Neuroimmune Interactions in Pain Flashcards by Bryony Hawkins

Describe the interactions between non-neuronal cells, neurones, and (neuro)inflammation after injury or insult. (3)

Non-neuronal cells drive (neuro)inflammation

and also release signalling molecules and inflammatory mediators

and these things all act on nociceptors to alter pain processing.

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True or false? Explain your answer if necessary. (1)

After injury or insult, non-neuronal cells release signalling molecules and inflammatory mediators which have very localised effects on the neurones in the area.

False - this is all true, however the signalling and inflammatory molecules can also have a widespread effect via the CSF and bloodstream

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Fill the gaps relating to inflammation and pain. (5)

……………………. cells are activated by …………….. or …………………, and release a host of mediators which can cause an ……………………. and …………………… peripheral nociceptors.

Non-neuronal

insult

injury

immune response

sensitise

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Give 11 examples of signalling molecules and inflammatory mediators which are released from non-neuronal cells and tissue after insult or injury. (11)

H+ ions
Adenosine
ATP
TNFa
IL1b
NGF
Bradykinin
PGE2
Histamine
5HT
Endothelin

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Do non-neuronal cells in inflamed/infected/damaged tissue provoke an innate or adaptive immune response by detecting the event and releasing signalling molecules? (1)

Innate

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Which of these mechanisms, which occurs after tissue damage or infection, activates neurones? (1)

Direct activation by damage and inflammation
Indirect activation by the systemic immune response produced by non-neuronal cells

Neurones are activated by both of these mechanisms.

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Name three non-neuronal cells (also non-glial cells) which play key roles in activating nociceptors after tissue injury or insult. (3)

Keratinocytes
Macrophages
Neutrophils

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What are keratinocytes, and what is their main role (not in pain)? (2)

Keratinocytes are the predominant cells of the epidermis.

Main role is to act as a physical barrier.

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True or false? Explain your answer if necessary. (1)

As well as acting as a physical barrier, keratinocytes also release neuroactive compounds in response to tissue injury or insult.

True

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Name 6 neuroactive compounds that can be released by keratinocytes after tissue injury or insult. (6)

CGRP
ATP
ACh
Glutamate
Cytokines
Growth factors

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Describe how keratinocytes may be stimulated to release neuroactive compounds after tissue injury. (2)

The signalling carried out by keratinocytes to the PNS is what type (endocrine/exocrine/paracrine)? (1)

They express ligand-gated ion channels

and voltage-gated ion channels.

This is paracrine signalling.

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Briefly describe a study that could test the effects of activating keratinocytes on activity in cutaneous sensory neurones and pain responses. (4)

Engineer mice to express ChR2 on keratinocytes
Or they could express halorhodopsin on keratinocytes
Shine blue light to activate (ChR2) or inhibit (halorhodopsin) keratinocytes
Measure responses in sensory neurones and by looking at pain behaviour

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Describe what you would expect the effect to be of expressing ChR2 on keratinocytes and shining blue light, on activity in cutaneous sensory neurones. (1)

Action potentials generated in multiple types of cutaneous sensory neurones.

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Describe what you would expect the effect to be of expressing halorhodopsin on keratinocytes and shining blue light, on pain responses in mice. (1)

Reduced pain responses

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Describe the general effect of activating keratinocytes on pain. (1)

Causes pain

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Fill the gaps relating to the recognition stage (first stage) of the pathway from tissue damage to inflammatory pain. (6)

There is initiation of an inflammatory response, because cells recognise ……………….. and ………………… interacting with their receptors, called ………………………

This interaction leads to the release of pro-inflammatory cytokines, mainly ………………, ………………….., and ……………….

PAMPs

DAMPs

PRRs

TNFa

IL-6

TGFb

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Fill the gaps relating to the recruitment stage (second stage) of the pathway from tissue damage to inflammatory pain. (3)

………………….. in the damaged tissue are activated. These cells produce ………………….. and ………………… which allow for cellular trafficking.

Leukocytes

cytokines

chemokines

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Fill the gaps relating to the response stage (third stage) of the pathway from tissue damage to inflammatory pain. (2)

The …………………….. cells begin the process of ………………….. to control foreign attacks to external or internal antigens.

leukocyte/immune

phagocytosis

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Fill the gaps relating to the resolution stage (fourth stage) of the pathway from tissue damage to inflammatory pain. (5)

…………………… cells undergo apoptosis, and this is followed by …………………….. and ……………………
…………………….. and …………………… that remain elevated for a long time are the driving factors in inflammatory mediated chronic pain.

Neutrophil

tissue remodelling

scar formation

PRRs

adhesion factors

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True or false? Explain your answer if necessary. (1)

The immune system interacts with peripheral nerve endings in the skin, but the rest of the pain pathway is not affected by the immune system.

False - the immune system interacts with pain processing throughout the neuroaxis, including the periphery, DRG, and spinal cord

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Inflammatory stimuli broadly fall into two categories.

Name/describe these two categories of stimuli. (2)

Pathogen-associated molecular patterns (PAMPs; from infections like bacteria).

Damage-associated molecular patterns (DAMPs; from tissue damage).

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Name one well-known PAMP, and describe where it originates from. (2)

Lipopolysaccharide (LPS)

found on the outer cell wall of gram-negative bacteria.

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Give three cell types or situations which cause release of DAMPs. (3)

Tumour cells
Dead or dying cells
Released from cells in response to signals such as hypoxia

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Are DAMPs derived from host cells or foreign materials? (1)

Host cells

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Name the specific type of inflammatory response produced from DAMPs, and explain the meaning behind the name. (2)

Sterile inflammatory response because they are derived from host materials and not foreign materials.

Name the general type of receptor to which PAMPs and DAMPs bind. (1)

Pattern recognition receptors (PRRs)

Name five types of pattern recognition receptors which bind to PAMPs and DAMPs. (5)

Toll-like receptors (TLRs) Cytoplasmic NOD-like receptors (NLRs) Intracellular retinoic acid inducible-gene I receptors (RLRs) Transmembrane C-type lectin receptors Absent in melanoma 2-like receptors (AIM2)

True or false? Explain your answer if necessary. (1) Pattern recognition receptors are expressed on immune cells and activation triggers a cascade of downstream signalling.

True and false - it is true, but PRRs are also expressed on non-immune cells

Which type of transmembrane proteins are TLRs? (1)

Type I

Describe the three structural domains present in TLRs. (3)

Leucine-rich repeats (LRRs) motif Transmembrane domain Cytoplasmic Toll/IL-1 receptor (TIR) domain

Give another name for the TLR4 receptor. (1) How heavy is this protein in KDa? (1)

CD284 100KDa

Give three cell types which express the TLR4 receptor. (3)

Neurones Macrophages Microglia

Describe the specific stimulus which binds to and activates the TLR4 receptor. (1)

Bacterial endotoxin (lipopolysaccharide)

Describe the intracellular effects of activation of the TLR4 receptor. (3)

- Increases transcription of genes associated with inflammation - For example pro-inflammatory cytokines, interferons, etc - By increasing activity of NFkB (translocates from cytoplasm to nucleus)

Fill the gaps relating to macrophages. (3_ Macrophages are a type of .................. blood cell. They ...................... debris and .....................

white engulf/clear invading cell types (pathogens)

Describe the relationship between monocytes and macrophages. (3)

Macrophages are specialised forms of monocytes. Monocytes are WBCs in the circulation, and macrophages are is tissue.

Describe how infection or tissue damage leads to monocytes in the blood to become macrophages in tissue. (5)

Resident cells (eg. tissue-resident macrophages, keratinocytes etc) detect infection/damage They release cytokines etc which results in fenestration of the capillary barrier So circulating cells migrate into the tissue Monocytes will chemotax towards damaged tissue, up cytokine concentration gradients When monocytes enter the tissue they specialise (polarise) to become macrophages

True or false? Explain your answer if necessary. (1) Macrophages are only found in damaged tissue - monocytes enter damaged tissue and specialise to become macrophages.

False - this does happen, but there are also tissue-resident macrophages, which reside in tissue that is not damaged

What is meant by 'polarisation' when talking about monocytes becoming macrophages? (1)

Monocytes specialise to become either M1 or M2 macrophage.

Macrophages exist in M1 or M2 form. Name another cell type that can exist as these forms. (1)

Microglia

Give two polarising stimuli for M1 microglia. (2)

IFN-y LPS (or both)

Give five polarising stimuli for M2 microglia. (5)

IL-4 IL-13 IL-10 Glucocorticoids TGF-b

Are M1 microglia pro-inflammatory or anti-inflammatory? (1)

Proinflammatory

Are M2 microglia pro-inflammatory or anti-inflammatory? (1)

Anti-inflammatory

Describe the in vitro morphologies of both M1 and M2 macrophages. (2)

M1 = round/oval M2 = elongated (fibroblast-like)

Give five products released from M1 macrophages. (5)

TNFa IL-1b IL-6 IL-12 IL-23

Describe the phagocytic activities of both M1 and M2 macrophages. (2)

M1 = high phagocytic activity M2 = low phagocytic activity

Describe the levels of antigen presentation shown by M1 and M2 macrophages. (2)

M1 = high M2 = low

Describe the arginine metabolism, and therefore vasodilation response, shown by M1 and M2 macrophages. (2)

M1 = iNOS makes NO from arginine (potent vasodilator) M2 = Arg1 makes ornithine from arginine (not much vasodilation)

Describe the antibacterial capacity of both M1 and M2 macrophages. (2)

M1 = high M2 = low

Describe the effect on tumours of both M1 and M2 macrophages. (2)

M1 = tumouricidal M2 = protumourigenic

M1 microglia contain iNOS, which makes NO from arginine. What is the effect of this NO on the immune response? (3)

NO dilates blood vessels so there is increased blood flow and more immune cells can enter the damaged area.

Describe the type of 'activation' that produces M1 macrophages. (1)

Classical activation

True or false? Explain your answer if necessary. (1) M1 macrophages express NF-kB, and release a range of pro-inflammatory molecules that sensitise nociceptors.

True

M1 macrophages cause inflammation. Why are they helpful to us? (1)

They are essential for host response to acute infections (get rid of infection).

Fill the gaps relating to M1 polarisation of macrophages. (3) The molecule .........................., which is produced by ..................... cells, is central to the production of M1 macrophages from ........................

IFNy T helper I (TH1) monocytes

Describe the type of 'activation' that produces M2 macrophages. (1)

Alternative activation

Describe the subdivisions of M2 macrophages. (3) Very briefly describe how each subtype is different. (1)

M2a, M2b, and M2c Each subtype is stimulated by different molecules.

Which type of macrophage is more involved in the resolution phase of inflammation? (1)

Fill the gaps relating to macrophages and inflammation. (3) Both M1 and M2 macrophages are produced at all stages of inflammation, however at the start, there are more ................, and in the resolution phase there are more ............ Also, all macrophages can produce both pro- and anti-inflammatory factors, it just depends on the .................... of factors which are produced as to whether the macrophage is 'pro-inflammatory' or 'anti-inflammatory'.

M1 M2 overall balance

What is complete Freund's adjuvant? (2) What receptor does it activate? (1)

Contains heat-treated bacteria so it is an inflammatory molecule. It activates TLR4

Is Complete Freund's Adjuvant a PAMP or a DAMP? (1)

PAMP

An experiment tested the different responses produced with CFA (PAMP) and incision (DAMP). Describe the different behaviours seen regarding heat and mechanical hyperalgesia with a PAMP and DAMP. (2)

Both PAMP and DAMP produce the same behaviour.

An experiment tested the different responses produced with CFA (PAMP) and incision (DAMP). Describe the difference in the inflammatory cells engaged and chemical mediators released with PAMP vs DAMP. (2)

PAMP and DAMP both engage different subpopulations of macrophages. And there are different cytokines released in PAMP vs DAMP.

An experiment tested the different responses produced with CFA (PAMP) and incision (DAMP). Describe the overall outcome of this study. (4)

There exist parallel systems which are activated by different stimuli (PAMPs and DAMPs) and engage different subpopulations of immune cells. There are also different chemical mediators released, but the same behavioural response is observed.

Are neutrophils granulocytes or agranulocytes? (1)

Granulocytes

Which WBC type is the most abundant? (1)

Neutrophils

Are neutrophils long-lived or short-lived? (1)

Short-lived (5-90 hours)

Fill the gaps relating to neutrophils. (2) Neutrophils are ...............(highly/hardly) motile, and they .................(can/cannot) enter tissues impenetrable to other cell types.

highly can

True or false? Explain your answer if necessary. (1) Neutrophils are phagocytes and the first major class of cell to respond to injury (within minutes), infection, and in some cases of cancer. They enter tissues chemotactically.

True

Fill the gaps relating to neutrophils and pain. (2) Neutrophils are essential for the .................... of some forms of inflammatory pain. Ablation of these cells can ..................... incisional pain.

initiation ameliorate

Name a molecule which is readily released from neutrophils, and describe its actions. (2)

PGE2 which acts to sensitise nociceptors (PGE2 is a very potent nociceptive molecule).

True or false? Explain your answer if necessary. (1) Subgroups of neutrophils release opioid peptides and can thereby increase pain.

False - subgroups of neutrophils release opioid peptides, but this would reduce pain

Fill the gaps relating to the blood-brain barrier. (4) The BBB plays the crucial role of limiting exposure of the central nervous system to ............................ Dysfunction of the BBB is critical in a broad range of CNS disorders including .........................., ....................................., and ......................

damaging molecules and cells neurodegeneration inflammatory or traumatic injury to the CNS stroke

Describe an experiment which would test how nociception and pain alters the permeability of the BBB. (3)

- Injure rats, stimulate C fibres, anaesthetise C fibres, etc - Intravenous injection with Evans blue or horseradish peroxidase - See how much has crossed the BBB or BSCB by measuring amount of dye in spinal cord (these molecules do not normally cross barriers)

An experiment injured rats with either chronic constriction injury (CCI) or spared nerve injury (SNI), and compared this with sham controls. They then injected Evans blue and tested how much entered the spinal cord. Describe the results you would expect to see with CCI or SNI compared to sham. (2) What conclusion can you draw from this? (1)

More Evans blue in spinal cord with both CCI and SNI. CCI and SNI somehow made the BSCB leaky (not through physical BSCB injury, but through release of a mediator).

An experiment injured rats with either chronic constriction injury (CCI) or spared nerve injury (SNI), and compared this with sham controls. They then injected Horseradish peroxidase and tested how much entered the spinal cord. Describe the results you would expect to see with CCI or SNI compared to sham. (2) What conclusion can you draw from this? (1)

Increased HRP in spinal cord with both CCI and SNI. Injuries made BSCB leaky.

An experiment injured rats with chronic constriction injury (CCI) and immediately injected lidocaine. They then injected Evans blue and tested how much entered the spinal cord. Describe the results you would expect to see with CCI + lidocaine vs naive or CCI without lidocaine. (1) What conclusion can you draw from this? (1)

CCI + lidocaine showed less Evans blue in spinal cord (about the same amount as naive, and much less than CCI + no lidocaine). Injection of lidocaine stops BSCB becoming leaky, so something released from peripheral nociceptors is causing the spinal cord to become leaky.

An experiment injured rats with chronic constriction injury (CCI). They then injected Evans blue and tested how much entered the spinal cord. They then performed a subsequent CCI. Describe the results you would expect to see regarding Evans blue in the spinal cord after subsequent CCI. (1)

Still increased levels of Evans blue in spinal cord.

An experiment took rats and stimulated the sciatic nerve first at A fibre intensity, then at C fibre intensity. They then injected Evans blue and looked to see how much entered the spinal cord. Describe the results seen with A fibre and C fibre intensity stimulation. (2) What conclusion can you draw from this?

A fibre stimulation did not cause increased Evans blue in spinal cord. C fibre stimulation did cause increased Evans blue in spinal cord. There is a factor released from C fibres when they are stimulated (no matter what injury has occurred), which makes the BSCB leaky.

An experiment took rats and stimulated the sciatic nerve first at A fibre intensity, then at C fibre intensity. They injected lidocaine before or after stimulation. They then injected Evans blue and looked to see how much entered the spinal cord. Describe the results seen with before and after block by lidocaine. (2)

Before = reduced Evans blue in spinal cord After = increased Evans blue in spinal cord

Describe how tissue injury/damage causes the BSCB to become leaky, and the consequences of this. (5)

- Damage to tissue = inflammation - Inflammation sensitises C fibres - A mediator released from C fibres causes the BSCB to become leaky - Blood contents can enter the spinal cord - Further inflammation and sensitisation can occur in the spinal cord

Electrically stimulating C fibres causes the BSCB to become leaky (shown as infiltration of Evans blue into the spinal cord). How does this change when applying capsaicin? (1)

Nothing changes - capsaicin also causes BSCB to become leaky

Very briefly describe the main mechanism which underpins acute pain. (1)

Activity in nociceptors

Is acute pain protective or not protective? (1)

Protective

How long does acute pain last for? (1)

<3 months

Describe the protective role of chronic pain. (1)

No protective role

How long does chronic pain last? (1)

>3 months

Very briefly describe the main mechanism which underpins chronic pain. (1)

Changes in the way that pain is processed in the PNS and CNS

True or false? Explain your answer if necessary. (1) Chronic pain is purely the result of changes in the way that neurones communicate with each other.

False - neurones also have interactions with other cells in the CNS which contribute to chronic pain

Name two cell types in the CNS which communicate with neurones and may influence chronic pain and central sensitisation. (2)

- Microglia - Astrocytes

Briefly describe the mechanisms leading to classical neuronal plasticity in the CNS. (3)

Pathological activity in the presynaptic primary afferent neurone induced changes in the strength of the synapse and this leads to the establishment of central sensitisation (wind-up, LTP, heterosynaptic potentiation).

Fill the gaps relating to glia and chronic pain. (4) There are billions of neurones in the CNS, but there are ...................... glial cells. Estimates vary, but there could be well over ...................... glia in the CNS compared to about 10 billion neurones. So it seems logical that they may play a role in the ......................... and ....................... of chronic pain states.

many more a trillion establishment maintenance

Fill the gaps relating to inflammation and chronic pain. (4) ..................... and ...................... induce inflammation in the peripheral nervous system by activating a range of cell types. However in the central nervous system, ....................... and ......................... play an important role.

PAMPs DAMPs microglia astrocytes

Which cells which contribute to inflammation in the CNS can be described as macrophage-like cells? (1)

Microglia

Which cells which contribute to inflammation in the CNS can be described as pseudoinflammatory cells? (1)

Astrocytes

Fill the gaps relating to glia and CNS inflammation. (3) There is a wealth of evidence for glia of all types playing a role in the ....................... function of the CNS, as well as being involved in ........................ Glia, although not involved in storing information relating to pain, are able to ............................ of neurones which do play a direct role in nociception and pain.

normal neuropathology modify the responses and function

Describe the appearance of astrocytes (not necessarily when activated). (2)

Star-shaped With many processes

Fill the gaps relating to astrocytes in the CNS. (5) Astrocytes maintain close, intimate contact with ....................... and .................... They regulate the .........................., help in .................... formation, and provide .......................... support.

blood vessels ependyma local environment scar structural

Fill the gaps relating to microglia in the CNS. (6) Microglia are the .................. cells of the nervous system. They ...................... damaged and foreign tissue, and are involved in ......................... disease and ...................... processing. They secrete both ....................-inflammatory agents and .................-inflammatory agents.

immune phagocytose neurodegenerative pain pro anti

What is minocycline and how does it affect microglia? (4) *Describe the exact mechanism by which it has its effects on microglia

Tetracycline antibiotic Inhibits the Jak-STAT pathway Reduces MHC-II expression So inhibits microglia (prevents them becoming activated)

Fill the gaps relating to minocycline and microglia. (3) Minocycline ..................... microglia, so microglia cannot alter their ...................., and they also cannot secrete ......................

inhibits morphology pro-inflammatory cytokines

What is the effect of minocycline on pain? (2) How does minocycline have this effect? (1)

Reduces pain (hyperalgesia) but does not completely remove it. It does this by inhibiting microglia.

Minocycline treatment reduces hyperalgesia, but does not completely remove it. What does this suggest about microglia and pain? (3)

Microglia do contribute to pain (hyperalgesia) but cannot fully be responsible for hyperalgesia (or else minocycline would have completely resolved hyperalgesia) so neuronal mechanisms such as classical heterosynaptic sensitisation also play a role.

Describe the change that microglia undergo to their appearance in persistent pain states. (4)

At rest microglia are RAMIFIED, with MANY THIN PROCESSES extending over a large area. Once activated they become AMOEBOID, swollen with FEW PROCESSES.

As well as a change in their appearance, describe another important cellular change that microglia make in persistent pain states. (2)

Alterations in gene expression and up-regulation of several important pro-inflammatory molecules.

True or false? Explain your answer if necessary. (1) Microglia are non-excitable electrically, so they do not get activated via propagation of action potentials.

True

Name three types of molecules which are released from damaged peripheral nerves which can lead to activation of pro-inflammatory microglia. (3)

- ATP - Chemokines - Proteinases

ATP is released from damaged peripheral nerves. Describe how ATP may lead to activation of microglia. (2)

Directly interact with receptors on microglia (and other cells) membrane. Binds to P2X receptor (which is a calcium channel).

Chemokines are released from damaged peripheral nerves. Very briefly describe the 'mechanism' by which chemokines are released from damaged nerves. (1) Describe how chemokines may lead to activation of microglia. (1)

Chemokines released due to spontaneous discharges. Directly interact with GPCRs.

Proteinases are released from damaged peripheral nerves. Very briefly describe the 'mechanism' by which chemokines are released from damaged nerves. (1) Describe how proteinases may lead to activation of microglia. (3)

Proteinases released due to spontaneous discharges. - Allow other signals to be cleaved - Such as chemokines (FKN) and cytokines (TNFa; IL-1b) - Which can then bind to their receptors on microglial membranes

What is fractalkine? (2) Give another name for fractalkine. (1)

A chemokine that sits in the neuronal membrane. Another name for fractalkine is CX3CL1.

True or false? Explain your answer if necessary. (1) Fractalkine's only known function is in pain.

False - it is involved in many processes, an important one being pain

Describe the structure of the fractalkine ligand in the neuronal membrane, and how this ligand interacts with proteinases. (3)

Ligand has a transmembrane section and a section which can be cleaved and become soluble so when proteinases are released from damaged neurones they can cleave fractalkine and release the soluble portion.

Describe the actions of the soluble (cleaved) form of fractalkine. (2)

Binds to its receptor (CX3CR1) which is only found on microglial membranes.

Name the receptor which fractalkine binds to. (1) Where is this receptor found? (1)

CX3CR1 Found on microglial membranes

Fill the gaps relating to nerve damage and microglial activation. (7) Cleavage of ........................., such as ........................; the release of neurotransmitters such as ....................... and .......................; or direct release of chemokines and cytokines such as .......................... and ........................; all interact with receptors on the surface of .......................... cells to activate them.

chemokines fractalkine glutamate ATP IL-1b TNFa microglial

Describe the general effects of substances released from damaged nerves binding to their receptors on microglial membranes. (6)

Activation of intracellular pathways eg. MAPK pathways which activate NF-kB and regulate gene transcription leading to production and release of several pro-inflammatory molecules and upregulation of receptors for inflammatory molecules.

Fill the gaps relating to microglial signalling in pain. (3) ATP is released from ...................... in response to painful stimuli. ATP interacts with various ....................... (type of receptor), one of which, .................., is known to be exclusively expressed by microglia in the spinal cord.

nociceptors purinoceptors P2X4

An experiment aimed to block ATP signalling from nociceptors to microglia in the spinal cord. Which receptor could be altered, and describe how they would do this? (2)

P2X4 receptor Knocked down using anti-sense oligonucleotides (asODN)

An experiment aimed to block ATP signalling from nociceptors to microglia in the spinal cord. They used anti-sense oligonucleotides to knock down the P2X4 receptor. Describe the results seen regarding mechanical and thermal hyperalgesia after spinal nerve transection in the following conditions: - baseline (before transection) - mismatch treatment - anti-sense treatment *They measured paw withdrawal thresholds* (3) Draw a conclusion. (1)

Baseline = high paw withdrawal threshold Mismatch = very low paw withdrawal threshold Antisense = higher WT than mismatch but still lower than baseline Blocking neurone/microglial ATP signalling is able to partially prevent mechanical and thermal hyperalgesia evoked by spinal nerve transection.

Which receptor, when blocked, blocks the robust mechanical allodynia produced by fractalkine? (1) How is this receptor blocked? (1)

IL-1 Blocked by an IL-1ra (IL-1 receptor antagonist)

True or false? Explain your answer if necessary. (1) IL-1ra blocks the allodynia seen with fractalkine. IL-1ra is produced naturally in the body, but adding more will block the actions of fractalkine.

True

Suggest how fractalkine signalling may interact with other signalling molecules and cells to contribute to pain. (3)

- Fractalkine activates microglia - And stimulates microglia to produce IL-1, IL-6, and potentially other inflammatory cytokines - These inflammatory cytokines contribute to pain

Fill the gaps relating to how fractalkine contributes to pain. (2) Blocking the actions of IL-1 prevents fractalkine-induced allodynia. The same occurs if the actions of .................. are blocked. This suggests that fractalkine stimulates the release of pro-inflammatory molecules from .....................

IL-6 microglia

Describe a major issue when looking at the results of blocking IL-1 and IL-6 signalling on pain. (4)

Results suggest that blocking these cytokines completely prevents pain. However there are many different factors which contribute to pain and pain sensations. Such as neurone to neurone interactions (LTP, wind up, classical sensitisation); and descending pain modulation (RVM). So it is unlikely that blocking the actions of just one cytokine will completely remove all pain (results may not be valid).

True or false? Explain your answer if necessary. (1) It is not just neuropathic pain and peripheral nerve damage which results in microglial activation - normally functioning, healthy sensory afferents can also activate microglia in the dorsal horn.

True - this was proven by stimulating the sciatic nerve at intensities high enough to activate A and C fibres

An experiment aimed to determine whether normal, healthy sensory afferents could activate microglia in the spinal cord. Briefly describe how this experiment was carried out. (5)

- Sciatic nerve dissected free of surrounding tissue - Electrodes used to deliver currents - Tested at lower currents which just recruited A fibres - And also at higher currents so that all fibres, including C fibres, were recruited - Controls performed to ensure no damage to the nerve took place

- Only if the electrical stimulation is at an intensity whereby C fibres are activated, do microglia in the SC change phenotype - Ipsilateral dorsal horn would show increased levels of activated microglia with C fibre stimulation despite no damage to the neurones - At lower intensities, at which only A fibres are activated, there are no changes in microglia morphology

An experiment aimed to determine whether normal, healthy sensory afferents could activate microglia in the spinal cord. They stimulated the sciatic nerve at intensities sufficient to recruit just A fibres, or to recruit all fibres, including C fibres. A significant increase in IBA-1 immunofluorescence is seen as stimulus intensity increases (showing increased activated microglia). How would the level of immunofluorescence change with minocycline? (1) How would pain behaviour be altered (not with minocycline)? (1)

IF would decrease. Only pain behaviour (lower withdrawal thresholds) on the ipsilateral side is altered (not on contra side or in sham controls).

SNI induces mechanical allodynia in mice. Describe the effect of injecting minocycline or Mac-1 + saporin (SAP) in male and female mice. (2) Hint: both drugs have same effect What conclusion can you draw from this? (1)

Minocycline and SAP reverse allodynia in male mice but not female mice. Females may not rely on microglia to cause allodynia and hyperalgesia like males do - allodynia may be caused by a different mechanism in females.

Where are T cells derived from? (1)

Thymus

T cells are typically subdivided into subclasses, but this is not a hard and fast rule and different nomenclatures are used. Name the three typical subclasses of T cell. (3)

- Helper - Killer - Regulatory

Fill the gaps relating to T cells. (4) T cells are able to differentiate between ...................... and ..................... cells in the body. They are usually ........................., and are not usually found in the ........................

healthy diseased systemic central nervous system

What have gene chip analyses shown us about T lymphocytes following neuropathic injury? (1)

Many markers of T lymphocytes are upregulated in the dorsal horn

True or false? Explain your answer if necessary. (1) T cells are known to increase in the dorsal horn after injury, but they are not thought to contribute to pain.

False - Costigan et al (2009) have shown that these cells can drive neuropathic mechanical hyperalgesia

True or false? Explain your answer if necessary. (1) After neuropathic injury, increased T lymphocytes are only seen in the ipsilateral side of the spinal cord dorsal horn.

False - a larger increase is seen ipsilaterally, but a smaller increase is also seen contralaterally

True or false? Explain your answer if necessary. (1) Neonates do not experience neuropathic pain like adults. Following nerve injury in adults it is thought that T lymphocytes infiltrate the spinal cord, however following nerve injury in babies T lymphocytes are not thought to be upregulated.

True

Briefly describe an experimental technique which could be used to eliminate T cells in mouse models. (1)

Use Rag1 deficient mice (which lack an immune system)

Describe the effect of Rag-1 deficiency on mechanical hypersensitivity after SNI. (1)

Significantly reduced

Fill the gaps relating to T cells and pain. (2) Mice with ........... deficiency, meaning they had no T lymphocytes, developed hyperalgesia to a ................. degree than rats that did have T lymphocytes in tact.

Rag-1 lesser

If microglia do not play as much of a role in female pain as they do in males, which cell type is suspected to take on this role? (1)

T cells

Fill the gaps relating to T cells and pain. (2) Females have a ............... level of T cells in the periphery than males, and .................... levels of T cell markers 7d after SNI.

(2-fold) higher higher

Describe the effects of minocycline, flurocitrate, and propentofyline on allodynia in CD-1 (normal) male and female mice. (2) Hint: all three drugs had the same effect

Male mice = all molecules reverse allodynia Female mice = do not reverse allodynia

Explain why you would expect the molecules: minocycline, flurocitrate, and propentofyline, to produce similar effects on allodynia. (1)

They all inhibit microglia

Describe the effects of minocycline, flurocitrate, and propentofyline on allodynia in nude (no immune response) male and female mice. (2) Hint: all three drugs had the same effect What conclusion can you draw from this? (1)

All drugs reversed allodynia in both males and females. While females preferentially use T cells to cause hypersensitivity, they can engage glial-mediated hypersensitivity in the absence of T cells (males always seem to use glia).

Fill the gaps relating to astrocytes and pain. (2) In much the same way that microglia have increasingly been recognised as being intrinsically involved in pain, so have ...................... Astrocytes are a .................. heterogenous population of cells which subserve numerous functions.

astrocytes highly

How can we show in experimental conditions that astrocytes become activated following neuropathic injury? (1)

Show increased expression of GFAP (glial fibrillary acidic protein)

Describe the morphological change that occurs in astrocytes following neuropathic injury. (2)

Become larger and extend their processes further.

Describe two functional changes that are seen in astrocytes following neuropathic injury. (2)

Reduction of glutamate re-uptake Release of various neuromodulatory molecules

Describe what happens to GFAP levels in the spinal cord after inducing CFA-mediated inflammation in the periphery. (1)

GFAP levels increased (and stayed increased for about a week)

Describe the mechanism by which CFA-mediated inflammation in the periphery is able to alter the morphology of astrocytes in the spinal cord. (2)

CFA in periphery alters activity in sensory neurones which terminate in the spinal cord.

Name a drug which could be used to inhibit astrocytes in pain studies. (1)

Fluorocitrate (FC)

Describe the effect of giving fluorocitrate after CFA on hyperalgesia. (1) Describe the mechanism involved. (1)

Less hyperalgesia in response to CFA. FC inhibits astrocytes to cause less hyperalgesia.

True or false? Explain your answer if necessary. (1) After injury, all immune cell types in the CNS are activated immediately.

False - they are activated at different time points

Which is the first cell type in the CNS to be activated following nerve injury? (1)

Microglia (T cells in females)

At what time point after nerve injury are microglia recruited in the spinal cord? (1)

Very rapidly (in males)

At what time point following nerve injury are T cells activated in the spinal cord? (2)

Very quickly in females. Within 7 days in males.

At what time point following nerve injury are astrocytes activated in the spinal cord? (1)

Around 10 days

Inhibition of microglia is effective at preventing hyperalgesia, but only if inhibition .............................. (1)

precedes injury

True or false? Explain your answer if necessary. (1) Astroglial inhibition is able to reverse pain that has already been established.

True - therefore, astrocytes maintain pain

Are microglia thought to establish or maintain pain? (1)

Establish

Name another system in the body, as well as the immune system (astrocytes and microglia), which contributes to establishment and maintenance of chronic pain. (1)

Descending modulatory systems

True or false? Explain your answer if necessary. (1) Glial-derived cytokines can be antinociceptive as well as pro-nociceptive.

True

Name two anti-hyperalgesic compounds which occur naturally (from glia) and are released in the spinal cord. (2)

IL-10 IL-1ra (IL-1 receptor antagonist)

An experiment aimed to test the effects of injecting anti-inflammatory cytokines on pain. Describe how they did this. (3)

Inject acetic acid into peritoneum measure writhes before and after anti-inflammatory cytokines.

An experiment aimed to test the effects of injecting anti-inflammatory cytokines on pain. Describe the effect of increasing concentrations of IL-4, IL-10, and IL-13 on the number of writhes after peritoneal acetic acid. (3)

All cytokines decreased number of writhes with increasing concentrations.

Describe the temporal pattern of pro- and anti-inflammatory cytokine release from microglia after a painful stimulus. (2)

Pro-inflammatory cytokines would normally be released immediately with anti-inflammatory cytokines being released at the end of the pain period.

Neuroimmune Interactions in Pain Flashcards

(168 cards)