J.D. Flashcards
What is the ultimate relevance/aim of classifying nociceptors?
Possibility of selectively targeting subpopulations
If separate populations that deliver acute protective pain from those that deliver central sensitisation & chronic disabling pain then may allow people to function without risk of hurting themselves
If you stimulate a whole nerve what fibres make up the AP?
First = large & fast (Aα [efferent] and Aβ neurons)
Small blip after = Aδ
After = C fibres
Which nerves do adaptation and sensitisation occur in?
Adaptation occurs in NON-NOCICEPTORs e.g. wooly jumper = adaption in low threshold mechanoreceptor
Sensitisation occurs in SOME nociceptors: can be injury (cuts/burns) or chemical
- can be primary sensitisation or secondary sensitisation, and can cause primary hyperalgesia & secondary hyperalgesia
What is the effect of capsaicin?
Agonist at TRPV1
Doesn’t necessarily activate all afferents - some biologists think TRPV1 is a ‘nociceptive marker’ i.e. marks all the nociceptors - this is an oversimiplification
What is primary hyperalgesia / how is it mediated?
Enhanced response to HEAT & MECHANICAL stimuli
Mediated by sensitisation of PRIMARY AFFERENTs (primary sensitisation)
What is secondary hyperalgesia / how is it mediated?
Enhanced response to MECHANICAL stimuli (mechanical allodynia + punctate mechanical hyperalgesia)
Initiated by primary afferent nociceptors, leading to sensitisation of spinal neurons (interneuron or projection neuron) - central sensitisation
Sensitised CENTRAL neurons amplifies the response from both NON NOCICEPTIVE afferents and NOCICEPTIVE afferents
What happens when you inject capsaicin? Different zones on skin
Algesic bleb: development of sensory distubance
Area of primary hyperalgesia (increased pain to heat and mechanical stimuli) via primary sensitisation
Flare (around primary area): mediated by class of nociceptors with efferent function (release substance P, cGRP - vascular changes & hyperaemia, seen as flare + temperature increase)
Outside flare: secondary area of MECHANICAL ALLODYNIA (mediated by low-threshold, non-nociceptive afferents) & PUNCTATE MECHANICAL HYPERALGESIA (mediated by nociceptive afferents passing impulse to sensitised spinal neurons)
What is teased fibre?
Anaesthetise rat, expose the saphenous nerve - can tease fine filaments of the nerve away from the rest of nerve, hang on recording electrodes & start to record single unit activity
Record extracellular action potentials arising from 1-3 primary afferent neurons
Stimulate receptive field e.g. thermal/electrical, or can stimulate whole nerve nearer to the teased filaments
If stimulate in periphery than can use distance between stimulating electrode and recording electrode to calculate latency / speed of conduction (e.g. A vs C)
Teased fibre - advantages and disadvantages
✔︎ Very stable recording, good for classifying afferents and finding out how they respond (but can’t be taken much further)
✔︎ Big signal:noise ratio (very clean trace)
╳ no measure of perception (but in rats, can only do this through behaviour anyway)
╳ cut central terminal - degree of nerve injury has occurred
╳ cannot easily relate unit you are recording from to a particular cell body within the DRG, so ability to apply molecular biology techniques or genetics is very limited
╳ ethical considerations - terminally anaesthetised rats
What is microneurography? What are the advantages and disadvantages?
Introduce fine micro-electrode into nerve body itself - again record extracellular action potentials
✔︎ Can be done in humans
╳ can’t physically separate units - always get multiunit activity
╳ signal to noise ratio is low (lower quality of recording)
Low quality has led to development of the marking technique..
What is the marking technique?
C fibres exhibit activity-dependent slowing (when stimulated, their conduction velocity slows down)
- If stimulating nociceptor, then have some physical stimulation e.g. thermal stimulus, mechanical stimulus - see the slow latency is slowed even further, then comes back to normal when thermal/mechanical stimulation is removed: *this indicates that the unit you have stimulated, is the unit you have recorded from
How can selective blocks & selective activation be used?
Manipulating populations of afferents to infer whether they are involved in what you are studying
1) Compression to cutaneous branch of radial nerve: sensory changes to skin: first lose input from large myelinated A fibres
- → thinner, lightly myelinated A delta fibres → C fibres
2) Lidocaine/LA: blocks smaller afferents before they block larger afferents (C fibres → A delta → A beta)
Temporal method of segregating conduction velocity classes
3) Capsaicin: small dose = selective activation of subpopulation, high dose = selective desensitisation/ablation of subpopulation (clinically Quitenza 8% - used on allodynia patches in NP pain)
Broadly - what techniques should be brought together to get a global picture of nociception from molecular level to spinal pathways?
Psychophysics: multi fibre / multi level
Selective blocks: separate fibre populations
Electrophysiology: single fibres (teased fibre & microneurography with marking technique)
What is the difference between volar forearm & palm?
VF: thinner, different nociceptor population, hairy
Palm: densely innervated by low threshold afferents, glabrous (not hairy)
What kind of experiment was used to distinguish between fibre types in hairy/glabrous skin?
Psychophysics (Campbell + LaMotte): CO2 laser thermal stimulus, asked what they felt
Two peaks of response (latency vs number of responses: very hot temps (48), clear and quick pain response, lower temps (40)- slower onset pain response.
In between temps (42-45?) = two distinct sensations
First heat pain: clear quick response (first heat pain): well-localised, sharp, pricking
Second heat pain: slower onset, less well localised, burn/ache
Temporal distinction fits with faster & slower conducting fibres, and with different spinal circuits (discriminatory vs general discomfort)
What do A fibres respond to?
Most to innocous stimuli
Most AB = tactile afferents
SOME A delta = thermoreceptors & low threshold non-nociceptive mechanoreceptors
(note - it is not a case of C = nociception and A = not, also not a case of ad = nociceptive and ab = not)
What sensations do A fibre nociceptors serve?
- First / fast heat pain in hairy skin
- Fast / sharp mechanical pain
Pathology = PRIMARY heat hyperalgesia in HAIRY skin & TRANSMISSION of PUNCTATE MECHANICAL HYPERALGESIA (onto sensitised spinal neurons)
How do we know what purpose A fibres serve?
Treede et al. teased fibre single fibre recording in monkeys: looked at A and C fibres in hairy and glabrous skin
Laser, quick onset heat simulus: subcut and surface temp increased - frequency histogram against timecourse of stimulus - showed two types of AMH fibre (A mechano-heat nociceptors) response in HAIRY skin:
- Type II AMH (heat sensitive)
- Type I AMH (heat ‘insensitive’)
In glabrous skin, only type I response found* - controversy
Based on Treede’s work - what are the characteristics of the AMH Type II fibres?
Fire quickly at pain onset, then activity diminishes (little bit of baseline activity then drop off even with stimulus)
- Slower conduction velocity (CV) = 15m/s
- Lower heat threshold = 46°C (easier to activate)
- ‘Higher’ mechanical threshold
- Fast utilisation time
- Not seen in glabrous skin? (CONTROVERSY - depends on experimental design)
Based on Treede’s work - what are the characteristics of the AMH Type I fibres?
At onset of temp ramp, not much activity, then slowly builds then drops off
- Faster CV = 25m/s (like Aβ nociceptor range)
- Higher heat threshold >53°C
- ‘Lower’ mechanical threshold (easier to activate)
- Slow utilisation time
Why is there controversy about whether Type II AMH are found in glabrous skin (& whether first heat pain exists in glabrous skin)
Growing consensus within psychophysics: 1st heat pain subserved by A fibres & the only population that could carry the sensation were type II AMH, as they fire early at stimulus onset
But: type II not very abundant & varies with experimental design
Mechanical search stimulus - (when recording APs, forceps in anaesthetised monkey to find mechanoreceptors - found type II AMH with expected properties but not many - how can such a sparse population perform such an important role?
Then: electrical search stimulus (tickle toe - find receptive field, then electrical stimulus to see if any slow conducting potential nociceptors in field)
What did Treede et al’s (1998) work with electrical stimulation show about AMHs?
76% of Type II AMH were mechanically insensitive (MIA) (therefore, those they found had very high mechanical thresholds)
58% of Type I AMH were mechanically sensitive (MSA) (remaining 40% had high temperature thresholds)
This shows that 2/3rds of the nociceptors within the A delta fibres were missed in the first set of experiments!?
How do A fibres behave after injury?
Type I AMH: sensitised to heat by burn injury
Type II AMH: DESENSITISED to heat by burn injury (heat threshold goes up)
How do we know which pathological process is brought about by Type I AMH?
Meyer and Campbell 1981: Psychophysics + neural recordings at glabrous skin (thenar eminence)
Apply thermal stimulus & report pain increases as temp increases. Then apply burn - pain increases disproportionately & starts earlier
In glabrous skin after burn injury: C fibre responses go down (desensitisation in glabrous skin), A fibre responses increase (sensitisation in glabrous skin)
We know sensitisation is mediated by A fibres, and type I A fibres exhibit sensation but type II fibres do not exhibit this sensation - therefore: Type I AMH mediate PRIMARY HEAT HYPERALGESIA in GLABROUS skin
What is the effect of capsaicin on A fibres?
Capsaicin injected then needle withdrawn: MIA (~type II) desensitised by capsaicin, MSA (~type I) have small response but not much
MSA = AMH Type I
MIA = AMH Type II
(mechanical vs thermal classification)
Therefore, AMH type II are CAPSAICIN SENSITIVE
(rough approximation that Type I = predominantely capsaicin insensitive, Type II = predominantly capsaicin sensitive)
How do we know which fibres mediate pin prick pain?
Magerl et al (2001)
Vehicle injection = standard stimulus response pain. Then apply A fibre compression block: perception of pin prick dramatically drops (over 80%): therefore A fibres mediate pin prick pain in acute setting (little bit must also be C fibre)
Capsaicin desensitises subpopulation of A-fibres: therefore add desensitising dose & repeat: percept of pin prick pain diminishes but not by very much
- PINPRICK pain is mediated by CAPSAICIN-INSENSITIVE A-fibre nociceptors (AMH I)
Then put A fibre compression back on - sensation drops off to normal (whatever C fibre population was also involved, is also capsaicin sensitive(?))
How do we know which fibres transmit punctate secondary hyperalgesia?
AMH I
Capsaicin desensitisation combined with model of capsaicin hyperalgesia
Day 1: apply high dose capsaicin (desensitise the capsaicin responsive afferents within rectangle)
Day 2: sensitising dose of capsaicin and put between capsaicin desensitised space and space containing vehicle
FLARE completely obliterated in capsaicin pretreated area: therefore flare mediated efferent activity of nociceptors, which is MEDIATED by CAPSAICIN-SENSITIVE population
Punctate area of hyperalgesia intact after the capsaicin desensitisation (symmetrical around the capsaicin injection)
Therefore: punctate secondary hyperalgesia is mediated by an A fibre population, and a population which is caspasicin-INSENSITIVE
Punctate secondary hyperalgesia transmitted by AMH type I
What do C fibres respond to?
Not all nociceptors
Some are innocuous cool & tactile afferents (positive/bonding)
often experiments look at small diameter fibres as assumed to be nociceptive as assumed to contain C fibres - oversimplification
What sensations do C-fibre nociceptors subserve
Second burning heat pain
Minimal role in acute pain
PRIMARY HEAT hyperalgesia in HAIRY skin
MECHANICAL hyperalgesia in JOINT
TONIC MECHANICAL pain in skin (sensitisation)
Axon flare
Ongoing/spontaneous pain
Sensitisation of spinal neurons
When were C-MiHi fibres discovered?
Perl 1969: Electrophysiology: cat saphenous nerve data
Were called unclassified or excitable - now understood better as better able to study them: C-fibre silent nociceptors / C-fibre mechanically / heat insensitive (C-MiHi)
Also found that C-MH fibres were POLYMODAL
Which fibres are responsible for acute heat pain in glabrous skin? (dull, burning)
How do we know?
C-MH. 1989 - some of first uses of human microneurography
Median nerve - inserted very fine (0.2mm diameter) electrode
Occasionally, stimulating specific points caused heat & burning sensation, then passed very small current through same electrode (that recorded APs) - same sensations elicited
Intraneural stimulation + conduction velocity stimulation of receptive field to characterise that C polymodals responsible for ACUTE HEAT PAIN in palm (but the diffuse SECOND PAIN, not the first, well localised heat pain)
Which fibres are responsible for primary heat hyperalgesia in hairy skin? (dull, burning)
How do we know?
Hairy skin; C-MH neurons SENSITISE (temperature threshold decreases with recurrent stimulation)
Therefore C-MH responsible for primary heat hyperalgesia in hairy skin (but not in glabrous skin)
- Remember: Mayer and Campbell (1981) found that C fibres DESENSITISE in response to burn injury in GLABROUS skin, therefore this contrasts with hairy skin where they sensitises
- If step up heat stimuli & give repeated heat stimuli; APs generated for any given stimuli increase, but also the temperature at which the unit begins to activate also decreases.response curve shift to lower temperatures and response is elevated (classic sensitisation)
Overall what are C-MH / polymodal nociceptors responsible for
Acute heat pain in glabrous skin
Primary heat hyperalgesia in hairy skin
Overall what are C-Mi nociceptors responsible for?
Tonic mechanical pain (?skin)
Contribute to mechanical sensitisation in inflammed joint
Mediate the axon reflex in humans (different to rats)
Difference between studying human a and c fibres?
Lots of electrophysiology on A fibres has come from cats/rodents/monkeys: microneurography: difficult to find A delta fibres in microneurography
In contrast, lots of C nociceptor data in humans (mostly using marking technique)
Role of CMi nociceptors in cats/rats?
Cat joint / rat joint (don’t know in humans yet) - population of nociceptors that don’t respond to anything in normal conditions, but activated when joint is insulted (inflammation/pro-algesic) - not just ongoing activity but also develop sensitivity to mechanical stimuli & sometimes also heat stimuli
Sleeping nociceptors - sentient, waiting to be triggered?
- Some evidence to suggest that they may be the afferents that have some efferent activity, leading to vascular response/flare/temperature increase
What are C-Mi nociceptors?
First alluded to by Bessau and Perl in late 60s
Best characterised in the joint (See Schiable HG)
Now also recognised to play crucial role in cutaneous pain
silent/sleeping nociceptors, CMi/CMiHi
What did Schiable & Schmidt find about CMi?
Cat joint: backfired teased fibre experiments: saw nociceptors in bundles but couldn’t excite them
Kaolin injection (irritant clay causes robust inflammation) - start to get bit of activity from same stimulus & 2 hours later, really robust response
Mechanical sensitisation: but different to previous because there was no activity originally
Then collected data from different arthritis models: compared frequency at which afferents were found that had ongoing activity/sensitivity to mechanical stimuli
In controls: 1/4-1/3 of units, couldn’t excite by mechanical activity at all, and only small number of these showed any ongoing activity
In arthritis models: enormous increase in units with ongoing activity (relationship of recruiting more afferents as noxious stimulus increased completely reversed - after inflammation, lots of units now have ongoing activity and shifted large population from high threshold to almost ongoing activity to non-noxious stimuli)
Loss of ‘insensitive’ afferents in inflammation i.e. they have been activated
How did Weidner et al (1999) identify CMis?
Tracking cutaneous C fibres in humans, via electrical stimulation: (poor signal to noise ratio - APs tiny & not necessarily recognisable - but by repeatedly stimulating and getting constant latencies - can say with some confidence they are APs)
Stimulating afferent slowly shows most units have constant latency (staying at constant conduction velocity)
Most units show SLOWING to nociceptive MECHANICAL stimulation, but one unit does not does NOT- instead has profound slowing with HIGH FREQUENCY (ELECTRICAL) stimulation - this is less so for the mechano-nociceptors
Increase the frequency of the electrical stimulus, the conduction velocity slows, then when back down, recovers slightly but still slow
Different profiles of activity dependent slowing: tied to underlie physiological characteristics of the units (receptive field characterisation)
C fibre which is mechanically insensitive* by showing excessive slowing to intermittent electrical stimuli - can identify the unit without having to apply any stimuli to it
How did Serra et al (2004) use marking technique to distinguish CMH from CMi?
0.25Hz stimulus then break, then 0.25KHz stimulus, then 2Hz stimulus - get characteristic ‘sharks tooth’ changes in latency
Then apply increasing increments of mechanical stimulation, then see recruitment of mechanically sensitive CMH nociceptors (C polymodals)
But don’t get any change in the response latency of the CMi nociceptor
How do we know that CMis encode tonic pressure?
Microneurography: record afferent activity in humans & then use psychophysics - huge advantage of microneurography
Increased pain rating after 2nd stimulus (2nd stimulus feels worse, therefore sensitisation occurring), however, the candidate afferent is not showing sensitisation (C polymodal) - exactly the same response for stimulus 1 and stimulus 2
CMi: no real response to mech stimulation in 1st stimulus, but second response shows increase in activity in response to mechanical stimulation that matches the psychophysics
- Therefore CMi may be afferent population underlying the INCREASE in PAIN after a PUNCTATE MECHANICAL STIMULUS
How do we know that CMi have large receptive fields?
Schmidt et al 2002
Find them by transcutaneous electrical stimulation (cannot search for them with classic mechanical stimulation)
Human skin: low currents used to elicit their activity (10-20mA)- this is about the pain threshold- when they increased the currents being used to stimulate the CMi fibres, the receptive fields (area of skin that was excited) increased and increased (this contrasts CMHs)
When sensitised CMi afferents (using capsaicin) - large receptive field that could be excited by electrical stimuli could also be excited by physical stimuli when in the inflamed state
Therefore, CMH have very small receptive fields (couple mm diameter) & CMi have large receptive fields with large arborisations
Important: e.g. joint pain usually achy debilitating pain - very difficult to get rid of- if have large unit with wide arborisation won’t give good info about spatial distribution of the stimulus
- therefore these units may be responsible not for sensory-discriminative pain but more for learning / negative affect associated with pain
How do we know that CMi have large receptive fields?
Schmidt et al 2002
Find them by transcutaneous electrical stimulation (cannot search for them with classic mechanical stimulation)
Human skin: low currents used to elicit their activity (10-20mA)- this is about the pain threshold- when they increased the currents being used to stimulate the CMi fibres, the receptive fields (area of skin that was excited) increased and increased (this contrasts CMHs)
When sensitised CMi afferents (using capsaicin) - large receptive field that could be excited by electrical stimuli could also be excited by physical stimuli when in the inflamed state
Therefore, CMH have very small receptive fields (couple mm diameter) & CMi have large receptive fields with large arborisations
Important: e.g. joint pain usually achy debilitating pain - very difficult to get rid of- if have large unit with wide arborisation won’t give good info about spatial distribution of the stimulus- therefore these units may be responsible not for sensory-discriminative pain but more for learning / negative affect associated with pain
How do we know CMi responsible for axon flare?
Transcutaneous electrical evoked stimulation in volunteers
C nociceptors have higher electrical threshold - stimulate skin with current that we know elicits pain and activity in CMH -
even at high frequency, this does not produce a flare response, even though increasing current
Once start to get to current that excites CMi nociceptors, then start to elicit flare as well
*Flare poorly localised / diffuse therefore likely to be caused by unit with wide arborisation
How do we know CMi are capsaicin sensitive?
CMi are capsaicin sensitive
The mechanically sensitive A fibres were particularly capsaicin sensitive - same seen in CMi
Inject capsaicin, quite far away from centre of receptive field of unit (12mm), still see spontaneous ongoing activity
- have activity dependent slowing, but spontaneous activity evoked by capsaicin also evokes these changes in latency
- wide arborisation picks up insult of capsaicin & leading to ongoing activity
Capsaicin also initiates mechanical sensitivity in CMi:
so, similar to A fibres, capsaicin initiates ongoing CMi activity and initiates mechanical sensitisation
Role of CMi in hyperalgesia?
LaMotte 1991
LaMotte (1991): injected caps under LA (proximal nerve block, away from experimental site)
Without LA: primary and secondary hyperalgesia developed
With LA: Caps still evokes flare (suggesting that flare is not a peripheral response mediated by primary afferents)
With LA: Caps does NOT cause punctate mechanical hyperalgesia (secondary)- no 2dary hyperalgesia in the area outside the block
Evidence against CMi mechanical sensitisation (i.e. primary sensitisation) underlying secondary punctate mechanical hyperalgesia
Ongoing capsaicin sensitive C fibre input may be required to initiate the central sensitisation (in spinal cord) that, with input from A fibre nociceptors, gives rise to punctate secondary mechanical hyperalgesia
Controversy - speculation that CMi are well placed take the message in from periphery into spinal cord, sensitise spinal neurons, and the spinal neurons are activated by the A fibre nociceptors to give rise to the percept of punctate mechanical hyperalgesia
Punctate mechanical hyperalgesia carried by A fibres. Now: we think CMi may the fibres that sensitise these afferents. Why do we think this?
- CMi have large arborisations (survey large areas of the skin)
- When activated, give flare response (that characterises intradermal capsaicin model)
- When activated by caps, develop ongoing activity (firing APs at 1-2Hz, transmitting via glutamate, SP, CGRP - setting up some process within sensitised spinal neuron causing it to amplifies the input from a fibres)
This requires input from the periphery - because of LaMotte et al (1991) - blocking neural transmission leaves the flare response but ablates the secondary hyperalgesia response that you would expect after LA wares off
- therefore CMi might be the input that is required for the initiation of mechanical secondary hyperalgesia
What inherited disease does CMi fibres have possible role in?
Erythromyalgia
Microneurography on patients: units that have CMi pattern (slowing profile) show mechanical sensitivity - absent in healthy patients (also occasional spontaneous activity that changes their latency)
Suggests CMi afferents are sensitised by this disease state - (respond to mechanical stimuli)
Erythromyalgia = NaV1.7 gain of function mutation, therefore fits idea that CMi driving ongoing pain in some peripheral neuropathies & because they have ongoing activity, they set up spinal / supra spinal loops involved in sensitisation in very negative affect
Role of CMi in peripheral neuropathies?
Patients with peripheral neuropathies - tend to have easily activated & spontaneously active CMis but less pronounced changes in CMH
Not just split between those with and without disease, also people with neuropathies who do and don’t have pain
(pain - more active CMi)
Presence of spontaneous activity & sensitisation within the CMi silent nociceptors: gives rise to phenotype with ongoing burning pain that cannot treat well?
Pain patients summary?
- Abnormalities demonstrated in patients with chronic pain
- NB: large proportion of chronic pain patients are not represented (these are small fibre neuropathies)
- Recurrent theme = C fibre nociceptors become sensitised, develop ongoing activity
- This has intuitive implications for hyperalgesia and ongoing pain
- If those units with ongoing activity are required/responsible for initiating central sensitisation, these findings are also important in the context of allodynia
