7%; P  0.05), and in all cases, only the small spines were seen to disappear. These observations suggest that despite an apparently random sampling of the parenchyma, microglial processes specifically target a subset of small, structurally dynamic and transient dendritic spines.

There is also some description of studies that seemed to indicate that the microglial/synapse interactions could be modified through environmental stimulus, two experiments were described involving sensory deprivation and consequent changes in microglia activity.  Other experiments described changes in microglial surveillance as a result of induced changes in neuronal excitability by chemical agonists or antagonists of glutamate receptors.  [Perhaps this is the basis of the curious findings in Neuroprotective function for ramified microglia in hippocampal excitotoxicity?]

In their concluding statements, Tremblay provides a good description of just how little we know, and in a style that I love, poses open questions for the newer rounds of literature to address.

Since the recent studies have barely scratched the surface (of the brain in this case), the modalities of microglial interactions with excitatory and inhibitory synapses throughout the CNS, much as their functional significance and particular cellular and molecular mechanisms still remain undetermined. For example, in which contexts do quiescent microglia directly phagocytose axon terminals and dendritic spines, use other mechanisms such as proteolytic remodeling of the extracellular space, or refrain from intervening?  How do surveillant microglia recognize and respond to the various molecular signals in their environment, including dynamic changes in neurotransmission and neuronal activity at individual synapses? How do these immune cells cooperate with other glial cells, as well as peripheral myeloid cells, in maintaining or shaping neuronal architecture and activity? And, as in the case of microglial memory of past immune challenges (see Bilbo et al., 2012), do surveillant microglia somehow remember their previous behavioral states, the flux of information processing in the brain, or the structural changes of synaptic elements in recent and not so recent windows of intervention?

The last sentence there, I think, is especially salient considered within a context of developmental programming.

So what we’ve learned is that decades after the discovery of microglia cells as the immune regulators in the CNS, they appear to also be participating in more fundamental maintenance of the neural structure of our brains; there is increasing evidence of direct relationships in synaptic and axonal removal as well as roles in neurotransmission and the regulation of excitability.   Is more on the horizon?

But what about autism and our apparent autism paradox of a static or improving behavioral state alongside conditions of immune activation within the CNS?

Well, I have also been thinking about two brain scanning studies that have come out not too long ago, Neuron Number in Children With Autism (Courchesne et all) , which found increased numbers of neurons in the autism cohort, and Differences in White Matter Fiber Tract Development Present From 6 to 24 Months in Infants With Autism (Wolff et all) which found that the autism group showed denser bundled of white matter, so called wiring, between different parts of the brain.  In both of these studies mention is made of the fact that it was possible that their findings, increased cell numbers could be the result of inappropriate removal of excess neurons during development.

Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons.

and

For example, differences in structural organization prior to a period of experience-dependent development related to social cognition (52–54) may decrease neural plasticity through limitations on environmental input, preventing typical neural specialization (52). These alterations could have a ripple effect through decreasing environmental responsiveness and escalating invariance*, thus canalizing a specific neural trajectory that results in the behavioral phenotype that defines ASDs. In typical development, the selective refinement of neural connections through axonal pruning (55) along with constructive processes such as myelination (56) combine to yield efficient signal transmission among brain regions. One or both of these mechanisms may underlie the widespread differences in white matter fiber pathways observed in the current study. 

* 😦

So, we have growing evidence of microglial participation of neural maintenance alongside growing evidence of impaired maintenance in the autism cohort.

Can our autism paradox be explained by microglia converging in the center of these related lines of thought?  Is the answer to our riddle that the ongoing immune response in the brain is not sufficiently powered, or targeted, to cause increasing loss of abilities, but instead, was enough to keep critical, once in a lifetime chances for brain organization from occurring?  Are increased neuron number and altered white matter tracts the result of microglia not performing the expected maintenance of the brain?  Are the findings from Courchesne and Wolff the opportunity costs of having a microglia activated during decisive developmental timeframes?

That is a pretty neat idea to consider.

Even without the Courchesne and Wolff, the findings that specifically mention impaired network maintenance as possible culprits, the findings of active participation of ‘non-active’ microglia in brain optimization and normal processes is a very problematic finding for another autism canard, the idea that findings of neuroinflammation may not be pathological.  The intellectually honest observer will admit that the crux of this defense lay in vaccine count trial testimony presented by John Hopkin’s researchers after their seminal neuroinflammation paper was published.  Unfortunately, the vigor with which this testimony is trotted out online does not match the frequency with which such ideas actually percolate into the literature.

But with the data from Tremblay, Paolicelli, and others, such an idea becomes even more difficult to defend, we must now speculate on a mechanism by which either microglia could be in an excited state and continue to perform streamlining of the neural structure, or insist that it is possible that microglia were not excited during development, and something else happened to interfere with neuron numbers, and then, subsequently the microglia became chronically activated.

This is unlikely, and unlikelier still when we consider that anyone proposing such a model must do so with enough robustness to overcome a biologically plausible pathway supported by a variety of studies.  And that is only if there was anything underneath the vapor!  Make no mistake, if you ever press someone to actually defend, with literature, the mechanisms by which a state of chronic neuroinflammation might be beneficial in autism, or even the result of something else that also causes autism, no further elucidation of that mechanism is ever forthcoming.  There isn’t anything there.

At some point, it becomes incumbent of people wishing to make an argument that they propose a biologically plausible mechanism if they wish to continue to be taken seriously.  If they cannot, if the literature cannot be probed to make such a case with more empirical support than it might be, the notion so add odds with available evidence should be summarily discarded, unless and until a transcendent set of findings is presented.  There should always be room for more findings in our worldview, but precious limited space for faith in the face of contradictory findings.

–          pD

Hello friends –

Lately I’ve found myself reading papers and knowing and owning several of the references; tragically I can’t tell if I’m reading the right research and am onto something, or I am chasing phantoms and my web of pubmed alerts and reading interests are funneling my reference list into a narrowing echo chamber of sorts.   With that warning in mind, we can proceed to poking around several papers, only some of which mention autism per se.  Along the way, we will see evidence supporting the possibility of a biologically plausible mechanism of developmental programming of the neuroimmune environment, a sequence of events that may lead to impaired synaptic pruning in (some cases of?) autism.

By now, everyone has seen/read/heard about one form or another of the ‘a massive asteroid is going to destroy the world’ story.  One of the common survival strategies from an asteroid strike involves altering the path of the asteroid so that it misses the Earth.  The thoughtful analysis of this problem allows for the physics based reality of the problem, moving an asteroid out of an extinction based trajectory involves just a little work when the asteroid is ten thousand gazillion miles away, but a lot more work when it is only a gazillion miles away.  Upon careful evaluation living organisms display similar behavior, relatively minor disturbances in early life can alter the developmental trajectory, while that same disturbance later in life is unable to materially affect the organism beyond a transient effect.   The accumulated evidence that early life experiences can shape the adult outcome is nearly impossible to dispute with any remaining intellectual honesty, the question is instead, is how large is the effect in autism?

This analogy adequately symbolizes one of the more beautiful and terrifying concepts I’ve come across researching autism, that of ‘developmental programming’, which I blogged some about here, but essentially is the idea that there are critical timeframes during which environmental impacts can have long term persistent effects on a wide range of outcomes.  The most robustly replicated findings involve changes to metabolic profiles in response to abnormal prenatal nutritional environments, but there is also evidence of various other effects, including neurological, and reputable speculation, that autism, may in fact, be in part, a disorder of developmental programming.

Secondarily, there has long been speculation of problems in the removal of ‘excess’ synapses, i.e., ‘synaptic pruning’ in the autism population.   This culling of synapses begins in fetal life continuing throughout adolescence and the repeated observations of increased head circumference during infancy as a risk factor for autism has resulted in the idea that altered synaptic pruning maybe involved in autism.

In the last month or so several rather serendipitously themed papers have been published with tantalizing clues about some of the finer grained mechanisms of synaptic pruning, the possibility of impaired synaptic pruning in the autism population, and a known risk factor for autism that models a developmental programming event sequence that may tie them together.

First off, we have Synaptic pruning by microglia is necessary for normal brain development, (Paolicelli et all) with a very straightforward title, that has this dynamite in the abstract: (snipped for length)

These findings link microglia surveillance to synaptic maturation and suggest that deficits in microglia function may contribute to synaptic abnormalities seen in some neurodevelopmental disorders.

This paper is short, but pretty cool, and very nice from a new territory perspective.  It also speaks directly towards one of the increasingly hilarious obfuscations you will sometimes see raised in online discussions about immunological findings in autism, namely, that we can’t know if the state of chronic inflammation in the CNS observed in autism is harmful or beneficial.   [hint: It might not be causative, but it isn’t beneficial.]

Here’s is a snippet from the Introduction:

Time-lapse imaging has shown that microglia processes are highly motile even in the uninjured brain and that they make frequent, but transient contact with synapses. This and other observations have led to the hypothesis that microglia monitor synaptic function and are involved in synapse maturation or elimination.  Moreover, neurons during this period up-regulate the expression of the chemokine fractalkine, Cx3cl1, whose receptor in the central nervous system is exclusively expressed by microglia and is essential for microglia migration. If, in fact, microglia are involved in scavenging synapses, then this activity is likely to be particularly important during synaptic maturation when synaptic turnover is highest.

Nice.  A time dependent participation by microglia in the critical process of optimization of neuron numbers, a process we are still very much groping our way in the dark towards untangling.  The researchers focused in on a particular molecular target, a chemical messenger of the immune system, fractalkine, and found that without fractalkine, the process of synaptic turnover was impaired.

A couple of tests were performed, first immunohistochemistry (i.e., exceedingly clever manipulation of antibodies to determine the presence or absence of proteins in very specific locations) which demonstrated that microglia were, in fact, ‘engulfing synaptic material’ in animals during periods of synaptic maturation.

Secondly, so called ‘knock out mice’ (i.e., genetically engineered mice constructed without the ability to make a specific protein, in this case, fractalkine) were used evaluate for changes in synaptic form and function based on a lack of fractalkine.  Changes in dendritic spine density were observed in the knock out mice group, with much higher densities in a very specific type of neuron during the second and third postnatal week of life.  The authors indicate this is a key timeframe in synaptic pruning, and state their findings are “suggesting a transient deficient synaptic pruning in Cx3cr1 knockout mice “.  The effect of not having fractalkine on spine density was time dependent as shown below.

Several other measurements were taken, including synaptic firing frequencies, which also implicated an increased surface area for synapses on dendritic spines, consistent with impaired pruning.  Time dependent effects on synaptic efficiency and seizure susceptibility were also found, which the led the authors to conclude that the findings were “consistent with a delay in brain circuit development at the whole animal level.”

For additional evidence of fractalkine participation in synaptic maintenance, we can look to the opposite direction, where researchers evaluating neuron loss in an Alzheimers model reported “Knockout of the microglial chemokine receptor Cx3cr1, which is critical in neuron-microglia communication, prevented neuron loss”.  Taken together, the conclusion that fractalkine processing is involved with neuron maintenance is highly likely, and correspondingly, highly unlikely to be a set of spurious findings.

There’s a couple paragraphs on potential mechanisms by which fractalkine could be interacting with microglia to achieve this effect, with the authors claiming that their data and other data generally supports a model wherein microglia were not effectively recruited to appropriate locations in the brain due to a lack of fractalkine, or, a ‘transient reduction in microglia surveillance.’

The conclusion is a good layman level wrap up that speaks toward the Interconnectedness of the brain and the immune system:

In conclusion, we show that microglia engulf and eliminate synapses during development. In mice lacking Cx3cr1, a chemokine receptor expressed by microglia in the brain, microglia numbers were transiently reduced in the developing brain and synaptic pruning was delayed. Deficient synaptic pruning resulted in an excess of dendritic spines and immature synapses and was associated with a persistence of electrophysiological and pharmacological hallmarks of immature brain circuitry. Genetic variation in Cx3cr1 along with environmental pathogens that impact microglia function may contribute to susceptibility to developmental disorders associated with altered synapse number. Understanding  microglia-mediated synaptic pruning is likely to lead to a better understanding of synaptic homeostasis and an appreciation of interactions between the brain and immune system

That’s all pretty cool, but there was precious little discussion of autism, except in the general sense of a ‘developmental disorder associated with altered synapse number’.   [But the references do speak to autism, the first reference provided, Dendritic Spines in Fragile X Mice displays a significant relationship to autism, and it describes how another flavor of knock out mice, this time designed to mimic Fragile-X, exhibit a ‘developmental delay in the downregulation of spine turnover and in the transition from immature to mature spine subtypes.’  Go figure!]

The other reason Paolicelli is of particular interest to the autism discussion is one of the major players in this study, the microglia (i.e., the resident immune cells of the CNS), have been found to be ‘chronically activated’ in the autism brain by direct  measurement in two studies (here, and here, [and by me, here]), and tons of other studies have shown indirect evidence of an ongoing state of immunological alertness in the autism brain.

Considering this is a brand new paper, I do not believe that there are any studies illuminating the results of a state of chronic activation of microglia on the process of synaptic pruning per se.  I will, however, go on the record that such an effect is very, very likely, and the logical leap is microscopically small that there will be some detrimental impact to such a state.  The inverse argument, a scenario wherein there could be a state of chronic microglial activation that does not interfere with microglia participation in the synaptic pruning requires logical acrobatics worthy of Cirque Du Soleil.  I am open to evidence, however.

So, from Paolicelli, we know that a ‘transient reduction in microglial surveillance’ induced by a reduction in the ability to production fractalkine can result in a condition ‘consistent with a delay in brain circuit development at the whole animal level’.

Next up, we have a paper that was all over the JerkNet in the days and weeks following its release, Neuron number and size in prefrontal cortex of children with autism.  This is a cool study, and likely a very important paper, but I must say that a lot of the online commentary exhibits an irrational exuberance towards one part of the findings.   Here is part of the abstract.

Children with autism had 67% more neurons in the PFC (mean, 1.94 billion; 95% CI, 1.57-2.31) compared with control children (1.16 billion; 95% CI, 0.90-1.42; P = .002), including 79% more in DL-PFC (1.57 billion; 95% CI, 1.20-1.94 in autism cases vs 0.88 billion; 95% CI, 0.66-1.10 in controls; P = .003) and 29% more in M-PFC (0.36 billion; 95% CI, 0.33-0.40 in autism cases vs 0.28 billion; 95% CI, 0.23-0.34 in controls; P = .009). Brain weight in the autistic cases differed from normative mean weight for age by a mean of 17.6% (95% CI, 10.2%-25.0%; P = .001), while brains in controls differed by a mean of 0.2% (95% CI, -8.7% to 9.1%; P = .96). Plots of counts by weight showed autistic children had both greater total prefrontal neuron counts and brain weight for age than control children.  [PFC == prefrontal cortex]

Essentially the authors used a variety of mechanisms to measure neuron number in a specific area of the brain, the prefrontal cortex, and found large variations (increases) in the autism group.   The prefrontal cortex is thought to be involved in ‘planning complex coginitive behaviors’, and ‘moderating correct social behavior’, among others, so this was a smart place to look.

The implicit hype on the internet is that this firmly indicates a ‘prenatal cause’ to autism, but if you read the paper, read what Courchense has said, and read recent literature, you know that the simplicity of this as a singular prenatal cause of autism is long broad strokes, and short on appreciation of the subtlety that textures reality.

A link @ LBRB sent me to the team at The Thinking Person’s Guide To Autism, who had a very nice transcription of a talk given by Courchesne at IMFAR 2011.  Here is a snipet that started my wheels turning.

What we see in autism is either an excess proliferation, producing an overabundance of neuron numbers, or the excess might be due to a reduced ability to undergo naturally occurring cell death. Or it could be both. We don’t know which and our data don’t speak to that, although our data do suggest that it’s probably both.

Finally, our evidence shows that across time, there’s a prolonged period of apoptosis, removal and remodeling of circuits. In order to get back to where neuron numbers are supposed to be, it takes a very long time for the autistic brain. In the normal developing brain, this takes just a few months. In autism, it’s a couple of decades.

[Note how well this fits within the model described by Paolicelli, i.e., “consistent with a delay in brain circuit development at the whole animal level”.  ]

I would highly recommend anyone who has read this far to go read the entire post @ TPGTA sometime.

As far as synaptic pruning goes, here is the associated segment of the paper:

Apoptotic mechanisms during the third trimester and early postnatal life normally remove subplate neurons, which comprise about half the neurons produced in the second trimester. A failure of that key early developmental process could also create a pathological excess of cortical neurons. A failure of subplate apoptosis might additionally indicate abnormal development of the subplate itself. The subplate plays a critical role in the maturation of layer 4 inhibitory functioning as well as in the early stages of thalamocortical and corticocortical connectivity development.inhibitory functioning and defects of functional and structural connectivity are characteristic of autism, but the causes have remained elusive.

Nearly half of the neurons in the area studied are expected to be removed through pruning, a process that extends well after birth.  That is something that you didn’t see referenced in too many places trumpeting this study as ‘proof’ that autism was caused by disturbances in the prenatal environment.  I’m not coming down on the prenatal environment as a critical timeframe for autism pathogensesis, just the difficult to defend underlying notion that this is the only time the environment should be evaluated, or the idea that if something is initiated prenatally other timeframes are therefore, unimportant.

So, I’d read that microglia were actively involved in proper synaptic pruning, contingent on utilization of fractalkine, and then read that impaired synaptic apoptotic mechanisms could be participating in autism, with a consequence of an over abundance of neurons.

Then, I got myself a copy of Microglia and Memory: Modulation by Early-Life Infection, which is another study in a growing body of evidence that immune challenges early in life can have unpredictable physiological consequences.  (This is another very cool paper with Staci Bilbo as an author, whom I think is seriously onto something.)  This study, in particular, focused on interactions microglia and formation of memories.   Here is the abstract:

The proinflammatory cytokine interleukin-1ß (IL-1ß) is critical for normal hippocampus (HP)-dependent cognition, whereas high levels can disrupt memory and are implicated in neurodegeneration. However, the cellular source of IL-1ß during learning has not been shown, and little is known about the risk factors leading to cytokine dysregulation within the HP. We have reported that neonatal bacterial infection in rats leads to marked HP-dependent memory deficits in adulthood. However, deficits are only observed if unmasked by a subsequent immune challenge [lipopolysaccharide (LPS)] around the time of learning. These data implicate a long-term change within the immune system that, upon activation with the “second hit,” LPS, acutely impacts the neural processes underlying memory. Indeed, inhibiting brain IL-1ß before the LPS challenge prevents memory impairment in neonatally infected (NI) rats. We aimed to determine the cellular source of IL-1ß during normal learning and thereby lend insight into the mechanism by which this cytokine is enduringly altered by early-life infection. We show for the first time that CD11b+ enriched cells are the source of IL-1ß during normal HP-dependent learning. CD11b+ cells from NI rats are functionally sensitized within the adult HP and produce exaggerated IL-1ß ex vivo compared with controls. However, an exaggerated IL-1ß response in vivo requires LPS before learning. Moreover, preventing microglial activation during learning prevents memory impairment in NI rats, even following an LPS challenge. Thus, early-life events can significantly modulate normal learning-dependent cytokine activity within the HP, via a specific, enduring impact on brain microglial function.

Briefly, the authors infected rats four days after birth with e-coli, and then challenged them with LPS in adulthood to simulate the immune system to evaluate if memory formation was affected.   As further evidence of an immune mediated effect, prevention of microglial activation in adulthood was sufficient to attenuate the effect.  Clearly the effect on memory formation was based on the immune system.  (Note:  Most of the studies I’ve read would indicate [i.e., educated guess] that a four day old rat is brain developmentally similar to the third trimester of a human fetus.)  While a terrifying and beautiful expression of developmental programming in its own right, there isn’t much to speak towards synaptic pruning in this paper, except maybe, potentially, one part of their findings.

In our study, CX3CL1 did not differ by group, whereas its receptor was decreased basally in NI rats, implicating a change at the level of microglia.

This is where things get either highly coincidental, or connected.  CX3CL1 is another name for fractalkine, i.e., animals that were infected in early life had decreased expression of the receptor for fractalkine compared to placebo animals, i.e., fractalkine is the same chemical messenger found to be integral in the process of synaptic pruning in Synaptic pruning by microglia is necessary for normal brain development!  From a functionality standpoint, having less receptor is very similar to having less fractalkine; as the animals in Microglial Cx3cr1 knockout prevents neuron loss in a mouse model of Alzheimer’s disease tell us.

If, if synaptic apoptotic processes are impaired in autism, perhaps this is one mechanism of action. The timeline would involve a prenatal immune challenge, which causes a persistent decrease fractalkine receptor expression, which in turn, causes a consequent impairment in synaptic pruning through interference in microglial targeting.  There is near universal agreement that immune disturbances in utero are capable of altering developmental trajectory undesirably, and here, in an animal model, we have evidence that infections are capable of reducing availability of receptors of ligands known to play a critical role in synaptic pruning, the absence of which leads to conditions which are “consistent with a delay in brain circuit development at the whole animal level”. 

Only time, and more research, will tell if this is a pattern, a phantom, or a little of both.

–          pD


Hello friends –

A new paper  looking for evidence of an ongoing immune reaction in the brain of people with autism landed the other day, Microglial Activation and Increased Microglial Density Observed in the Dorsolateral Prefrontal Cortex in Autism

BACKGROUND: In the neurodevelopmental disorder autism, several neuroimmune abnormalities have been reported. However, it is unknown whether microglial somal volume or density are altered in the cortex and whether any alteration is associated with age or other potential covariates. METHODS: Microglia in sections from the dorsolateral prefrontal cortex of nonmacrencephalic male cases with autism (n = 13) and control cases (n = 9) were visualized via ionized calcium binding adapter molecule 1 immunohistochemistry. In addition to a neuropathological assessment, microglial cell density was stereologically estimated via optical fractionator and average somal volume was quantified via isotropic nucleator. RESULTS: Microglia appeared markedly activated in 5 of 13 cases with autism, including 2 of 3 under age 6, and marginally activated in an additional 4 of 13 cases. Morphological alterations included somal enlargement, process retraction and thickening, and extension of filopodia from processes. Average microglial somal volume was significantly increased in white matter (p = .013), with a trend in gray matter (p = .098). Microglial cell density was increased in gray matter (p = .002). Seizure history did not influence any activation measure. CONCLUSIONS: The activation profile described represents a neuropathological alteration in a sizeable fraction of cases with autism. Given its early presence, microglial activation may play a central role in the pathogenesis of autism in a substantial proportion of patients. Alternatively, activation may represent a response of the innate neuroimmune system to synaptic, neuronal, or neuronal network disturbances, or reflect genetic and/or environmental abnormalities impacting multiple cellular populations.

This is a neat paper,  to my eye not  as comprehensive as the landmark paper on microglial activation, Neuroglial Activation and Neuroinflammation in the Brain of Patients with Autism Neuroglial Activation andNeuroinflammation in the Brain of Patientswith Autism, but still a very interesting read.  Here are the some areas that caught my eye.   From the introduction:

These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalities in the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening of processes (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators (13–16). These results provide evidence for microglial activation in autism but stop short of demonstrating quantifiable microglial abnormalitiesin the cortex, as well as determining the nature of these abnormalities. Somal volume increases are often observed during microglial activation, reflecting a shift toward an amoeboid morphology that is accompanied by retraction and thickening ofprocesses (13). Microglial density may also increase, reflecting either proliferation of resident microglia or increased trafficking of macrophages across a blood-brain barrier opened in response to signaling by cytokines, chemokines, and other immune mediators(13–16).

Tragically, my ongoing google based degree in neurology has yet to cover the chapters on specific brain geography, so the finer points, such as the difference between the middle frontal gyri and the neocortex are lost on me.  None the less, several things jump out at me from what I have managed to understand so far.  The shift to an ‘ameboid morphology’ is one that I’ve run into previously, notably in Early-life programming of later-life brain and behavior: a critical role for the immune system, which is a paper I really need to dedicate an entire post towards, but as applicable here, the general idea is that the microglia undergo structural and functional changes during times of immune response; the ‘ameboid’ morphology is associated with an active immune response.  Regarding increased trafficking of macrophages across the BBB, Vargas 2005  noted chemokines (MCP-1) increases in the CNS, so we do have reason to believe such signalling molecules are present.

The authors went on to look for structural changes in microglia, differences in concentration of microglia, and evaluated  for markers indicative of an acute inflammatory response.  Measurements such as grey and white matter volumes and relationships to microglia structural differences, and correlations with seizure activity were also performed.   There were three specimens from children under the age of six that were analyzed as a subgroup to determine if immune activation was present at early ages.   From the discussion section:

Moderate to strong alterations in Iba-1 positive microglial morphology indicative of activation (13,29) are present in 5 of 13 postmortem cases with autism, and mild alterations are present in an additional 4 of 13 cases. These alterations are reflected in a significant increase in average microglial somal volume in white matter and microglial density in gray matter, as well as a trend in microglial somal volume in gray matter. These observations appear to reflect a relatively frequent occurrence of cortical microglial activation in autism.

Of particular interest are the alterations present in two thirds of our youngest cases, during a period of early brain overgrowth in the disorder. Indeed, neither microglial somal volume nor density showed significant correlation with age in autism, suggesting long running alteration that is in striking contrast with neuronal features examined in the same cases (Morgan et al., unpublished data, 2009). The early presence of microglial activation indicates it may play a central pathogenic role in some patients with autism.

The authors evaluated for IL-1R1 receptor presence, essentially a marker for an inflammatory response, and found that the values did not differ between the autism population and controls, and that in fact the controls trended towards expressing more IL-1R1 than the autism group.  I think this was the opposite of what the authors expected to find.

While Iba-1 staining intensity increases modestly in activated microglia (30), strong staining and fine detail were apparent in Iba-1 positive resting microglia in our samples. Second, there is no increase in microglial colocalization with a receptor, IL-1R1, typically upregulated in acute inflammatory reactions (28). The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.

I don’t think I’ve seen this type of detail in qualitative measures of the neuroimmune response in autism measured previously, so I definitely appreciate the detail.  Furthermore, from a more speculative standpoint, we may have some thoughts on why we might see this in the autism population specifically that I’ll go into detail below a little bit.

The authors failed to find a relationship between seizure activity and microglial activation, which came as a surprise to me, to tell the truth.  Also discussed was the large degree of heterogeneity in the findings in so far as the type and severity of microglial morphological differences observed.  The potential confounds in the study included an inability to control for medication history, and the cause of death, eight of which were drowning in the autism cases.  There was some discussion of potential causes, including, of course, gene-environment interactions, maternal immune activation, neural antibodies, and the idea that “chronic innate immune system activation might gradually produce autoimmune antibodies via the occasional presentation of brain proteins as antigens”  (!)  There was also this snipet:

Microglial activation might also represent an aberrant event during embryonic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations (17), given the largely or entirely separate developmental lineage of microglia (13). Alternatively, alterations might reflect an innate neuroimmune response to events in the brain such as excessive early neuron generation or aberrant development of neuronal connectivity.

There is a short discussion of the possible effects of an ongoing microglial immune response, including damage to neural cells, reductions in cells such as Purkinjes, and increases in neurotrophic factors such as BDNF.

This is another illustration of an ongoing immune response in the CNS of the autism population, though in this instance, only some of the treatment group appeared to be affected.  It would have been nice to see if there were correlations between behavioral severity and/or specific behavior types, but it would seem that this information is was not available in sufficient quality for this type of analysis, which is likely going to be an ongoing problem with post mortem studies for some time to come.   I believe that an effort to develop an autism tissue bank is underway, perhaps eventually some of these logistical problems will be easier to address.   The fact that some of the samples were from very young children provides evidence that when present, the neuroinflammatory response is chronic, and indeed, likely lifelong.

Stepping away from the paper proper, I had some thoughts about some of these findings that are difficult to defend with more than a skeletal framework, but have been rattling around my head for a little while.  Before we move forward, let’s be clear on a couple of things:

1) The jump from rodent to human is fraught with complications, most of which I doubt we even understand.

2) We can’t be positive that an activated neuroimmune system is the cause of autistic behaviors, as opposed to a result of having autism.  I still think a very strong argument can be made that an ongoing immune response is ultimately detrimental, even if it cannot be proven to be completely responsible for the behavioral manifestation of autism.

3) At the end of the day, I’m just Some Jerk On The Internet.

Those caveats made, Morgan et all spend a little time on the potential cause of a persistent neuroinflammatory state as referenced above.  One of the ideas, “an aberrant event during embyronic monocyte infiltration that may or may not also be reflected in astroglial and neuronal populations  given the largely or entirely separate developmental lineage of microglia”  struck me as particularly salient  when considered alongside the multitude of data we have concerning the difficult to predict findings regarding an immune insult during critical developmental timeframes.

We now have several papers that dig deeper into the mechanism by which immune interaction during development  seem to have physiological effects with some parallels to autism; specifically, Enduring consequences of early-life infection on glial and neural cell genesis within cognitive regions of the brain (Bland et all), and Early-Life Programming of Later-Life Brain and Behavior: A Critical Role for the Immune System (Bilbo et all) ; both of which share Staci Bilbo as an author and I think she is seriously onto something.  Here is the abstract for Bland et all:

Systemic infection with Escherichia coli on postnatal day (P) 4 in rats results in significantly altered brain cytokine responses and behavioral changes in adulthood, but only in response to a subsequent immune challenge with lipopolysaccharide [LPS]. The basis for these changes may be long-term changes in glial cell function. We assessed glial and neural cell genesis in the hippocampus, parietal cortex (PAR), and pre-frontal cortex (PFC), in neonates just after the infection, as well as in adulthood in response to LPS. E. coli increased the number of newborn microglia within the hippocampus and PAR compared to controls. The total number of microglia was also significantly increased in E. coli-treated pups, with a concomitant decrease in total proliferation. On P33, there were large decreases in numbers of cells coexpressing BrdU and NeuN in all brain regions of E. coli rats compared to controls. In adulthood, basal neurogenesis within the dentate gyrus (DG) did not differ between groups; however, in response to LPS, there was a decrease in neurogenesis in early-infected rats, but an increase in controls to the same challenge. There were also significantly more microglia in the adult DG of early-infected rats, although microglial proliferation in response to LPS was increased in controls. Taken together, we have provided evidence that systemic infection with E. coli early in life has significant, enduring consequences for brain development and subsequent adult function. These changes include marked alterations in glia, as well as influences on neurogenesis in brain regions important for cognition.

Bland et all went on to theorize on the mechanism by which an infection in early life can have such long lasting effects.

We have hypothesized that the basis for this vulnerability may be long-term changes in glial cell function. Microglia are the primary cytokine producers within the brain, and are an excellent candidate for long-term changes, because they are long-lived and can become and remain activated chronically (Town et al., 2005). There is increasing support for the concept of ‘‘glial priming”, in which cells can become sensitized by an insult, challenge, or injury,  such that subsequent responses to a challenge are exaggerated (Perry et al., 2003).

The authors infected some rodents with e-coli on postnatal day four, and then evaluated for microglial function in  adulthood.

We have hypothesized that the basis for early-life infection-induced vulnerability to altered cytokine expression and cognitive deficits in adulthood may be due to long-term changes in glial cell function and/or influences on subsequent neural development. E. coli infection on P4 markedly increased microglial proliferation in the CA regions of the hippocampus and PAR of newborn pups, compared to a PBS injection (Figs. 3 and 4). The total number of microglia, and specifically microglia with an ‘‘active” morphology  (amoeboid, with thick processes), were also increased as a consence of infection. There was a concomitant decrease in non microglial newborn cells (BrdU + only) in the early-infected rats, in the same regions.

Check that shit out! Rodents infected with E-coli during the neonatal period had an increased number of active microglia when compared to rodents that got saline as neonates.   Keep in mind that the backbone of these studies, and studies from other groups indicate that this persistence of effects are not specific to an e-coli infection, but rather, can be triggered by any immune response during critical timeframes.  In fact, at least two studies have employed anti-inflammatory agents, and observed an attenuation of effect regarding seizure susceptibility.

A final snipet from Bland et all Discussion section:

Although the mechanisms remain largely unknown, the ‘‘glial cell priming” hypothesis posits that these cells have the capacity to become chronically sensitized by an inflammatory event within the brain (Perry et al., 2003). We assessed whether glial priming may be a likely factor in the current study by measuring the volume of each counted microglial cell within our stereological analysis. The morphology of primed glial cells is similar to that of ‘‘activated” cells (e.g., amoeboid, phagocytic), but primed glial cells do not chronically produce cytokines and other pro-inflammatory mediators typical of cells in an activated state. There was a striking increase in cell volume within the CA1 region of adult rats infected as neonates (Figs. 2 and 8), the same region in which a marked increase in newborn glia was observed at P6. These data are consistent with the hypothesis that an inflammatory environment early in life may prime the surviving cells long-term, such that they over-respond to a second challenge, which we have demonstrated at the mRNA level in previous studies (Bilbo et al., 2005a, 2007; Bilbo and Schwarz, in press).

The concept of glial priming, close friends with the ‘two hit’ hypothesis (or soon to be, the multi-hit hypothesis?),  has some other very neat studies behind it, the coolest ones I’ve found so far are from a group at Northwestern, and include “hits” such as  Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of proinflammatory cytokine upregulationEnhanced microglial activation and proinflammatory cytokine upregulation are linked to increased susceptibility to seizures and neurologic injury in a ‘two-hit’ seizure model and Minozac treatment prevents increased seizure susceptibility in a mouse “two-hit” model of closed skull traumatic brain injury and electroconvulsive shock-induced seizures.   Also the tragically, hilariously titled, Neonatal lipopolysaccharide and adult stress exposure predisposes rats to anxiety-like behaviour and blunted corticosterone responses: implications for the double-hit hypothesis. (!)  These are potentially very inconvenient findings, the details for which I’ll save for another post.

Moving on to Bilbo et all, though a pure review paper than an experiment, it provides additional detailed theories on the mechanisms behind persistent effects of early life immune challenge.  Here’s the abstract:

The immune system is well characterized for its critical role in host defense. Far beyond this limited role however, there is mounting evidence for the vital role the immune system plays within the brain, in both normal, “homeostatic” processes (e.g., sleep, metabolism, memory), as well as in pathology, when the dysregulation of immune molecules may occur. This recognition is especially critical in the area of brain development. Microglia and astrocytes, the primary immunocompetent cells of the CNS, are involved in every major aspect of brain development and function, including synaptogenesis, apoptosis, and angiogenesis. Cytokines such as tumor necrosis factor (TNF)α, interleukin [IL]-1β, and IL-6 are produced by glia within the CNS, and are implicated in synaptic formation and scaling, long-term potentiation, and neurogenesis. Importantly, cytokines are involved in both injury and repair, and the conditions underlying these distinct outcomes are under intense investigation and debate. Evidence from both animal and human studies implicates the immune system in a number of disorders with known or suspected developmental origins, including schizophrenia, anxiety/depression, and cognitive dysfunction. We review the evidence that infection during the perinatal period of life acts as a vulnerability factor for later-life alterations in cytokine production, and marked changes in cognitive and affective behaviors throughout the remainder of the lifespan. We also discuss the hypothesis that long-term changes in brain glial cell function underlie this vulnerability.

Bilbo et all go on to discuss the potential for time sensitive insults that could result in an altered microglial function.  Anyone that has been paying attention should know that the concept of time dependent effects is, to my mind, the biggest blind spot in our existing research concerning autism and everyones favorite environmental agent.

Is there a sensitive period? Does an immune challenge early in life influence brain and behavior in a way that depends on developmental processes? Since 2000 alone, there have been numerous reports in the animal literature of perinatal immune challenges ranging from early gestation to the juvenile period, and their consequences for adult offspring phenotypes (see Table 1). It is clear that the timing of a challenge is likely a critical factor for later outcomes, impacting the distinct developmental time courses of different brain regions and their underlying mechanisms (e.g., neurotransmitter system development, synapse formation, glial and neural cell genesis, etc; Herlenius and Lagercrantz, 2004; Stead et al., 2006). However, the original question of whether these changes depend on development has been surprisingly little addressed. We have demonstrated that infection on P30 does not result in memory impairments later in life (Bilbo et al., 2006), nor does it induce the long-term changes in glial activation and cytokine expression observed with a P4 infection (Bilbo et al., unpublished data). The factors defining this “sensitive period” are undoubtedly many, as suggested above. However, our working hypothesis is that one primary reason the early postnatal period in rats is a sensitive or critical period for later-life vulnerabilities to immune stimuli, is because the glia themselves are functionally different at this time. Several studies have demonstrated that amoeboid, “macrophage-like”, microglia first appear in the rat brain no earlier than E14, and steadily increase in density until about P7. By P15 they have largely transitioned to a ramified, adult morphology. Thus, the peak in density and amoeboid morphology (and function) occurs within the first postnatal week, with slight variability depending on brain region (Giulian et al., 1988; Wu et al., 1992).  [emphasis theirs]

[Note:  The authors go on to state that this time period is likely developmentally equivalent to the late second, to early third trimester of human fetal development.]

We seem to have a growing abundance of evidence that immune stimulation in utero can have neurological impacts on the fetus that include schizophrenia, and autism.   In some instances, we have specific viral triggers; i.e., the flu or rubella, but  I’d further posit that we have increasing reason to believe that any immune response can have a similar effect.  The Patterson studies involving IL-6 in a rodent model of maternal activation seem to make this point with particular grace, as the use of IL-6 knockout mice attenuated the effect, as did IL-6 antibodies; and direct injection of IL-6 in the absence of actual infection produced similar outcomes.  In animal models designed to study a variety of effects, we have a veritable spectrum of studies that tell us that immune insults during critical developmental timeframes can have lifelong effects on neuroimmune activity, HPA-axis reactions, seizure susceptibility, and ultimately, altered behaviors.  I believe that we are rapidly approaching a point where there will be little question as towards if a robust immune response during development can lead to a developmental trajectory that includes autism, and will instead be faced with attempting to detangle the more subtle, and inconvenient, mechanisms of action, temporal windows of vulnerability, and indeed if there are subgroups of individuals that are predisposed to be more likely to suffer from such an insult.

Another thing that struck me about Morgan was the speculation that an increased presence of IL-1R in controls may have been suggestive of an attempt to muzzle the immune response in the case group; repeated from Morgan “The trend toward an increase in colocalization in control cases may also hint at downregulation of inflammatory signal receptors in a chronically activated system.” In other words, for controls it wasn’t a big deal to be expressing IL-1R in a ‘normal’ fashion, because the immune system is in a state of balance.  Another way of looking at our observations would be to ask the question as towards what has caused the normally self regulating immune system to fail to return to a state of homeostasis?   Ramping up an immune response to fight off pathogens and ratcheting back down to avoid unnecessary problems is something most peoples immune systems do with regularity.  Is the immune system in autism trying to shut down unsuccessfully?

There are clues that the homeostatic mechanisms are trying to restore a balanced system.  For example, in Immune transcriptome alterations in the temporal cortex of subjects with autism, researchers reported that the genetic pathway analysis reveals a pattern that could be consistent with “an inability to attenuate a cytokine activation signal.” Another paper that I need to spend some read in full, Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression describes a genetic and expression based study that concludes, in part, that downregulation of PRKCB1 “could represent a compensatory adjustment aimed at limiting an ongoing dysreactive immune process“.

If we look to clinical evidence for a decreased capacity to regulate an immune response, one paper that might help is Decreased transforming growth factor beta1 in autism: a potential link between immune dysregulation and impairment in clinical behavioral outcomes, the authors report an inverse dose relationship between peripheral levels of an important immune  regulator, TGF-Beta1,  and autism severity; i.e., the less TGF-Beta1 in a subject, the worse the autism behaviors [the autism group also, as a whole, had less TGF-Beta1 than the controls].

And then, in between the time that Morgan came out, and I completed this posting, another paper hit my inbox that might provide some clues,a title that is filled to the brim with autism soundbytes, “Effects of mitochondrial dysfunction on the immunological properties of microglia“.  The whole Hannah Poling thing seemed so contrived to me, basically two sets of people trying to argue past each other to reach a predetermined conclusions, and as a result, I’ve largely shied away from digging too deeply into the mitochondrial angle.  This may not be a luxury I have anymore after reading Ferger et all.   For our purposes, lets forget about classically diagnosed and acute mitochonrdrial disease, as Hannah Poling supposedly has, and just acknowledge that we have several studies that show that children with autism seem to have signs of mitochondrial dysfunction, as I understand it, sort of a halfway between normal mitochondrial processing and full blown mitochondrial disorder.  Given that, what does Ferger tell us?  Essentially an in vitro study, the group took microglial cells from mice, exposed some of them to toxins known to interferre with the electron transport chain, and exposed the same cells to either LPS or IL-4 to measure the subsequent immunological response.  What they observed was that the response to LPS was unchanged, but the response to IL-4, was blunted; and pertinently for our case, the IL-4 response is a so called ‘alternative’ immune response, that participates in shutting down the immune response.  From the conclusion of Ferger:

In summary, we have shown that mitochondrial dysfunction in mouse microglial cells inhibit some aspects of alternative activation, whereas classic activation seems to remain unchanged. If, in neurological diseases, microglial cells are also affected by mitochondrial dysfunction, they might not be able to induce a full anti-inflammatory alternative response and thereby exacerbate neuroinflammation. This would be associated with detrimental effects for the CNS since wound healing and attenuation of inflammation would be impaired.

If our model of interest is autism, our findings can begin to fit together with remarkable elegance.  And we haven’t even gone over  our numerous studies that show the flip side of the immunological coin; that children with autism have been shown time and time again to have a tendency towards an exaggerated immune response, and increased baseline pro-inflammatory cytokines when compared with their non diagnosed peers!

Anyways, those are my bonus theoretical pontifications regarding Morgan.

– pD


Enter your email address to subscribe to this blog and receive notifications of new posts by email.

Join 34 other followers


    • TomorrowWorld 2017 tickets: We come from all components of the nation and meet up in Atlanta for a weekend of music and memories.
    • bizi sikin pornosu izle: What's up to every body, it's my first pay a visit of this web site; this weblog consists of remarkable and actually good data in favor of visitors.
    • Emlak: Nice weblog here! Additionally yolur web site quite a bit up fast! What host aree you using? Can I gget your associate hyperlink for your host? I w