Glial Cells – the future or just more garlic bread ?

1. Dr Stephen May Garlic Bread Garlic and Bread Together Are You sure ? That’s the future that is Glial Cells – the future or just more garlic bread ?

2. Glial Cells – What Are They • 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

3. Glial Cells – What Are They • Initially believed that glial cells did not have synapses or release neurotransmitters • Not the case • 4 types • Microglia (specialised macrophages) • Macroglia • Schwann cells and satellite cells in PNS • Astrocytes, oligodendrocytes, ependymal cells and radial glia in CNS astrocytes

4. Glial Cells – What Are They

5. How did glia become of interest to the pain field ? • 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

6. How did glia become of interest to the pain field ? • 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

7. How did glia become of interest to the pain field ? • 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

8. What are microglia and astrocytes • 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

9. What are microglia and astrocytes • 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

10. What are microglia and astrocytes • 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”

11. Microglia • 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)

12. Microglia • “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

13. Astrocytes • 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 perivascularendfeet to contact capillaries and form a border layer in the meninges

14. 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 glialfibrillary acidic protein (GFAP) expression, morphology and proliferation upon persistent activation

15. Microglia – astrocyte interactions • 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

16. Microglia – astrocyte interactions • 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

17. Microglia – astrocyte interactions

18. Glial dysregulation of pain • 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

19. Glial dysregulation of pain • 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

20. Glial dysregulation of pain • 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

21. How do glia become activated ? • Sickness induced hyperalgesia involves a vagus-nucleus tractussolitarius-ventromedial 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

22. How do glia become activated ? • 4 potential classes of mediators being considered • 1) Neurotransmitters released by activated sensory afferents bind to and activate gliaeg. Glutamate, ATP • 2) Neuromodulators released by activated neurons eg. NO, PG’s • 3) Neurons may release glial excitatory chemokineseg. 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

23. How do glia become activated ? • 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

24. 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

25. Mechanisms of glial enhancement of pain • 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

26. Mechanisms of glial enhancement of pain • 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

27. Mechanisms of glial enhancement of pain • Glia release other neuroexcitatory substances • D-serine more potent than glycine at NMDA glycine site • Others also

28. 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

29. Mechanisms of glial enhancement of pain

30. Interaction of glial cells and opiates • ? 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

31. Interaction of glial cells and opiates • 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

32. 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

33. Interaction of glial cells and opiates • 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

34. Interaction of glial cells and opiates • ? 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, upregulatesproinflammatory 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

35. Interaction of glial cells and opiates • ? 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

36. 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

37. 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 non-stereoselective 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.

38. Potential for new therapies • Animal studies have used a broad array of compounds to inactivate and/or disrupt glial function • Effects are there • BUT • Most are nowhere near appropriate for human use

39. 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

40. Potential for new therapies • 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

41. Potential for new therapies • IL-1 and TNF antagonists • Animal studies show effect • BUT • Need intrathecal administration (as none cross BBB) • Especially problematic for chronic use

42. Potential for new therapies • 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

43. 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)

44. 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)

45. 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

46. Potential for new therapies • 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)

47. References (best ones) Neuroimmune Interactions and Pain: The Role of Immune and Glial CellsPsychoneuroimmunology (Fourth Edition), 2007, Pages 393-414Linda 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

48. Questions ?