passionless Droning about autism

Archive for the ‘Autism’ Category

Hello friends –

There used to be a poker room about twenty miles from my home; it sat above a run down greyhound racing track and smelled like an old shoe on the best day.  But they had poker.   They hosted an accumulating jackpot hand, usually worth a couple of thousand dollars, sometimes quite a lot more, which you could win if you got a royal flush in the current suit; i.e., if the suit was hearts, and you wound up with 10-J-Q-K-A hearts, you’d win the Jackpot.  This could lead to some unusual cost/reward analysis scenarios.

Let’s say you sit down to play and buy in for a hundred dollars.  Then, three hands later, you look at your two hole cards and you have 10-J hearts.  Not really a great hand, but if the board winds up showing Q-K-A hearts somewhere in the next five cards, you win fifteen thousand dollars (or whatever the Jackpot had accumulated to).   Almost everyone folds, but before you get a chance to see the next three cards for the two measly dollars you put up as a blind, an aggressive, serial over-better to your right raises to fifteen dollars.  You are in a tough spot, you know the guy bets like crazy anytime he thinks he can steal a pot, but you still are losing to anyone with a queen.   If you had 10-J spades, or clubs, or mixed, or (nearly) whatever else, this is easy; you dump your shitty cards.  But with your two royal heart cards, you *could* win the jackpot; your odds still totally suck, even if you were getting paid off a thousand to one you still didn’t have the ‘right’ odds to make the call, but if you inhabit a place where losing fifteen dollars won’t kill you, but winning fifteen thousand would definitely be a game changer, the magnitude of the potential winnings must be part of your decision making process.

I called the raise a few times, but never hit the jackpot.  Or even came close.

I keep coming back to the idea of incorporating the scale of potential outcomes when I think about the non event of the hilarious prevalence numbers that came out a while, one in fifty with ‘autism’.  Nobody outside of Journey Autism fucking cared and the responses were depressingly predictable; the media and the Internet skeptics went ‘full awareness’, and found nothing of any alarm in these numbers, the Internet vaccine crazies went ‘full autism’, and assumed the numbers were solely comprised of individuals who would need 24×7 assistance for forever.  It was all a big joke.  Haha.

I don’t know how large the real increase in autism is (the older parental age data tells us unambiguously that some of the increase is non-imaginary), but I do know that as our best efforts at figuring this thing out has left us skipping from one in two hundred and fifty, to one in a fifty in eight short years.  To my eye, this means a real increase of fifty percent (or more!) could easily be hiding in the static and we’d never know.  Most everyone doesn’t seem to care, that is the way of the Prevalence Hookup, quickly embracing whatever prevalence numbers come out, coupling until a set of newer, bigger, even more ‘greater awareness’ numbers come along.

But my thoughts continue to be formed by concept of a sort of missed jackpot opportunity when I see a sense of complacency about our ever growing autism population; it isn’t that I don’t believe that diagnostic changes and the watering down of what a diagnosis means in terms of life skills aren’t affecting rates, those factors are clearly at play, but the ramifications of just “some” of the increase being real seems like a big, big, big deal to me.  When your population of interest is every child, a small real increase means a lot of individual children are affected.  Sure, it is, possible that older parental age is the only recent development that is affecting rates upward, with all of the rest being diagnostics, but I find little comfort in this notion.  If the soft social scientists are wrong, even a little, and there is a true increase in incidence, we may come to regret the solace provided by our collective bobbleheading at the mantra of ‘greater awareness’, for it enabled us to waste a great amount of precious time.

The thing is, it doesn’t really cost us that fucking much to apply more resources to the unimportant, nagging question on the neurodevelopment of a generation of infants.  In 2006, Bush signed the ‘Combating Autism Act’, a bill included a billion of dollars for ‘research, surveillance, and treatment’.  That’s two hundred million a year.  Last year, The Avengers, a stupid and shitty movie, made over a billion dollars.  Now, I know there are other funding sources for research, surveillance, and treatment, but there were also a lot of other stupid movies.

I believe that this prioritization is the equivalent of folding 10-J hearts to a dinky four dollar raise; the knowledge we could gain from a relatively small outlay is worth a lot.  We shouldn’t be worrying about the cost, we should be considering the payoff; the question we are trying to understand, “are today’s infants neurobiologically different than infants of the last generation?” has a difficult to understate payoff. We shouldn’t be embracing reasons to stop playing, we should chomping at the bit to see the next three cards.  This is an easy call.

And yet, there was a collective yawn when the CDC announced 2%.

Funny enough, it was just a few years ago that the UK NHS study of adults found a prevalence of 1%, a finding which was heralded as remarkably strong evidence that autism rates are stable (at the time, 1% was the general value for US children.  Oh well.).  For some reason, the robustness of the NHS adult findings didn’t cause anyone to exclaim that there is a sort of epidemic-lite, what with US kids having autism as twice the rate as NHS adults.  It was a classic case of doublethink; US kids have autism at 2%, England adults have autism at 1%, and autism rates are stable.  (Believing that any of the numbers have validity might be closer to triplethink!)

A while ago I saw an interview with Fombonne on the SFARI site that contained the unsurprising byline: ‘Eric Fombonne says that the new CDC report does not necessarily mean that prevalence is increasing’.   [Note: This was BEFORE the 2% numbers were reported!]  Anyway, he made some interesting points about the messiness of the autism data showing how silly the state by state numbers are; Utah has four times the cases that Alabama does, and utilized different diagnostic methods.  In the text of the interview, he reveals Utah also had very low levels of MR (~ 13% instead of ~ 28%), AND had a creepy low male to female ratio.  Either there is something really weird going on in Utah, or the ‘numbers’ from Utah and Alabama are not measuring the same thing.  It could also be that the numbers are measuring some of the same thing, and there are a couple of weird things going on in Utah (heh).  But the bigger point should be that we shouldn’t expect to get a decent understanding of autism rates at a national level by clumping together Alabama numbers, Utah numbers, and whatever other numbers, shaking up them up, and averaging them out.  Maybe the headline ought to read, ‘Pretty much somewhere between half a percent, and two percent of children might have something a psychologist, or a doctor, or both, have something called autism, the manifestations and lifelong impact of which vary considerably individually and regionally’, or maybe ‘Autism Rates: Your guess is as good as ours!’.

I don’t trust any set of numbers more than an educated stab in the dark.

[Note: for a slightly different take on ADDM numbers, you can see this interview on SFARI, where Walter Zahorodny reports that detailed analysis of NJ data indicates a likely real increase in rates.  Doh!]

I began to wonder; if almost nobody really seems worried about an ‘epidemic lite’, if no almost no one is alarmed that the confidence intervals in our data could incorporate huge numbers of actual people, why am I so concerned?  Is my version of the precautionary principle overly cautious?  I don’t know the answer to these questions, but I think that part of the answer lies within my journey autism, watching my son’s challenges (and triumphs) unfold, and the knowledge that whatever we find about autism incidence, he will be reliant on other people for his survival for his entire life.  That is the gift autism has given him; it doesn’t mean he can’t be happy, it doesn’t mean he can’t experience love, but so far, we cannot detect that autism has provided him anything other than near debilitating OCD, an imperfect sense of dangerous situations, and a lifelong requirement of the kindness and capabilities of others.

I am filled with a pervasive and soul crushing sadness at the possibility of one ‘extra’ child having the same challenges because of changes we have collectively made to the environment, and that is the heart of the semantic dance over how much of the increase is real.  That is the Jackpot.

But, your mileage may vary.  I know that there are some parents and people out there who have challenges as heavy as my son’s, and they don’t share my sense of panic over the issue.  A lot of people credit their autism with benefits.  I won’t discount their experiences.  Part of the reason we don’t see eye to eye may be that we look at the same question, but see different risks, and different payoffs.

– pD

Hello Friends – There are (at least) two big
classifications of microglia findings in autism, an altered
morphology (i.e., shape and function, or ‘activated’ versus
‘quiescent’), and an increased number (i.e., more), with both
parameters varying with each other and spatially.  In other
words, disparate parts of the brain have different numbers of
microglia in them, and the functional profile of those microglia
also varies from one area to another. 
[Note: There is ongoing
discussion
regarding the appropriate definition of
‘activation’ of microglia, with evidence of (at least) four states
of microglial morphology.] Recently I saw a discussion on the SFARI
site about the fancy in
vivo study of microglia numbers in high functioning males with
autism
.  (I believe I am growing
increasingly jaded, as it occurs to me that radio tracing against
[11C](R)-PK11195) to
show microglial activation is a fancy trick, but one leaving us
open to detecting other stuff too.)  In any case, the findings
are not especially unexpected by now, well not to me anyways, but a
comment at the SFARI site really got me thinking about the chain of
events that could lead to different spatial and
morphological characteristics of microglia.  Perhaps we could
gain insight into the question of what the microglia are doing by
trying to understanding how they got there. Do we have any
biologically plausible models that might educate us on how a
different morphology and distribution of microglia could be
achieved? 
A while ago I got a copy of a few
articles that don’t have autism in them per se, but they kept
coming to the forefront of my mind when I thought about that
question.  The first is Distribution
of microglia in the postnatal murine nigrostriatal
system
,
which had a disease focus on
Parkinson’s, but what really grabbed my attention is what they
learned about the developmental pathway microglia took to populate,
and then depopulate the substantia nigra (SN), a little wedge of
brain involved with motor skills, reward seeking, and addiction.

Interestingly, the SN
has been shown to contain more microglia than
adjacent 
structures. We have analysed
changes in microglia numbers and in microglial morphology in the
postnatal murine nigrostriatal system at various stages ranging
from postnatal day 0 (P0) up to 24 months of age. We
clearly show that the microglia numbers in the SN and in the
striatum dramatically increase from P0 to P15
and
significantly decrease in both areas in 18-month-old and
24-month-old animals.

[Note: There seems to be
some variance in the appropriate ‘rat-to-human-age’ approximations;
especially when trying to do something as
expeditionary as comparing brain development.  We should
extrapolate only with caution.] The part that makes me grin is that
it illustrates our nascent understanding of the process of
microglial colonization into the CNS, the hows,
whens
, wheres, and whys are still
shrouded in mystery. The broadest outlines tell us that microglial
penetration into the brain is a long running, dynamic process; the
microglia are slow infiltrators, gaining access into parts of the
brain in concert with a swath of proliferating and inhibitory
factors, all at a time of once in a lifetime neurodevelopmental
modifications. Regulation
of postnatal forebrain amoeboid microglial cell proliferation and
development by the transcription factor Runx1
paints a
beautiful portrait of functionality.  Runx1 is a chemical
messenger that participates in phenotyopic determination of blood
cell progenitors into mature cells.  The researchers observed
spatial, time dependent expression of Runx1 in the developing
forebrain, and differential levels following injury.

Here we show that the mouse
transcription factor Runx1, a key regulator of myeloid
cell proliferation and differentiation, is expressed in forebrain
amoeboid microglia during the first two postnatal weeks
.
Runx1 expression is then downregulated in ramified microglia. Runx1
inhibits mouse amoeboid microglia proliferation and promotes
progression to the ramified state. We show further that
Runx1 expression is upregulated in microglia following nerve injury
in the adult mouse nervous system.
These findings provide
insight into the regulation of postnatal microglia activation and
maturation to the ramified state and have implications for
microglia biology in the developing and injured brain.

It doesn’t really tell us much about a
persistent change in microglia per se, but it does render a picture
of proliferation and differentiation as an easily
disrupted
symphony.  When we think about the
developing brain, I won’t pretend to have more than a lightyear
close guess at what microglia might be doing
differently between amoeboid and ramified
morphologies in this locale, at this time, but I
very highly doubt there isn’t
a functional impact on microenvironment neurodevelopment; our
developing brains are using opportunities like the Indians used the
buffalo, no waste, no excess, and because balance is important,
everything is important. Moving back to the
question of the plausibility of a pathway to the autism state,
luckily (or unluckily?) the literature is veritably littered with
insults that perturb microglial development, leading to
 persistent changes to microglial morphology, ultimately
percolating up to behavioral changes. Prenatal stress is a bad, bad
thing, and here is a study that finds that extreme mice stress can
persistently alter the mice activation profile of mice microglia.
Prenatal
stress increases the expression of proinflammatory cytokines and
exacerbates the inflammatory response to LPS in the hippocampal
formation of adult male mice
, was just published, and
comes wrapped up with a double hit, and
different resting and stimulated neuroimmune environments.

Under basal conditions,
prenatally stressed animals showed increased expression of
interleukin 1ß and tumor necrosis factor-a (TNF-a) in the
hippocampus and an increased percentage of microglia cells with
reactive morphology in CA1 compared to non-stressed males.
Furthermore, prenatally stressed mice showed increased TNF-a
immunoreactivity in CA1 and increased number of Iba-1
immunoreactive microglia and GFAP-immunoreactive astrocytes in the
dentate gyrus after LPS administration. In contrast, LPS did not
induce such changes in non-stressed animals. These
findings indicate that prenatal stress induces a basal
proinflammatory status in the hippocampal formation during
adulthood that results in an enhanced activation of microglia and
astrocytes in response to a proinflammatory
insult.

Note: I have not read this
paper so I do not know if a qualitative number of microglia, or
just more immune-targeted microglia were found, but likely the
latter. A similar, full free paper, Prenatal
stress causes alterations in the morphology of microglia and the
inflammatory response of the hippocampus of adult female
mice
, found broadly similar results; perturbed resting
and stimulated states in the treatment group.

Prenatal stress, per se,
increased IL1ß mRNA levels in the hippocampus, increased the total
number of Iba1-immunoreactive microglial cells and increased the
proportion of microglial cells with large somas and retracted
cellular processes. In addition, prenatally stressed and
non-stressed animals showed different responses to peripheral
inflammation induced by systemic administration of LPS.
LPS induced a significant increase in mRNA levels of IL-6,
TNF-a and IP10 in the hippocampus of prenatally stressed mice but
not of non-stressed animals.
 

Going back to my
ramblings on glial priming
, it seems that here we have an
example of a type of cross system priming (sweet!), where
disturbing the stress response system changed the immune system;
such is the way of the polyamorous chemical families interacting in
our brain.  It also occurs to me that given the
delicate nature of the developing brain, and the
crazy important
tasks going on in there, we might want to think very
carefully before we ‘induced a significant increase in
mRNA levels of IL-6, TNF-a and IP10 in the hippocampus‘

on subgroups who might be environmentally predisposed to react with
exaggerated vigor.  But what do I know? Of course, the
prenatal immune challenge arena holds a ton of studies on
persistent microglial function, and ‘consequences’.  There are
way too many to list, but a quick overview of some very recent ones
would include: Enduring
consequences of early-life infection on glial and neural cell
genesis within cognitive regions of the brain
, an early
life real infection model with e coli that
concludes, “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
.”  (Staci Bilbo!)  This paper was sort
of a quinella, as it showed both changes in immune responsiveness
into adulthood; it also demonstrated the ability
of an immune insult to alter
the developmental trajectory of the
microglia, i.e., 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.
Neonatal
lipopolysaccharide exposure induces long-lasting learning
impairment, less anxiety-like response and hippocampal injury in
adult rats
very directly blasted rats with some LPS
immune activation action, and includes, ”Neonatal LPS
exposure also resulted in sustained inflammatory responses in the
P71 rat hippocampus, as indicated by an increased number of
activated microglia and elevation of interleukin-1ß content in the
rat hippocampus.”  
(Sound familiar?) Interleukin-1
receptor antagonist ameliorates neonatal lipopolysaccharide-induced
long-lasting hyperalgesia in the adult rats
took the
extra step of adding a set of animals that got inhibited
inflammatory responses.  Results are increasingly
unsurprising.

Neonatal
administration of an IL-1 receptor antagonist (0.1mg/kg)
significantly attenuated long-lasting hyperalgesia induced by LPS
and reduced the number of activated microglia in the adult rat
brain. These data reveal that neonatal intracerebral LPS
exposure results in long-lasting hyperalgesia and an elevated
number of activated microglia in later life. This effect is similar
to that induced by IL-1ß and can be prevented by an IL-1 receptor
antagonist

I love how (once again) we
can see how interrupting the immune response can have an effect.
Environmental impacts outside of the immune
activation realm may also find a place within the ‘big tent’ of
microglial agitation with consequent developmental impacts. 
The people who made the first big neuroimmune / autism splash at
Johns Hopkins later came out with Neuroinflammation
and behavioral abnormalities after neonatal terbutaline treatment
in rats: implications for autism
, which found that an
agent used to prevent labor in some situations could
produced a robust increase in microglial activation on PN
30 in the cerebral cortex”
in treatment animals. 
The drug in question, terbutaline, has been weakly associated with
increased incidence of autism, i.e., Prenatal
exposure to ß2-adrenergic receptor agonists and risk of autism
spectrum disorders
, and beta2-adrenergic
receptor activation and genetic polymorphisms in autism: data from
dizygotic twins
. And now, in 2013, Beta-adrenergic
receptor activation primes microglia cytokine production
,
displays another example of cross system
priming.

To determine
if ß-AR stimulation is sufficient to prime microglia, rats were
intra-cerebroventricularly administered isoproterenol (ß-AR
agonist) or vehicle and 24h later hippocampal microglia were placed
in culture with media or LPS. Prior isoproterenol treatment
significantly enhanced IL-1ß and IL-6, but not TNF-a production
following LPS stimulation. These data suggest that central
ß-AR stimulation is sufficient to prime microglia cytokine
responses.

In other words, they gave
the rats a drug in the class of terbutline, and subsequently
observed an increased microglia responsiveness in cultured
cells.  What a crazy coincidence. Detecting total
populations
of microglia in adulthood, either regionally
or in the brain as a whole is a little more difficult, the little
buggers are a lot easier to detect when we light them up with neon
green tracers that stick to proteins expressed at ‘activation’
time, and it just doesn’t look like the question has been asked too
many times.  I did, however, find something that has a sort of
chip shot on this analysis, Prenatal
stress alters microglial development and distribution in postnatal
rat brain
, which looked at regional microglia populations
and phenotypes at two time periods following prenatal stress
events.

Prenatal
stress consisting of 20 min of forced swimming occurred on
embryonic days 10–20. On postnatal days 1 and 10, stressed and
control pups were killed. Microglia were identified using Griffonia
 simplicifolia lectin and quantified in the whole encephalon.
In addition, plasma corticosterone was measured in dams at
embryonic day 20, and in pups on postnatal days 1 and 10.
At postnatal day 1, there was an increase in number of
ramified microglia in the parietal, entorhinal and frontal
cortices, septum, basal ganglia, thalamus, medulla oblongata and
internal capsule in the stressed pups as compared to controls, but
also there was a reduction of amoeboid microglia and the total
number of microglia in the corpus callosum.
By postnatal
day 10, there were no differences in the morphologic type or the
distribution of microglia between the prenatal stress and control
groups, except in the corpus callosum; where prenatal stress
decreased the number of ramified microglia. The stress procedure
was effective in producing plasma rise in corticosterone levels of
pregnant rats at embryonic day 20 when compared to same age
controls. Prenatal stress reduced the number of immature
microglia and promoted an accelerated microglial differentiation
into a ramified form.

They did a lot
of clever stuff at analysis time, taking samples from several
locations after birth and ten days later, and
also did some fine grained classification of the
shape of the microglia.  They include spatial and temporal
mappings of four microglial developmental profiles.  It looks
as if prental stress was able to alter the developmental speed of
microglia from one morphology to another in different parts of the
brain.  There was as small section in the discussion that
speculated on what such changes might mean for neurodevelopment.

Given that during the
early postnatal period occur numerous brain developmental processes
(e.g. neurogenesis, myelination, synaptogenesis, astrogliogenesis,
neuronal cell death and blood–brain barrier maturation) [6, 19, 22,
25, 36, 52] it is possible that altered microglial
development induced by in utero stress may affect other
developmental processes either changing microenvironment molecular
constitution or triggering earlier inflammatory changes secondary
to the blood–brain barrier opening induced by prenatal
stress
.  Although punctual, the altered microglial
development might alter extensively the other
neurodevelopmental processes
ensuing perdurable
structural changes
; for example it is possible that the
change in the distribution pattern of microglia in the prenatal
stress group may render vulnerable some neuroanatomic
regions due to the reduction of neurotrophic factors
,
such as the corpus callosum where there is a continuous axonal
growth

No kidding! [There is also some very
interesting notes regarding microglial participation in purkinje
cell death that deserves and entire post. . .] This should be the
point that any rational observer must accept that we several lines
of evidence that early life experiences can persistently alter
microglial function with plausible mechanisms that could affect
neurodevelopment.  Our data concerning total population
numbers in adulthood is a lot more difficult to come by, but I
think this will probably be getting looked at soon enough. Of
course, in any particular individual it is difficult (or
impossible?) to know how they may have arrived at a state of
increased microglial activation, but at the same time, it is not as
if we have no clue on possible pathways to this destination; our
short list of environmental factors includes immune insult, stress,
and chemical agents. If the question is, ‘what are the microglia
doing in the autism population?’, one plausible answer is ‘their
phenotype was persistently altered by an early life event through a
developmental programming model’. As I was mulling all of this
over, two things happened.  First, a maternal CRP
study
came out, and found a pretty strong relationship
between direct measurements of mommy inflammation with increased
risk of baby autism.  The nice part is that they had a
gigantic data set (1.2M births!) to work with thanks to a few
decades of single payer medicine.  (Very
nice!
)

For maternal CRP
levels in the highest quintile, compared with the lowest quintile,
there was a significant, 43% elevated
risk.
This finding suggests that
maternal inflammation may have a significant role in autism, with
possible implications for identifying preventive strategies and
pathogenic mechanisms in autism and other neurodevelopmental
disorders.

Just after that paper came out, I
made some Fred Flintstone style beef ribs.  I ‘primed’ the
meat with a Moroccan inspired spice rub overnight, then
slow, slow, slow cooked them with a
low, low, low heat all day
long
, and blasted away with a date glaze under the
broiler just before go time and they were caveman style
primal fucking awesome
.  The key to arriving there
was the slow cooking. The rib preparation
process got me thinking about our population wide experiment
in replacing infection with inflammation
where we have
traded in death by pathogens or other once fatal ailments in
exchange for a longer life frequently plagued by conditions
associated with higher inflammation.  Our analysis on long
term alterations to microglial proliferation and morphology is
largely comprised of studying acute insults
(sound familiar?), i.e., injection of purified bacterial cell
components known to trigger a robust immune response, ten sessions
of mouse based pregnant forced swimming, or exposure to chemicals
with rare and particular exposure routes in humans.  Mostly I
think this is due to the black swan nature of the developmental programming
model
alongside the very new idea that microglia are
doing jobs other than responding to infections; our models are
crude because of our relative ignorance.  What will we find
when our filters are appropriately powered to detect for chronic,
but subtle insults? It occurs to me that there may be a ribs model
of altered microglial colonization of the fetal brain; it seems
clear that proliferation and differentiation of microglia can
clearly be changed by powerful inputs, but the
chemical messengers that impact that change are closely related (or
the same) as the measurement points in the maternal CRP study.
Could a slow cooking of slightly higher but not acutely
increased
maternal inflammation be participating in the
genesis of autism (in some children) through altering the migration
and proliferation of microglia into the neonatal brain?  Could
the same chemical messengers of inflammation be subtly
priming
the microglia to respond with increased vigor to
insults later in life?  Has our replacement of infection with
inflammation included an unanticipated effect that alters the
developmental pathway of the very cells that help shape our
children’s brains? I don’t think we are (quite) clever enough to
answer these types of questions yet, but we are at least starting
to generate the right kind of data to inform us on how to get
started.  I don’t know what we will find, but the initial data
doesn’t look very good.  In the meantime, I am recommending
you go get some ribs and let them cook all day long.      

pD

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 –

We keep on finding things that seem to very gently alter developmental trajectory towards (or away from) an eventual diagnosis of autism; a genetic variant here or there, an environmental exposure, or one of our very many experiments in cultural engineering.  When these nudges are founded on genetic variances, they are often referred to as “low penetrance” risk factors; here is a snipet from the wiki definition for “Penetrance

An allele with low penetrance will only sometimes produce the symptom or trait with which it has been associated at a detectable level.

I would argue, and have previously on this blog, that there isn’t a good reason that the descriptive of low penetrance should be relegated solely to genetic inputs.  The ‘non-genetic’ factors we seem to have associated with autism risk, or protection, seem to inherit the same quality of a low grade impact; the risk of an autism diagnosis isn’t altered by too much, but instead, just a little. 

There are a great number of examples of environmental impacts that seem to follow a low penetrance model of effect; maternal obesity, paternal age, cesarean section, maternal asthma, maternal folate ingestion [protective!], maternal use of anti-depressants (or being depressed?), low birth weight, and some perhaps some drugs given during pregnancy.

[Please, please note:  I’m not “blaming the mother” here, but we do not have the luxury of invoking Bettleheim as a mechanism for avoiding evident truths.   A dispassionate analysis of the data mandates we accept that the prenatal environment is critical.

If you think that some percentage of the autism ‘epidemic’ is real, you should realize that this issue is too important to be scuttled by emotional hotspots.  You cannot blame yourself for things that were unknown to you during your pregnancy.  If, instead, you don’t think autism rates have changed, none of the above impacts can be meaningful.  Finally, if you believe that autism is more gift than disorder, then you aren’t getting blamed for anything anyways.]

Unfortunately, a mixture of subtle changes makes for a messy situation for our researchers for a few reasons; environmental studies contain a difficult to contend with set of confounders; knowing what to measure, when to measure it, and the often times necessary evil of usage of self reporting, computer models, or other proxies for exposure measurements.  Making things even worse, it is biologically plausible, indeed, mandatory, that low penetrant effects operate with each other.  What we will eventually need to be working on, for example, is determining the specific genetic dispositions that act in concert with a low birth weight and with gestational anti-depressant exposure to perturb neurodevelopment toward autism.  That’s a tough thing to do.

Throwing this kind of disparate data into a blender at study time looks to be largely beyond our current capacities; researchers are struggling to identify single gene-environment interactions, for example, MET-C/pollutants, or the terrifying notion of RORA demythlation/endocrine disruptors interacting together.  Looking at more, or a handful, as is likely necessary, is a long ways off.

I’ve been thinking about the intersection of these two things lately; our relative inability to evaluate for several, subtle, interacting forces, with the growing evidence that a great many mysterious conditions, including autism, seem to be governed by lots of small things occurring differently.   I am left with the idea that are woefully unready to understand the participating factors in any particular case of autism, with similar reservations regarding our ability to know how much, if any, of the autism ‘epidemic’ is real.

A few weeks ago, there was an Op-Ed in the New York Times that speculated on the link between an in-utero environment characterized by increased inflammation and an eventual diagnosis of autism.  I was largely in agreement with Moises Velasquez-Manoff on a the basic premise of his argument; especially regarding the state of the science on the immune findings in the autism realm, the use of helminths, not so much. A very widely read response by Emily Willingham accused the author of the piece of invoking a naturalist theory of the past:

Whether he means to or not, Velasquez-Manoff then echoes one of the favorite refrains of the anti-vaccine movement, that back when the world was a beautiful place of dirty, worm-infested children, clean water, 100% breastfeeding, and no television, it was a place where the immune system could do its work peacefully and with presumably Zen-like calm, weeding out the weak among us and leaving behind the strong.

I don’t think that the NYT article did anything of the sort, the author merely stated that there seem to be fewer signs of immune dysregulation and autoimmune conditions in some types of living conditions.

Then, a few weeks later, a widely publicized metadata study on organic eating came out.  Again, the skeptics were ready to pummel the bruised body of the naturalistic fallacy, in this case, Stephen Novella at SBM:

Environmental claims for organic farming are complex and controversial – I will just say that such claims largely fall prey to the naturalistic and false dichotomy fallacies.

Stephen Novella’s version here is terse, but I think it is fair to say that in this context, the idea is that that if something is labeled as ‘natural’, that it then must be somehow superior to a ‘non-natural’ alternative, is a fair characterization of a naturalistic fallacy.

[The masochist could read through a few comments on that thread to see my take on the organic/non organic study; but the TL;DR version is, the study could have just as easily been titled, “Evaluations of Organic Eating Insufficiently Powered Or Designed To Know More Than The Most Primitive Endpoints, At Best”.  Here is an NPR transcript where the presenter is a little more up front in that the state of the science is that health benefits have not been evaluated for.

But what I should point out here is that the studies of people were very limited. They were short-term and, like, narrowly focused. So they would look at pregnant women, for instance, and say, are pregnant women eating organic, are their children – did their children have left eczema or allergic conditions? So these are sort of narrowly focused studies. They were short-term, and there weren’t very many of them.

One of the few human studies in this metadata analysis involved a dietary intervention of one apple.  What we have is a lack of evaluation, as opposed to a lack of findings, a familiar situation.]

Even so, it must be stated: The naturalistic fallacy(ies), as presented by the skeptics, and as believed by some fraction of grape-nut-eating-tarot-card-flipping people out there, is bogus.  Things weren’t better way back then.  Just because something is ‘natural’ doesn’t mean it is better, or without unknown consequences.  Washing your hands is good, but antibiotics are also good, and work better when necessary.  Breastfeeding is good, but it doesn’t keep your infant from getting cholera.  Vaccines work.  Modern agriculture is feeding a lot more of us than we used to be able to feed, and the hard truth be told, it is policies and habits that are leaving lots of people hungry.  I don’t know if eating a organic diet is better for you or not, but I do know that I do like supermarkets.

But.

Our history is littered with the discarded arguments of people just as smart as us using rudimentary tools to understand complicated systems, declaring a lack of effect and throwing a contemptuous look over their shoulder at the rubes who long for the hilariously outdated solutions of yesteryear.  We shouldn’t be concerned with the fact that the naturalistic fallacy is intellectually bankrupt; we should be concerned with the fact that our incredibly stupid species is changing our environment with reckless abandon on the assumption that we are smart enough to understand what we are doing.  If the naturalistic fallacy is bad, the perfection-of-progess fallacy is almost as bad, with bonus negative points of being invoked by people who should know better.

How many examples do we need of our previous hubris until we realize that we are just barely less dumb now than we were then? 

First we thought lead was safe as a pesticide, in paint, and as a gasoline additive.  Then, we figured out it was only safe for paint and gasoline; then just in gasoline.  Now, we know that any amount of measurable levels of lead are associated with cognitive effects.  Any individual reader of this column was very likely an adult in 2002, and at that time, the state of our knowledge didn’t tell us that any amount of lead was less safe than no amount of lead.  Ten goddamn years ago, the FDA thought there was a level of lead that in the bloodstream that did not affect cognitive function in children.

We have been performing increasingly optional cesarean sections for decades before starting to figure out that they are associated with adverse health effects for the lifespan.  Only within the past few years have we discovered that this procedure is associated with altered microbiomes,  obesity, and asthma.

We have been so successful at distributing products with based on plastic  that over 90% of every human on the planet has detectable levels of component chemicals in their bloodstream.  Only now that we have insured that nearly every human has been touched, we consistently find associations with metabolic and reproductive changes.

After near thirty years, the recommendations over administering Tylenol to infants was changed.  In the 1980s we saw Reyes syndrome, made the association with aspirin, failed to observe any acute differences in infants given Tylenol, and pulled the trigger on global recommendation to replace aspirin with acetaminophen.  It took decades before we were clever enough realize that eliminating Reyes might not have been the only thing we did, because we were too stupid to realize that effects do not have to be immediately obvious in order to have profound outcomes.

Human bodies were forged through the crucible of evolution, thousands of generations of adaptation, to be ready to start reproducing by the teens, and we have decided to start putting that process of for a decade, or two.

All of these examples are founded of the specificity of our analytical abilities, or rather a relative lack of specificity.  We weren’t clever enough to understand to look for associations, so they remained invisible to us.  A question never asked is never answered.  Even worse, some of these are discrete events, disturbances orders of magnitude more simplistic to analyze compared to ‘eating organic’.

A lot of the skeptical sites will utilize the idea that humans are ‘pattern seekers’, especially when it comes to people reporting temporal associations with development of autistic behaviors and vaccination.  I kind of like the idea of the pattern seeking human in general; the biggest pattern we seem to be seeing is the one that tells us that our current state of knowledge gives us enough information to understand what we are doing, a type of uber-pattern.

The idea that we have a decent understanding the effect of ingesting increased pesticide residue, a finding included in the organic metadata study, is a joke.  The idea that we have the faintest clue of the outcomes of replacing infection with inflammation, a practice we have embraced with great enthusiasm, is a total fucking joke.  We have barely bothered to look.  Do not believe anyone who tells you otherwise.

This is what bothers me so much about a casual wielding of the naturalistic fallacy; it is so frequently a feint from critical questions.  The discordance with reality of the naturalist fallacy has been established.  It is great how much less suffering there is now, compared to then, but let’s not rest on our laurels.  Am I the only one worried about how wrong we are here, now? 

I don’t know if eating less pesticide is better than eating more pesticide, and I also can’t be sure that a lifestyle characterized by increased inflammation is a risk factor for developmental differences.  I do know that the rules implemented by the natural world have no care for our hubris.  Those same rules have violated our once pristine knowledge so dispassionately and with such regularity that I can find no pleasure in hurling the accusation of the naturalistic fallacy at anyone.  Instead, the idea fills me with a sense of honorable mention at best; we are more capable than last century, last generation, last year, but we remain at the mercy of machinations which hold no regard for such incremental progress in knowledge in the face of unprecedented changes to our environment.

–       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

I’ve been thinking a lot lately about the beauty and trials of the tightly coupled systems, the interconnected pathways that keep popping up when pubmed tells me something that might be of interest on journey autism.  One theme bubbling to the top of my thoughts is that there is a large set of inputs capable of tweaking the areas we see altered in autism; broken isn’t necessarily appropriate, but the research increasingly tells us that a delicately balanced set of connected processes is readily changed, and the way that the physics work out, there is no way to change just one thing when you have a polygamous marriage of chemical systems.

Imagine a orchestra where all of the musicians were physically bound to one or more of their counterparts, a system of wires, pulleys, springs and levers such that the musicians are actually participating in the playing of each other, not soccer players doing synchronized flips so much as a set of violin-em-cello-em robots, connected to play their instruments in unison, wind them up and create a symphony.  Different orchestras might have a tighter wire from one member to another, or an older spring, but when they worked together, you could tell what composition they were playing.  In this analogy, you cannot have the drummers start beating harder and faster without also changing how hard the French horn players blow.  The situation only gets more complicated if some of our musicians were connected to several other musicians simultaneously.  There would still be music if the cellist couldn’t keep a steady rhythm, but it would be different music, not just a different cello.

The communication between a lot of our “systems”, immune, endocrine, stress response and central nervous systems are a lot like musicians in the orchestra, interdependent and intimately connected.

The funny thing is, this same message is being blared to me, and to you, all the time, damn near every time you turn on the TV, but it is hidden in plain sight by legislatively mandated doublespeak.  Consider how many advertisements each of us have seen for pharmaceutical drugs where the number of complications and contra-indicated conditions far, far exceed the number of desired effects?

Here is a list of common side effects of Viagra:

Diarrhea, dizziness, flushing, headache, heartburn, stuffy nose, upset stomach

So right off the bat, besides what we are looking for, we can see it is common to expect Viagra to also affect your GI system, immune system, and/ or brain function.  These are the types of things that are “common”.  (One wonders how Viagra would sell if it always caused headaches and diarrhea, and sometimes transiently ameliorated erectile dysfunction? )  A list of ‘severe’ side effects includes memory loss and a sudden decrease in hearing or vision.  Even after decades of work by a lot of exceptionally smart people and hundreds of billions of dollars, the interlocked complexity of our bodies are continuing to prove very difficult to adjust in only the way we’d like, and seemingly minor perturbations in one area can pop up in very unpredictable fashion in other functions.

Trying to put my mind around the implications of this in regards to autism often leaves me with a sense of being profoundly humbled and woefully underprepared, not unlike a lot of my experiences with autism in the real world.  Secondarily, again with great similarity to personal experience, I (eventually) come to the (re-)realization that we should rejoice in opportunities to be challenged and learning more about something makes us richer in ways more important than dollars.

A superb example of all of this and more landed in my inbox the other day, Environmental enrichment alters glial antigen expression and neuroimmune function in the adult rat hippocampus (Williamson et all).  [Also on this paper, blog favorite, Staci Bilbo]

Williamson reported that animals given a so called ‘enriched environment’ exhibited significantly decreased immune responses in certain portions of the brain following immune challenge, with reduced levels of several chemokines and cytokines in the hippocampus in the treatment group. (A previous discussion about environmental enrichment on this blog can be found here)   In this instance, the treatment group got to spend twelve hours a day in a different area, a housing unit with “a running wheel, a PVC tube and various small objects and toys”, while the control group of animals stayed in their drab, Soviet era proletariat cages all day and all night long.  Here is the abstract:

Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Environmental enrichment (EE) in rodents increases neurogenesis, enhances cognition, and promotes recovery from injury. However, little is known about the effects of EE on glia (astrocytes and microglia). Given their importance in neural repair, we predicted that EE would modulate glial phenotype and/or function within the hippocampus. Adult male rats were housed either 12h/day in an enriched environment or in a standard home cage. Rats were injected with BrdU at 1week, and after 7weeks, half of the rats from each housing group were injected with lipopolysaccharide (LPS), and cytokine and chemokine expression was assessed within the periphery, hippocampus and cortex. Enriched rats had a markedly blunted pro-inflammatory response to LPS within the hippocampus. Specifically, expression of the chemokines Ccl2, Ccl3 and Cxcl2, several members of the tumor necrosis factor (TNF) family, and the pro-inflammatory cytokine IL-1ß were all significantly decreased following LPS administration in EE rats compared to controls. EE did not impact the inflammatory response to LPS in the cortex. Moreover, EE significantly increased both astrocyte (GFAP+) and microglia (Iba1+) antigen expression within the DG, but not in the CA1, CA3, or cortex. Measures of neurogenesis were not impacted by EE (BrdU and DCX staining), although hippocampal BDNF mRNA was significantly increased by EE. This study demonstrates the importance of environmental factors on the function of the immune system specifically within the brain, which can have profound effects on neural function.

Total interconnectedness kick ass!

Considering the wide ranging and predominantly ‘rather-not-have-than-have’ properties of ‘extra’ TNF-alpha and IL-1beta in the CNS, this is a pretty interesting finding.  Not only that, animals ‘protected’ through environmental enrichment also showed increased levels of growth factors known to be altered in autism, again in the hippocampus.  In a very real and measurable sense, it was possible to shuffle the neuroimmune cocktail of the brain by changing things like the availability of quality leisure time.  As we have seen in other areas, altering the chemical milieu of immunomodulatory factors in the brain isn’t trivial, and is increasingly associated with a variety of conditions classically diagnosed through the study of behaviors.

It should be noted that there were unexpected, and generally negative findings from this study, namely, a relative lack of biomarkers indicative of increased neurogenesis in the environmental enrichment group; something that I think took the authors by a bit of surprise.

There is a short discussion on the possibilities on why the findings of differential neuroimmune responses were found only in the hippocampus, with reference being made to previous studies indicating that this area of the brain has been found to be more susceptible to a variety of insults.

There were some other findings that struck me as particularly intriguing; something that has been hinted at previously in other studies (or transcripts), but not yet well described, likely due to the fact that the area is still largely unknown to us.  Specifically, the authors reported a state of glial activation, somewhat the opposite of what they expected.

The data instead suggest that EE changes the phenotype of glia, altering their activation and attenuating their pro-inflammatory response to peripheral LPS, although this remains to be directly tested. Interestingly, the blunted neuroinflammatory response within the DG of EE rats occurring concomitant with the increase in classical glial ‘‘activation’’ markers runs counter to our initial prediction. However, we believe these data simply highlight the fact that little is known about the function of these markers. Moreover, there is a growing literature that distinguishes classical versus alternative activation states in microglia, the latter of which is associated more strongly with repair (Colton, 2009; Colton and Wilcock, 2010).

And

Thus, it is possible that EE shifts microglia into an alternatively activated phenotype, an intriguing possibility that we are currently exploring.

(Totally sweet!)

The authors discuss the fact that their findings were highly spatially specific within the brain, involved a subset of cytokines and chemokines, and environmental enrichment did not seem to affect immune response in the periphery.

The immune response within the hippocampi of EE rats was markedly attenuated for a subset of cytokines and chemokines measured in our study. Importantly, not all measured immune molecules were blunted in the hippocampi of EE rats. Furthermore, the immune response was similar for each housing group in the parietal cortex as well as in the periphery. Within the hippocampus, however, EE rats had an attenuated response of interleukin-1b (IL-1b), the TNF family of genes, and several chemokines involved in the recruitment of leukocytes and monocytes. These families of genes indicate an altered hippocampal milieu in EE rats that may be less pro-inflammatory, more neuroprotective and less permeable to peripheral infiltrating immune cells.

There is a short discussion on the existing knowledge concerning IL-B and TNF-alpha in normal and pathological conditions, and how these findings are consistent with other findings involving environmental enrichment and cognition.

Tumor necrosis factor alpha (TNFa) is well characterized for its roles in inflammation and host defense, sepsis and, most intriguing for this study, apoptosis cascades (for review, see Hehlgans and Pfeffer, 2005). The observed attenuation after an immune challenge of TNFa and several associated genes in EE rats compared to HC controls indicates a potential enduring change in the hippocampal microenvironment of enriched rats, such that one mechanism by which EE may increase neuroprotection following insults to the CNS (Briones et al., 2011; Goldberg et al., 2011; Young et al., 1999) is via altered TNF tone and function, increasing the likelihood of cell survival by reducing apoptotic signaling. In addition to attenuated IL-1b and TNF responses, EE rats showed blunted responses for several chemokines known to influence the recruitment of circulating monocytes and leukocytes to the CNS.

Finally, the authors conclude how their findings add to the literature on environmental enrichment and brain function.

In summary, environmental enrichment is a relatively simple manipulation that results in robust beneficial outcomes for the brain. While previous studies have shown a role in post-insult rehabilitation for EE, our study provides evidence that enrichment need not follow the insult in order to be beneficial. Indeed, neuroinflammatory disease states might be attenuated or delayed in their onset in the face of ongoing EE. The translational reach of this manipulation remains to be explored, but in animal models of neuroinflammation, EE may provide a simple preventative measure for negative outcomes.

The bottom line is that a fuller rat life experience resulted in different neuroimmune profiles, findings with some consistency with previous observations that an enriched rat house resulted in improved behavioral manifestations of cognitive performance.  The qualities of these different neuroimmune profiles are also consistent with chemical profiles associated with positive outcomes in several conditions.

There is a deceivingly startling realization hidden in these finding, startling because it reveals the malleable nature of the seemingly different, but basic systems interacting and deceptive because it is so obvious.   How many of us have known someone who deteriorated upon entering a nursing home, or even retiring from working?  How many of us have kept their children inside for a week due to weather and watched their children go crazy after the already inferior indoor entertainment options are long exhausted?  Those changes in emotion, in behaviors and function, just like the findings from this study, are founded by chemistry.

But seeing evidence that relatively simple environmental modifications can rejigger the molecular atmosphere of the brain is still more than a little awe inspiring.   Knowing there is machinery underneath the hood is a little different than observing the cogs of cognition swell , shrink, or slow down; nothing less than a deeper understanding of the chemical basis of thought.  And that is pretty cool.

–          pD


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