Inflammation and Neuropathic Pain

Question 1

Define pain as defined by the international association for the study of pain.

Answer 1

an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.

Question 2

What is nociceptive pain?

Answer 2

− Nociceptive pain results from the detection of intense or ‘noxious’ stimuli by specialized high-threshold sensory neurons (nociceptors), transduction of this stimulus to an electrical impulse, conduction of action potentials to the spinal cord and onward transmission of the warning signal to the brain where it is perceived.
− It is imperative that the body is aware of potentially damaging stimuli to guard against tissue injury
− So, noxious stimuli are stimuli that threaten to damage tissues of the body.
− Loss of nociception, as in hereditary disorders associated with congenital insensitivity to pain, leads to repeated injury.

Question 3

Describe the organisation of the sensory nervous system

Answer 3

• A sensory axon (part of the peripheral nervous system) has peripheral terminals with the receptors
• The cell body of the sensory axon resides in the dorsal root ganglia
− One axon from the peripheral terminal to the cell body
− One axon projectes from the cell body up to the spinal cord
• In the brain, it will synapse with motor neurons to respond to the stimulus.

Question 4

Describe the 3 forms of sensory neurons

Answer 4

Aa/Ab
Large, 40-80um
Myelinated
Fast, 40-120m/s dude to saltatory conduction
Proprioception → sensing of movement, joints and joint position. Enables us to know where we are in space without having to look
Low threshold mechanoreception – response to mechanical pressure or touch.

Ad	
Small-medium 
15-50um	
Thinly myelinated
Medium, 10-30m/s	
High threshold mechanoreception (higher stimulus)

C	
Small 10-25um	
No myelination
Slow, 0.5-2m/s	Thermoreception
High threshold mechano/thermo/chemoreception
Silent polymodal nociceptors

Question 5

Describe the nociceptors

Answer 5

• Cold → TRPM8
• Heat → TRPV1 & TRPV3
• H+ → TRPV1 & ASIC]
• Mechanical → P2X/P2Y & TRPA1
• Capsaiin in chillies is a TRPV1 agonist – we feel heat when we eat chillies because they activate the heat receptor
• When we have menthol we feel cold because it activates the cold receptor

Question 6

How do nociceptors respond to pain (direct and indirect)

Answer 6

• Peripheral terminals are not alone – there exists around them a variety of other cell types
• In physiological pain, nociceptors respond to acute tissue damaging stimuli either directly through transduction of the stimulus energy by nociceptors, or indirectly through activation of TRP channels on cells such as keratinocytes and/or the release of intermediate molecules such as ATP which in turn act on sensory neuron receptors.
• Sensory afferents are mostly glutaminergic (releasing glutamate) and peptidergic (releasing substance P)

Question 7

Describe the laminar organisation of the spinal cord and primary afferent inputs

Answer 7

• Primary afferents terminate at discrete areas of the spinal cord
• Nociceptors (Ad and C-fibres) terminate right at the top of the dorsal horn (the superficial dorsal horn)
• The faster, myelinated Ab afferents terminate lower down
• Most neurons in the spinal dorsal horn receive inputs from several sensory afferents (wide dynamic range) – respond to brush, touch, tap, pinch, pressure etc…
• Others are nociceptor specific neurons – only respond to noxious stimuli like the pinpricks and pinch

Question 8

What are the 3 types of pain

Answer 8

Acute nociceptive
Inflammatory
Neuropathic

Question 9

Describe acute nociceptive pain

Answer 9

• High threshold, stimulus dependent pain (thermal, mechanical or chemical stimuli)
• Mediated by high-threshold, unmyelinated C-fibres or thinly myelinated Ad fibres feeding into nociceptive pathways of the CNS
• Pain evoked in a graded response by appropriate high intensity (noxious) stimuli
• For nociceptive pain to subserve its protective function, the sensation must be so unpleasant that it cannot be ignored.
• Nociceptive pain occurs in response to noxious stimuli and continues only in the maintained presence of noxious stimuli
• Adaptive, and serves a protective purpose → good pain!

Question 10

Describe inflammatory pain

Answer 10

• Active inflammation – occurs in response to tissue injury and the subsequent inflammatory response.
− TNFa, IL-1B, IL-6 ,NO, Bradykinin etc… released by inflammatory cells activated in response to tissue damage can all activate nociceptors.
• Sensitisation → Heightened sensitivity occurs within the inflamed area and in contiguous noninflamed areas as a result of plasticity in peripheral nociceptors and central nociceptive pathways. Because the pain system after inflammation is sensitized, it no longer acts just as a detector for noxious stimuli but can be activated also by low-threshold innocuous inputs.
− Here we have the involvement of second messenger systems – Isnt a straightfoward transit of information – second messengers can amplify the response à this is known as peripheral sensitisation.
• Adaptive
• Pain evoked by low and high intensity stimuli BUT
• This is protective during healing responses – To aid healing and repair of the injured body part, the sensory nervous system undergoes a profound change in its responsiveness; normally innocuous stimuli now produce pain and responses to noxious stimuli are both exaggerated and prolonged. For example, if you had broken your risk, you may experience pain when your wrist is just lay on a surface – this wouldn’t normall trigger pain, but after tissue injury, innocuous stimuli are painful. It is alerting you that there has been a problem, and not to damage the body more.
• Reversible → typically disappears after resolution of the initial tissue injury. However, in chronic disroders such as RA, the pain persists for as long as inflammation is active.

Question 11

Define neuropathic pain

Answer 11

• Pain initiated or caused by a primary lesion in the nervous system
• Ongoing, non-stimulus dependent pain

Question 12

Name the 6 causes of neuropathic pain with examples.

Answer 12

1. Traumatic → e.g. nerve entrapment, axotomy
   − eg, from carpal tunnel syndrome or amputation.
2. Central → e.g. stroke, spinal cord injury, multiple sclerosis
3. Neurotoxic → eg. microtubule-stabilizing agent (MTSA) induced neuropathy
   − Chemotherapy agents can cause microtubules and peripheral nerves to die back, causing neuropathic pain.
4. Infectious → e.g. HIV-neuropathy and post-herpatic neuralgia
   − Shingles (post herpatic neuralgia) can persist for up to a year
5. Metabolic → e.g. diabetic neuropathy and alcohol-induced neuropathy
6. Idiopathic (unknown cause).

Question 13

Give an example of neuropathic pain.

Answer 13

50% of diabetic patients and ~ 30% of HIV-positive patients will present with some degree of DPN (from mild-chronic)
• Distal axon degeneration and reduced ability of nerves to regenerate.
• Numbness and loss of protective reflexes
• Neuropathic pain with a glove-stocking distribution (arms and legs)
• In diabetic paients, there is a decrease in the number of nerve fibres as you move from thigh to calf. This not only causes numbness, it can cause neuropathic pain. It is hard to treat – most analgesics will not work.

Question 14

What are the features of neuropathic pain?

Answer 14

• Marked neuroimmune component
• Sensititisation → Once neuropathic pain is generated, the sensory hypersensitivity typically persists for prolonged periods, even though the original etiological cause may have long since disappeared, as after nerve trauma. The syndrome can nevertheless progress if the primary disease, such as diabetes mellitus or nerve compression, continues to damage the nervous system.
• Spontaneous and evoked by low and high intensity stimuli → even in the absence of stimuli, patient will report ongoing pain.
• Maladaptive nad persistent
• Abnormal amplification
• Serves no useful purpose
• Not well managed → bad pain!

Question 15

Describe the prevalance for neuropathic pain and potential risk factors

Answer 15

• It is not an inevitable consequence of neural lesions → the pain associated with acute neural damage usually transitions to chronic neuropathic pain in the minority of patients
• For damage of a small neve, risk of persistant pain is 5%, whereas a large nerve such as the sciatic nerve is 30-60%
• Understanding why one individual may develop chronic pain and another doesn’t is crucial to developing strategies to abort such transitions
• Injury such as brachial avulsion during birth does not produce pain in neonates, but produces chronic neuropathic pain in 40% of adults → may depend on the maturity of the nervous system
• Assocaited risk factors for the development include gender, age, anatomic site of injury and severity of pain at the time of event
• Emotional and cognitive factors influence how patients react to chronic pain, but less certain if these contribute to its development.

Question 16

What are the two types of neuropathic pain

Answer 16

• Stimulus-evoked pain:
− Hyperalgesia → enhanced pain evoked by a noxious stimulus –thermal or mechanical
− Allodynia → pain evoked by a normally innocuous stimulus
• Spontaneous pain:
− Often described as burning, tightness accompanied with shooting or stabbing pains.

Question 17

Why are co-morbidities common with neuropathic pain?

Answer 17

− Imagine an excruciating pain every time clothes touch your skin, spontaneous burning that feels like boiling water, bursts of “pins and needles” in your feet when you walk, a continuous crushing pain after an amputation as if your phantom foot is being squeezed, a band of searing pain around your body at the level at which you have lost all sensation after a spinal cord injury.

Question 18

What are the pre-clinical models of neuropathic pain, and how can we assess for them pre-clinically.

Answer 18

Experimental models of neuropathic pain typically involve unilateral injury to rodent peripheral nerve.

1. Chronic constriction injury
2. Full or partial sciatic nerve ligation/crush
3. Spinal nerve ligation
4. Nerve transection (axotomy)

But, there are also animal models of specific neurotoxic, infectious and metabolic neuropathies.
− eg) Streptozocin induced diabetics for diabetic neuropathy, paclitaxol (chemotherapy neuropathies).

Most people with neuropathic pain have specific neuropathies, like diabetes. So models should focus more on this rather than simple models of neuropathic pain.

Assessing pre-clinical models of neuropathic pain:
We can test thresholds using eg):
− Von Frey Filaments (mechanical sensitivity) → if hypersensitive, very fine filaments will be enough to evoke a response.
− measure the existence of pain through behaviors such as withdrawal, licking, immobility, and vocalization.
− the filaments are pieces of nylon rod of varying diameters
− Is a test of mechanical nociception
− The animal stands on an elevated mesh platform, and the filaments are inserted through the mesh to poke the animal’s hindpaw
− Hargreaves IR/hot plate (thermal sensitivity)
− A heat-conductive surface, such as porcelain or metal, is heated to a temperature that will induce a nociceptive response in an animal subject – normally 50–56 °C.[2] The subject is then placed onto the surface and prevented from leaving the platform by blockades. The latency to pain-reflex behavior is measured

These measure stimulus-evoked thresholds.
• Paw withdrawal threshold or latency – test for presence of hypo/hypersensitivity
• Left versus Right thresholds for nerve injury, or vs sham or naïve animal depending on model

• Non-stimulus evoked measures are not easy to quantify in animals, and this is really what we want to understand – the chronic ongoing pain.
• However, you can look at behavioural changes, e.g. thigmotaxis/open field, burrowing, changes in gait, expression), place preference test

Question 19

How do we assess pain clinically?

Answer 19

• Easier to quantify pain in patients than rodents
• Most common method is McGill quesionairre approach
− Rates in terms of feeling, eg) throbbing, shooting, stabbing, sharp, cramping
− Rates each of these as mild, moderate, severe
• Can also use the Wong Baker face scale (hurts a little bit, hurts a little more…)
• Frida Kahlo approach – painted portraits of herself with thorns in her neck to describe her pain

• Functional magnetic resonance imaging (FMRI) use enables blood oxygen level dependent (BOLD) signals to detect changes in cerebral activity in patients with neuropathic pain, and this technique allows the study of responses in particular regions of the brain and brain stem.
• Patients with chronic pain show strong activation of the prefrontal cortex, as well as disruption in resting functional connectivity of widespread cortical areas (Baliki et al. 2008).

Question 20

What are the 4 mechanisms of neuropathic pain?

Answer 20

1. increased inflammatory mediators in the CNS/PNS.
2. Altered nociceptor activity
3. ALtered spinal processing
4. Altered central processing (central inhibition)

Question 21

Describe the role of mast cells in neuropathic pain

Answer 21

• First to be activated

eg) After partial sciatic nerve ligation
• Resident population of mast cells in the peripheral nerve are activated and degranulated at the site of nerve damage
• Release histamine, serotonin, cytokines and proteases
• Histamines seem to be a key mast cell mediators, having sensitized effects on nociceptors and capable of inducing severe burning pain when applied to the skin of patients suffering post-herpatic neuralgia

• The site of primary hyperalgesia is confined to where the injury took place
− Here, tissue damage activates mast cell degranulation
− Histamine binds to receptors on the sensory afferent terminal, sending an action potential
− However, sensory afferents have many collateral branches
− The action potential can travel down the axon, but also down the collateral branch, cuasing substance P release
− This can bind to receptors on mast cells, causing more histamine release
− This can cascade → what started as local injury results in secondary hyperalgesia – the feeling is being perceived in a wide area

• Stabilisation of mast cells by sodium cromoglycate attenuates the development of allodynia
• Treatment with histamine receptor antagonists suppress mechanical allodynia in neuropathic rats

1. Direct receptor mediated excitatory effects of mast cell degranulation:

Histamine
• Usually, recordings from nociceptors are silent, unless activated by a stimulus
• Injection of histamine results in them no longer being silent, they have constant activity
• If a noxious stimulus is introduced, gives an amplified signal → hyperalgesia
• This is long-lasting, even when the histamine is taken away, it doesn’t go back to normal

TNFa
• Rapidly upregulated within hours of nerve injury
• Has direct receptor effects → binds to the TNFR1 on sensory neurons, causing an increase in firing rate, and can increase the conductance of TTX-R sodium channels and ATP receptors (P2X) via p38 MAPK → leads to potentiation of nociceptor activity
• Studies show large increase in firing rate from Ad and C fibre afferents post-injection with TNF
• Anti-TNFa (Etancercept) can reduce sensitivity following in vivo nerve injury model
• Anti-TNFR1 can reduce sensitivity
• Hyperalgesia is induced by TNFa injection

So TNF important – the first wave is from the mast cells, the second from neutrophils

Question 22

Describe the role for neutrophils in neuropathic pain

Answer 22

• Normally, neutrophils aren’t present in nerves
• Following damage, they are the first to infiltrate
• They have a role in phagocytosis
• They increase production of inflammatory mediators, inc. TNF and chemokines, which activate and attract other inflammatory cell types, most notably macrophages → so although neutrophil infiltration is short lived, its effects can be longer lasting through recruitment of macrophages
• Depletion of neutrophils following nerve injury via administration of an anti-neutrophil antibody decreases thermal hyperalgesia

Question 23

Describe the role for macrophages in neuropathic pain

Answer 23

• Macrophage activation is a central component of Wallerian degeneration distal to axonal injury
• Wallerian degeneration is a process that results when a nerve fibre is damaged, in which part of the axon separated from the neuron cell body degenerates itself distal to the injury
• This is also known as anterograde degeneration
• 2-3 days after nerve injury, resident macrophages infiltrate to the injury site → orchestrated by CCL2 & CCL3 acting on CCR2, CCR1 and CCR4
• They function to phagocytose damage tissue and increase production of inflammatory mediators, eg) IL-1B
• Reducing the number of circulating monocytes via IV liposome encapsulated ciodronoate reduces hypersensitivity

Question 24

Describe other peripheral inflammatory reactions to nerve lesions (MMPs, neuregulin & ERBB2, NGF and GDNF etc..)

Answer 24

• Activated macrophages and denervated Schwann cells secrete MMPs that attack the ECM → leads to an interruption of the BBB
• Vasoactive mediators including CGRP, substance P, bradykinin and nitric oxide are released from injured axons to cause hyperemia and swelling. These vascular changes support the invasion of circulating immune cells
• Within minutes of a nerve lesion, neuregulin, a growth and differentiation factor present on the axonal membrane, induces activation of the tyrosine kinase receptor ERBB2, which is constitutively expressed on Schwann cells causing demyelination. Later in the course of wallerian degeneration, ERBB2 upregulation and activation is associated with Schwann cell proliferation.
• In the reverse direction, Schwann cells release chemical signals that promote axonal growth and remyelination13. These include NGF and GDNF. NGF and GDNF also directly activate and sensitize nociceptors, contributing to the initiation of pain in response to nerve injury - this can result in persistent pain.

Question 25

Describe altered nociceptor activity as a mechanism of neuropathy

Answer 25

Altered sodium channel activity in neuropathic pain:
• Voltage-gated sodium channel genes are upregulated in injured sensory neurons –↑ excitability/ectopic firing.
• Activity can be modulated
• Important in pain? YES
• Channelopathies:
• Paroxysmal extreme pain disorder is caused by gain-of-function mutation in voltage-gated sodium channel
• Rare mutations of the Nav1.7 gene causes a congenital insensitivity or indifference to pain.
• Pharmacological blockade of sodium channels provide pain relief, e.g.
• Carbamazepine: trigeminal neuralgia.
• Lamotrigine: HIV sensory neuropathy and central poststroke pain.
• Topical lidocaine: PHN.

Question 26

Describe altered spinal processing (spinal hyperexciteability) and descending inhibition as a mechanism for neuropathy

Answer 26

• Activation of the C-fibre (blue) by immune mediators in the periphery leads to massive release of glutamate.
• There are lots of glutamate receptors on the neurons in the DRG. If we get sustained glutamate release, we can begin to activate the NMDA receptors and activation of the nociceptive specific neuron (yellow).
• We also have Ab fibres (red) – the large myelinated, fast conducting fibres. These can connect in via sprouting. The result of this is that Ab fibres that normally finish further down, put out collateral branches and may terminate further up. As a result, stimulation of an Ab fibre can directly activate a nociceptive fibre through synaptic sprouting/reorganisation.
− We know allodynia is a low threshold, innocuous stimulus suddenlyy being perceived as painful. This may be because we have a network were Ab fibres are activating the nociceptive fibres.
− AB fibres can also help dampen down pain response by overloading the network with non-painful stimuli (Eg, when you bang your hand, you rub it – overloading with the non-nociceptive stimuli, and overloading the Ab fibres).
• The other way is that there is also input from Ab fibres via interneurons. These can be excitatory (glutaminergic) or inhibitory (gabaminergic). If inhibitory, this would be good – it could provide a way to dampen down the response.
• We also have descending neurons from higher centres, these secrete neurotransmitters such as noradrenaline and are inhibitory (descending inhibition). So we have a network by which we try to control spinal hyperexciteability.

Question 27

Describe the role of microglial activation in spinal hyperexciteability

Answer 27

• Spinal microglial activation in both dorsal and ventral horns peaks 1 week after injury, followed by a slow decline over several weeks.
- Pathways recruiting microglia involve the chemokine fractalkine (CX3CL1) signalling through CX3CR1, and CCL2 signalling through CCR2 and TLRs.
- Intrathecal injection of fractalkine produces mechanical allodynia and thermal hyperalgesia (increases spinal exciteability), whereas administration of a neutralizing antibody against CX3CR1 delays the development of mechanical allodynia.
• CCL2 potentiates glutaminergic signalling. Injection of CCL2 or the transgenic over expression of CCL2 increases the nociceptive response.
• Mice lacking CCR2 show substantially less hypersensitivity to mechanical stimulation after partial sciatic nerve ligation
-A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Microglial cells express P2X and P2Y receptors and ATP is a potent stimulator of microglia in vitro.
• When injected intrathecally, ATP provokes sustained mechanical hypersensitivity in the rat.
• Potentially, ATP is actively released from injured primary afferents
- Microglial activation increses synthesis of IL-1B, TNF, IL-6, IL-10. IL-1 directly activates nociceptors, and transactivate Trpv1.
-Chemokines stimulate Ca2+ release, so are directly excitatory.

Question 28

Describe the pain processing pathways in the brain

Answer 28

• Pain fibres synapse in the spinal cord, and cross the midline in the spinal cord. They then go up to the thalamus, and then up to the somatosensory cortex.
• The larger, myleinated proprioceptive fibres send collateral afferents up to the medulla, and cross over in the medulla. Again, they then pass through the thalamus and up to the cortex.
• The somatosensory cortex is the sensory-discriiminative component. It is here that the painful stimulus is identified according to location, time and intensity, and decides what action should be taken.

However, this isn’t the only story.
• We can also have transmission to the hypothalamic and limbic system, and this gives us the behavioural and emotional responses to pain.
• Remember, we also do have descending inhibition – detecting pain may increase output down to the spinal cord to try and dampen down the spinal exciteablity.
• However, PET and FMRI studies reveal many regions are activated by pain, and this has been termed the ‘pain matrix’. There isn’t just one pain centre.

A multitude of factors can either amplify or attenuate the pain experience, eg:
• Context → pain beliefs, expectation, placebo (many drugs for pain don’t get approved because of placebo effect, just the thought that things might get better).
• Mood → depression, catastrophorizing, anxiety
• Cognitive set → hypervigilance, attention, distraction
• Chemical and structure → neurodegeneration, metabolic, maladaptivit plasticity

Question 29

As well as inflammatory cytokines leading to spinal hyperexcitability, what is the other factor contributing to neuropathic pain?

Answer 29

Endogenous inhibitory pain modulating pathways are reduced in neuropathic pain:
Spinal cord:
• Inhibitory interneurons (GABAergic and glycinergic) presynaptically modulate afferent input

Brain:
• Descending inhibitory pathways originate in the anterior cingulate gyrus, amygdala, and hypothalamus and are relayed to the spinal cord
• Inhibitory neurotransmitters include NA, 5-HT, and opioids.

• Melzack and Wall (1965) proposed the ‘Gate control theory of pain’ – a balance of large Ab and small C fibre activity. You can get pain relief by increasing Ab input (as discussed before).

Question 30

What are the genetic risks for developing neuropathic pain?

Answer 30

• Because many causes of neural damage associated with neuropathic pain are sporadic, it is rarely possible to rely on family history and classic genetic techniques to evaluate the degree to which genetics is involved
• However, two recent twin studies using experimental (nociceptive) pain models in healthy volunteers have estimated the impact of inherited heritability of pain sensitivity with a broad range 20%-60%.
• In humans, the genetic risk of developing neuropathic pain likely to result from multiple risk-conferring genes.
• Candidate gene association studies have preliminarly identified polymorphisms in catecholO-methyltransferase (COMT) that modulate nociceptive and dysfunctional (temporomandibular joint disorder) pain. COMT is an enzyme in the metabolism cascade for dopamine, norepinephrine, and epinephrine. Higher COMT activity leads to lower transmitter and pain levels.

Question 31

Treatment for neuropathic pain

Answer 31

• analgesics - for neuropathic pain aim to reduce the excitability of neurons in the peripheral nervous system or the CNS by modulating the activity of ion channels (gabapentin, pregabalin, carbamazepine, lidocaine and capsaicin) or by mimicking and enhancing endogenous inhibitory mechanism (tricyclic antidepressants, duloxetine and opioids).
• Stepwise management of TCAs, SNRIs, Opiods, Cannabinoids….

Preclinical studies have explored several routes of immune and glial modulation84. Global inhibitors of glial metabolism reduce cytokine release and attenuate pain-responsive behavior in several animal models of neuropathic pain:
• Fluorocitrate inhibits aconitase, blocking the citric acid cycle.
• Propentofylline reduces proliferation and activity of microglia and astroyctes by inhibiting extracellular adenosine transporters, which increases cAMP.

More targeted interventions have been aimed at purinoceptors, cannabinoid receptors, MAP kinases, TNF and interleukins.
• Recently developed selective antagonists of P2RX2 and P2RX3 reduce spontaneous discharges and evoked responses of primary sensory neurons, decrease cytokine release and attenuate mechanical hypersensitivity after nerve injury.
• Etanercept, a soluble TNF receptor fusion protein, and anakinra, a recombinant form of human IL-1 receptor antagonist, have been tested in animal models of peripheral nerve injury and reduce neuropathic pain-like behaviour.