Download Microglia important in initiation of pain facilitation

Dr Stephen May
Glial Cells – What Are They
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Glia meaning glue (Greek)
Non-neuronal cells
Surround neurons – holding them in place
Supply nutrients and O2 to neurones
(homeostasis)
Insulate neurons from each other (myelin)
Destroy pathogens and remove dead neurons
Modulate neurotransmission
 Outnumber neurons 10:1
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Glial Cells – What Are They
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Initially believed that glial cells did not have
synapses or release neurotransmitters
Not the case
4 types
Microglia (specialised macrophages)
Macroglia
astrocytes
Schwann cells and satellite cells in PNS
Astrocytes, oligodendrocytes, ependymal cells
and radial glia in CNS
Glial Cells – What Are They
How did glia become of interest
to the pain field ?
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2 independent and distinct lines of research (non pain)
led to the recognition of glial modulation of pain
The first (mid 80’s) looking at brain to immune
communication
In 1990’s recognised that pain facilitation was part of this
sickness response
Pain facilitation enhanced survival – decreased activity
with propensity to curl up, rest and heal
How did glia become of interest
to the pain field ?
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Then evidence of inflammatory cytokines (TNF, IL-1,
IL-6) critically involved in every sickness response
studied
Glia were then implicated as a major source of these
proinflammatory substances
Later confirmed that glia and proinflammatory
cytokines were central to generation of sickness
induced hyperalgesia
How did glia become of interest
to the pain field ?
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2nd line of research began in 1970’s
Found that CNS microglia and astrocytes become
activated following trauma to peripheral nerves
Motor nerve trauma – glial activation surrounding
axotomised motor neurons
Sensory nerve trauma – glial activation in the central
region where sensory terminals were degenerating
This work also demonstated that MK801 blocked
glial activation and neuropathic pain behaviours
Therefore neuropathic pain and glial activation were
at least correlated
What are microglia and
astrocytes
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The literature concentrates on microglia and astrocytes
Unlikely that these are the only non-neuronal cells
involved in pain enhancement (other cells harder to study
due to lack of expression of upregulatable expression
markers)
When these cells become activated they :
A) upregulate cell type specific activation markers (can
be seen with immunohistochemistry)
B) release a variety of substances (eg. proinflammatory
cytokines, chemokines, ATP, NO, excitatory AA’s, etc)
these enhance pain by excitating surrounding cells
What are microglia and
astrocytes
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These are not troublesome cells that we should be
aiming to “knock out” they have important
homeostatic functions unrelated to pain modulation
When they become of relevance to pain is when they
are activated
Different from nerve activation (end point is to
increase AP discharge)
Activation is multidimensional (glia perform multiple
tasks)
Multiple activation states related to various
components expressed with different intensities and
time courses dependent on the triggering stimulus
What are microglia and
astrocytes
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Unknown if glia are involved in loss of neurons
observed under neuropathic pain conditions – but is
possible
Substances released can interact eg. Synergy
Glia do not have axons – cannot relay sensory
information from spinal cord to brain
Pain modulation role must be indirect
? Substances released act to increase AP
discharge/neurotransmitter release in neural cells
If we can target glial activation – can we “turn down
the gain in pain”
Microglia
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5–12% of all cells in the CNS and 5–10% of all glia.
Under basal conditions, microglia perform immune
surveillance
Stimuli that can activate microglia include trauma,
infection, ischemia and neurodegeneration
Activation results in changes in morphology (e.g.,
retracted processes), proliferation, upregulated
receptor expression (e.g., complement receptors,
scavenger receptors)
and changes in function (e.g., migration to sites of
damage, phagocytosis, release of proinflammatory
mediators)
After resolution of a challenge – either return to basal
state or remain primed (can be for prelonged period)
Microglia
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“Primed” microglia do not actively produce
proinflammatory cytokines (and indeed may instead
release anti-inflammatory cytokines)
but now over-respond to new challenges, both in the
speed and magnitude of release of proinflammatory
products
Microglial priming may prove to be important in
explaining why some people greatly over-respond, in
intensity and duration, to a pain-evoking event
Evidence to date from animal models suggests that
prior microglial activation can leave these cells
primed, such that a later pain-evoking stimulus
produces both enhanced spinal proinflammatory
cytokine production and enhanced pain
Astrocytes
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40 – 50% of glial cells
Outnumber neurons
Astrocytes tightly enwrap the vast majority of synapses in
the CNS and actively modulate neuron-to-neuron synaptic
communication
The dynamic astrocyte regulation of synaptic
communication has given rise to the term “tripartite
synapse” as astrocytes are integral parts of neuronal
signalling
astrocytes are important contributors to synaptic “memory”,
as prior synaptic activity leads, at later times, to greater
astrocyte responses to subsequent synaptic input
Beyond the synapse, astrocytes also are in intimate contact
with neuronal cell bodies, dendrites, and nodes of Ranvier.
They extend perivascular endfeet to contact capillaries and
form a border layer in the meninges
Astrocytes
Under basal conditions :
 astrocytes provide neurons with energy
sources and neurotransmitter precursors,
provide trophic support, regulate extracellular
ions and neurotransmitters, and regulate
neuronal survival and differentiation, neurite
outgrowth, and formation of synapses
 Astrocytes are activated by trauma,
inflammation or infection, with progressive
upregulation in glial fibrillary acidic protein
(GFAP) expression, morphology and
proliferation upon persistent activation
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Microglia – astrocyte interactions
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In vivo, astrocytes and microglia interact
Their released products can synergize, and
substances released by one can activate the other
Regarding synergy, proinflammatory cytokines can
synergize with each other as well as with
neurotransmitters and neuromodulators, such as NA,
PGE2, NO
Pain relevant examples include the synergy of TNF
and IL-1 with ATP to enhance PGE2 release
nitric oxide potentiating IL-1 induced PGE2
production and substance P release from sensory
afferent terminals in spinal cord
and substance P potentiating IL-1 induced release of
IL-6 and PGE2 from human spinal cord astrocytes
Microglia – astrocyte interactions
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Regarding cross-stimulation between glia:
(a) astrocytes release substances that stimulate microglial
activation, proliferation, and production of nitric oxide
(b) microglia release substances that induce astrocyte
activation, expression of adhesion molecules, functioning
as antigen presenting cells, and release of glutamate, TNF,
IL-1 and NO
These microglia–astrocyte interactions are consistent with
the developing pain literature that suggests that microglia
are the first glial type to become activated
and that their activation leads in turn to the recruitment of
nearby astrocytes, such that both cell types can contribute
to observed downstream alterations in pain
Microglia – astrocyte interactions
Glial dysregulation of pain
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Normally in basal state not affecting pain modulation
Different when activated
Having said this, basal cytokine levels are integral to
maintaining neuronal plasticity.
It is now known that glial regulation of pain extends
well beyond enhanced pain responses that occur as
a natural component of the sickness response
Both astrocytes and microglia in spinal cord are
activated (as inferred from glial activation markers) in
response to inflammation or damage to peripheral
tissues, peripheral nerves, spinal nerves, or spinal
cord
The generally accepted view has developed that
microglia are activated first, followed by astrocytes
Glial dysregulation of pain
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While microglia were thought to fade, and astrocytes
to increase, in prominence over time, a prolonged
role for microglia has recently been proposed
Many studies have demonstrated that spinal glial
activation and proinflammatory cytokines have been
implicated in enhanced pain associated with almost
every animal model examined to date
based on the facts that intrathecal (into the
cerebrospinal fluid [CSF] space surrounding the
spinal cord) injection of proinflammatory cytokines
enhances pain, and glial and cytokine inhibitors
prevent and/or reverse such pain enhancements as
does knocking out IL-1 signaling
Glial dysregulation of pain
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ongoing inhibition of spinal cord proinflammatory cytokines
can reverse established nerve injury-induced pain
facilitation (i.e., neuropathic pain):
(a) for 3+ months suggestive that other mechanisms will
not reinstate pain facilitation in the absence of these
cytokines
(b) even after pain facilitation has been maintained
continuously for 1–2 months
strong support for the conclusion that glia and
proinflammatory cytokines do not just induce pain
facilitation but, rather, are key mediators of pain
maintenance as well
increases in proinflammatory cytokines and/or decreases
in anti-inflammatory cytokines have been found in spinal
CSF samples from chronic pain patients diagnosed with
complex regional pain syndrome, fibromyalgia and
neuropathic pain
How do glia become activated ?
Sickness induced hyperalgesia involves a
vagus-nucleus tractus solitariusventromedial medulla-spinal cord circuit
 Spinal cord glia are thought to be activated
by release of medullospinal
neurotransmitters eg. Substance P, CCK,
glutamate
 Direct pathway from periphery to spinal
cord doesn’t explain this
 After peripheral nerve injuries : probable
that factors released by sensory afferents
(projecting to spinal cord) activate glia
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How do glia become activated ?
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4 potential classes of mediators
being considered
1) Neurotransmitters released by
activated sensory afferents bind to
and activate glia eg. Glutamate,
ATP
2) Neuromodulators released by
activated neurons eg. NO, PG’s
3) Neurons may release glial
excitatory chemokines eg.
Fractalkine
4) Glial excitatory signals may be
released from sensory afferent
fibres within spinal cord following
damage to their peripheral nerve
axons eg. HSP 27, TLR 4
How do glia become activated ?
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Do triggers for acute versus chronic pain differ ?
Glial activation is involved in pain enhancement
induced in acute inflammation and in pathological
pain such as neuropathic pain
Seems that microglia are activated first
Their activation induces the initiation of
exaggerated pain responses
Fits as normal glial function is to sensor
microenvironment
Consequent to microglial activation astrocytes
become activated and are prominently involved in
chronic stages of neuropathic pain
How do glia become activated ?
If neuronal release of substances were
the key :
 Astrocytes are better positioned
anatomically to react than microglia
 ? Astrocytes respond first to to acute
injury or infection where key is
enhanced activity in neurons
 ? Microglia respond first in pathological
conditions where non-synaptically
released substances readily reach
microglia
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Mechanisms of glial
enhancement of pain
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Only activated glia are
important
Glial activation can occur
under physiological and
pathological conditions
Reduced pain enhancement
(animal models) by :
Disrupting glial activation
Disrupting spinal cord
proinflammatory cytokine
actions
Ongoing research
Mechanisms of glial
enhancement of pain
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Neurons express receptors for
proinflammatory cytokines
Neuronal excitability increases in response to
known glial products
? Direct effect
IL-1 enhances neuronal NMDA conductance,
induces ATP (neuroexcitant) release
TNF increases neuronal AMPA receptor no. &
conductance, increases glutamate response
Proinflammatory cytokines induce production
of various neuroexcitatory substances eg. NO,
PG’s, reactive oxidants
Mechanisms of glial
enhancement of pain
Glia release other neuroexcitatory
substances
 D-serine more potent than glycine at
NMDA glycine site
 Others also
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Mechanisms of glial
enhancement of pain
Glia now implicated in spread of pain
beyond site of injury
 ? Astrocyte gap junction communication
involved – movement of neuroexcitatory
products
 Disrupting gap junction found to abolish
mirror image pain and not territorial pain
ipsilateral to site of injury
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Mechanisms of glial
enhancement of pain
Interaction of glial cells and
opiates
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? Glial cells involved in morphine tolerance
? Counterregulatory response dampening
opiate effect
Glia can express receptors for mu,kappa,delta,
orphan opiod receptor-1
Exposure increases receptors for the agonist
Opiates also stimulate glial production of NO,
superoxide, TNF, IL-1, IL-6
All have neuroexcitatory effects in pain
pathways
Interaction of glial cells and
opiates
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Intracerebroventricular IL-1 blocks systemic
morphine analgesia
Reversed by IL-1 antagonist
IL-1 can inhibit binding of opiate ligands
IL-1 can stimulate CCK release (suppresses
acute morphine analgesia and enhances
development of morphine tolerance)
IL-1 antagonists potentiate analgesic effect of
acute morphine, delay development of
morphine tolerance and reduce tolerance
associated pain facilitation
Interaction of glial cells and
opiates
Chronic morphine exposure downregulates
glial glutamate transporters (including dorsal
horn)
 ? Morphine induced PKC activation
 Overlap of drugs that suppress morphine
tolerance and glial function
 eg. NMDA antagonists
 ? Glial involvement in analgesic tolerance
extends beyond morphine
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Interaction of glial cells and
opiates
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Opiods appear to cause direct glial
activation in nonclassical receptor fashion
Act on pattern recognition receptors
Toll-like receptors (TLR’s)
TLR activation has similair downstream
effects to IL-1 activation
Other studies have implicated TLR’s in
chronic pain states
? Role for BBB permeable molecules that
block TLR4 and/or TLR2
Interaction of glial cells and
opiates
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? Naloxone binds to TLR’s as well as classical opiate
receptors (only bind (-) isomers)
(+) naloxone can reverse neuropathic pain
? Blocking TLR4
(+) methadone (no effect at classical opiate receptors)
activates glia, upregulates proinflammatory cytokine
production, causes allodynia, hyperalgesia
Supports concept that neuron to glia signalling of neuropathy
induced “danger signals” is important for ongoing
neuropathic pain state
Seems that immunomodulation by opiate receptors is not via
classical opiate receptors
? Opiates are agonists of TLR 4
Interaction of glial cells and
opiates
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? Opiate induced glial activation involved in morphine
tolerance and withdrawl
Glial activation inhibitor significantly reduces
naloxone induced withdrawl behaviour
Also reduced glial activation in brain nuclei
associated with opiate action
Same inhibitor (AV411) protected against
dependence behaviour and spontaneous withdrawl
(+) naloxone attenuates (-) naloxone precipitated
withdrawl in morphine dependent animals
AV411 reduces “morphine reward” – reduced
dopamine levels in nucleus accumbens
Interaction of glial cells and
opiates
A) Classical view : (−)-opioid agonist
isomers stereoselectively bind to
classical opioid receptors producing an
inhibitory influence on nociceptive
signal transmission
B) ignores an important nociceptive
modulatory influence driven by opioid
induced glial activation resulting from
opioid agonists binding to glial opioid
binding receptors which in turn
increase proinflammatory cytokine
expression and release leading to a
decrease in opioid efficacy at the
neuronal component
C) (−)-Opioid antagonists bind to both
the neuronal and glial components
resulting in blockade of any potential
opioid analgesia and glial activation
Interaction of glial cells and
opiates
D) only the (−)-isomer of opioid
agonists and antagonists are able to
bind. Combination of an opioid (+)antagonist and an opioid (−)-agonist
are introduced to this system, the (+)antagonist is unable to bind to the
stereoselective neuronal opioid
receptor, but able to block the nonstereoselective glial site. The opioid
(−)-agonist can act freely at the
neuronal opioid receptor, but is unable
to bind to the glial site due to its
blockade by the (+)-antagonist.
Therefore, this situation produces
opioid receptor mediated analgesia
without the opposing force of opioid
induced glial activation, thereby
potentiating opioid analgesia
E) Based on the above hypothesized series of events this
schematic diagram demonstrates how analgesia decreases
over time as a direct result of increased glial activation.
Potential for new therapies
Animal studies have used a broad array
of compounds to inactivate and/or
disrupt glial function
 Effects are there
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 Most are nowhere near appropriate for
human use
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Potential for new therapies
Big obstacle :
 A systemic drug must penetrate BBB to
effect central glial function
 However potential to effect brain and
peripheral immune/glial function
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Potential for new therapies
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Fluorocitrate is a glial cell inhibitor but effects
other critical glial cell functions
Minocycline : selectively targets microglia –
disrupting microglia activation and production of
proinflammatory cytokines and NO
Animal studies suggest it is far more effective at
prevention rather thn treatment of chronic pain
states
? Microglia important in initiation of pain
facilitation
? Astrocytes more important in pain
maintenance
? Targeting microglia worthwhile
Potential for new therapies
IL-1 and TNF antagonists
 Animal studies show effect
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 Need intrathecal administration (as none
cross BBB)
 Especially problematic for chronic use
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Potential for new therapies
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Inhibit proinflammatory cytokine synthesis
Propentofylline
Orally active, crosses BBB, reverses and prevents
pathological pain states in animal models
BUT
Problematic food/drug interactions
Thalidomide and derivatives (lacking teratogenicity)
Evidence for effect on peripheral immune cell
production of proinflammatory cytokines and pain
facilitation
Some cross BBB - ? Effect on central glial cells
Potential for new therapies
p38 MAP (mitogen activated protein) kinase
inhibitors
 Activated p38 MAP important in cascade of
proinflammatory cytokine production
 Some cross BBB
 Less effect on established pain (like
minocycline)
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Potential for new therapies
IL-10 (anti-inflammatory)
 Doesn’t cross BBB and peripheral actions
would be detrimental to normal immune
function
 Effective in reversing pain facilitation but
short lived effect
 ? Role for nonviral gene therapy approach
(early clinical trials suggest 90 day effect)
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Potential for new therapies
AV411 (ibudilast) – anti-inflammatory and
cerebral vasorelaxant properties
 Relatively selective PDE inhibitor
 Reduces proinflammatory cytokine production
by glial cells
 Effect in reducing allodynia in animal models
 Seems to have effect on pain modulation
outwith PDE effect
 ? Adjuvant prep with morphine or gabapentin
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Potential for new therapies
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Perfect pharmaceutical
1. Orally active
2. No effect on immune system or other systems
3. BBB permeable
4. No effect on the brain
5. Target only proinflammatory functions of
spinal cord glia
6. Be reversible (proinflammatory functions of
spinal glia could be imp in other circumstances
eg. Infection)
References (best ones)
Neuroimmune Interactions and Pain: The Role of
Immune and Glial Cells
Psychoneuroimmunology (Fourth Edition), 2007,
Pages 393-414
Linda R. Watkins, Julie Wieseler-Frank, Mark R.
Hutchinson, Annemarie Ledeboer, Leah Spataro,
Erin D. Milligan, Evan M. Sloane, Steven F. Maier
Glial Dysregulation of Pain and Opiod Actions :
Past, Present, and Future
Pain 2008 an updated review, 2008,
Pages 249-268
Mark R. Hutchinson, Kirk W. Johnson, Linda
Watkins
Questions ?