Nerve Injury, Wallarian Degeneration, and Primary Mediators

Following injury, Wallerian degeneration of severed axons is associated with neutrophil, macrophage, and T-lymphocyte invasion as well as activation of Schwann cells, fibroblasts, mast cells, keratinocytes, and epithelial cells. ^{Reference Scholz and Woolf14–Reference Marais, Light, Mason, Paterson, Olson and Marshall16} Once activated, these immunocompetent cells generate and release pro-inflammatory primary mediators. These include tumor necrosis factor (TNF-α), ^{Reference Scholz and Woolf14,Reference Leung and Cahill17} interleukins 1β,15,17 and 18 (IL-1β, IL-15, IL-17, and IL-18), ^{Reference Scholz and Woolf14,Reference Boakye, Tang and Smith18–Reference Kleinschnitz, Hofstetter, Meuth, Braeuninger, Sommer and Stoll21} nerve growth factor, ^{Reference Scholz and Woolf14,Reference Pezet and McMahon22} monocyte chemoattractant protein 1 (MCP-1/CCL-2), ^{Reference White and Wilson23} chemokine (C-X-C motif) ligands 1 (CXCL-1) ^{Reference Scholz and Woolf14,Reference Silva, Lopes, Guimaraes and Cunha24} and 12 (CXCL-12), ^{Reference Yu, Huang, Di, Qu and Fan25} histamine, prostaglandins, serotonin, and substance P ^{Reference Scholz and Woolf14,Reference Kaur, Singh and Jaggi26,Reference ObaraI Telezhkin, Alrashdi and Chazot27} as well as the secreted glycoproteins Wnt3a (wingless-type mammary tumor virus integration site family member 3a) and Wnt5a. ^{Reference van Vliet, Lee and van der Poel28}

Structural Remodeling of Injured Peripheral Nerves

Following SNI of rodent peripheral nerves, degeneration of the axons of low threshold non-nociceptive afferents can lead to loss of sensation. Peripheral nociceptors then sprout into territories that were previously occupied by low threshold afferents. These nociceptors are transformed to exhibit a low activation threshold so that mild tactile stimulation now produces mechanical allodynia. ^{Reference Gangadharan, Zheng and Tabener29}

In many cases, injury also provokes the sprouting of perivascular sympathetic fibers so that they interact and excite sensory nerve terminals and DRG cell bodies. ^{Reference McLachlan, Janig and Michalis30,Reference Yen, Bennett and Ribeiro-da-Silva31} These processes are especially relevant to the etiology of complex regional pain syndrome II. ^{Reference Drummond, Drummond and Dawson32}

Injury-Induced Peripheral Sensitization, the Importance of Spontaneous Activity, and the Generation of Secondary Mediators

Immune cell-derived primary mediators sensitize peripheral nerve endings, axons, and cell bodies of primary afferents. ^{Reference Scholz and Woolf14} Mediators also promote plasma extravasation and increase the permeability of the blood–brain barrier ^{Reference Xanthos, Pungel, Wunderbaldinger and Sandkuhler33} and the blood–nerve barrier in the periphery. ^{Reference Lim, Shi and Martin34} This and the chemoattractant profiles of various mediators facilitate the recruitment of immunocompetent leucocytes and lymphocytes to the site of injury. ^{Reference Moalem and Tracey15,Reference Gomez-Nicola, Valle-Argos, Suardiaz, Taylor and Nieto-Sampedro20} These myeloid and lymphoid cells themselves release a host of cytokines and chemokines thereby instigating a positive feedback process in the initiation and maintenance of neuroinflammation and pain. Neuroinflammation is defined as activation of the brain’s innate immune system in response to an inflammatory challenge. ^{Reference DiSabato, Quan and Godbout35,Reference Milatovic, Zaja-Milatovic, Breyer, Aschner, Montine and Gupta36}

Satellite glial cells and resident macrophages in DRG ^{Reference Noh, Mikler, Joy and Smith37–Reference Xie, Strong and Zhang39} represent yet another source of inflammatory mediators. The actions of primary mediators such as IL-1β and TNF-α on DRG neurons culminate in marked changes in genes coding for neuropeptides, cytokines, chemokines, receptors, ion channels, signal transduction molecules, and synaptic vesicle proteins. ^{Reference Zhang and Xiao40,Reference Biber and Boddeke41} Some of these gene products also function as secondary mediators that are released and effect the transfer of information between damaged peripheral nerves and various cell types in the spinal dorsal horn. ^{Reference Boakye, Tang and Smith18}

Primary mediators also control the expression of long non-coding RNA’s ^{Reference Baskozos42} and microRNA’s in DRG. The latter are also upregulated by nerve injury ^{Reference Finnerup, Kuner and Jensen6} and post-transcriptionally regulate the protein expression of hundreds of genes in a sequence-specific manner. ^{Reference Liu, Xu and Wang43} Transfer of microRNAs between cell types may be brought about by the release and uptake of exosomes. ^{Reference Zhang, Ye and Zhao44}

Importantly, altered function of ion channels as a result of the action of primary mediators leads to increased excitability of primary afferent neurons ^{Reference Noh, Stemkowski and Smith45–Reference Chen, Pang and Shen49} and the generation of stimulus-independent spontaneous activity. This incessant spontaneous activity in primary afferents is absolutely crucial for the onset and persistence of pain. ^{Reference Pitcher and Henry50–Reference Yatziv and Devor53} This is illustrated by the effectiveness of topically applied lidocaine in the clinic. ^{Reference Dworkin, O’Connor and Backonja54} Altered ion channel function and peripheral hyperexcitability may even be involved in spinal cord injury ^{Reference Ritter, Zemel, Hala, O’Leary, Lepore and Covarrubais55} and central post-stroke pain. ^{Reference Haroutounian, Ford and Frey56} Although Na_v1.7, K_v7.2, Ca_v2.2, Ca_v3.2, and HCN2 channels have emerged as potential therapeutic targets for drug development, with the notable exception of gabapentinoid action on voltage-gated Ca²⁺ channels, ^{Reference Alles and Smith9} pharmacological manipulation of these channels has failed to identify new therapeutic approaches. ^{Reference Alles and Smith57}

The observation that peripherally generated pain is often not suppressed by rhizotomy ^{Reference Eschenfelder, Habler and Jannig58} seems at odds with the idea that stimulus-independent spontaneous activity is required for pain maintenance. It is possible, however, that pain seen after rhizotomy is related to deafferentation. This deafferentation pain may replace that which previously resulted from ectopic primary afferent activity. ^{Reference Eschenfelder, Habler and Jannig58}

As would be expected, the population of ion channels affected by primary mediators is similar to that affected by peripheral nerve injury ^{Reference Noh, Stemkowski and Smith45,Reference Stemkowski, Noh, Chen and Smith47,Reference Stemkowski and Smith59} and in animal models, blockade of the actions of primary mediators abrogates signs of injury-induced pain. ^{Reference Scholz and Woolf14,Reference Moalem and Tracey15,Reference Boakye, Tang and Smith18,Reference Wolf, Gabay, Tal, Yirmiya and Shavit60–Reference Grace, Hutchinson, Maier and Watkins63} In general however, attempts to block the action of inflammatory mediators to limit neuropathic pain in the clinic have met with limited success. ^{Reference Yekkirala, Roberson, Bean and Woolf64}

Bidirectional Signalling between the Nervous and Immune Systems and “Neurogenic Neuroinflammation”

The relationship between immune cells and neurons is bidirectional. In addition to the well-documented actions of immune mediators on neurons, ^{Reference Boakye, Tang and Smith18,Reference Noh, Stemkowski and Smith45–Reference Binshtok, Wang and Zimmermann48,Reference Gustafson-Vickers, Lu, Lai, Todd, Ballanyi and Smith65–Reference Vikman, Siddall and Duggan67} neuronal activity has a direct effect on immune cells. ^{Reference Talbot, Foster and Woolf68–Reference McMahon, La Russa and Bennett72} This “neurogenic neuroinflammation” ^{Reference Xanthos and Sandkuhler73} is brought about by the release of neuropeptides and glutamate from primary afferents and their interaction with their cognate receptors on immune cells, astrocytes, and microglia. ^{Reference McMahon, La Russa and Bennett72,Reference Shi, Wang, Li, Peymen, Kingerly and Clark74}

Actions of Secondary Mediators and Transfer of Information from the Periphery to the Spinal Cord

Most secondary mediators are released from primary afferent terminals. Substances such as colony-stimulating factor 1 (CSF-1), the chemokines CCL-21, CXCL-12, and Wnt3a and Wnt5a ^{Reference Finnerup, Kuner and Jensen6,Reference Boakye, Tang and Smith18,Reference van Vliet, Lee and van der Poel28,Reference Okubo, Yamanaka and Kobayashi75–Reference Dong, Xu, Xia and Zhang78} activate their cognate receptors on spinal microglia and/or astrocytes and alter their properties. Activated glia thereby detects and mount an enduring response to peripheral nerve injury. Spinal microglia are affected in male rodents ^{Reference Malcangio77} whereas invading macrophages and adaptive immune cells such as T-lymphocytes are involved in females. ^{Reference Halievski, Ghazisaeidi and Salter79–Reference Sorge and Mapplebeck81} CCL-21 and CXCL-12 signal to activate astrocytes. ^{Reference Dong, Xu, Xia and Zhang78,Reference van Weering, de Jong, de Haas, Biber and Boddeke82} The inflammatory mediator, IFN-γ is increased in spinal cord following peripheral nerve injury ^{Reference Costigan, Moss and Latremoliere83} and this may originate from invading T-lymphocytes.

Generation and Release of Tertiary Mediators in the Dorsal Horn and Central Sensitizaton

Glial activation and proliferation leads to the generation and release of tertiary mediators such as BDNF, IL-1β, TNF-α, and IFN-γ. ^{Reference Boakye, Tang and Smith18,Reference Biggs, Lu, Stebbing, Balasubramanyan and Smith84,Reference Smith85}

BDNF is released from microglia in response to the secondary mediators CSF-1 ^{Reference Boakye, Tang and Smith18,Reference Guan, Kuhn and Wang76,Reference Yu, Basbaum and Guan86,Reference Boakye, Rancic and Whitlock87} and/or Wnt3a. ^{Reference Zhang W.Shi and Peng88} BDNF release requires activation of P2X4 receptors by ATP. ^{Reference Alles and Smith9,Reference Trang, Beggs, Wan and Salter89} As a mediator of the effect of nerve injury, ^{Reference Coull, Boudreau and Bachand90–Reference Chen, Balasubramanyan, Lai, Todd and Smith92} BDNF facilitates excitation ^{Reference Biggs, Lu, Stebbing, Balasubramanyan and Smith84,Reference Lu, Ballanyi K.Colmers and Smith93–Reference Hildebrand, Jian and Dedek95} and attenuates inhibition in the superficial dorsal horn. ^{Reference Alles and Smith9,Reference Coull, Beggs and Boudreau96} These changes, which lead to central sensitization, spontaneous activity, and the misprocessing of sensory information, ^{Reference Peirs and Seal97–Reference Prescott, Ma and De Koninck100} involve at least four cellular mechanisms.

Microglial-derived BDNF increases excitatory drive to excitatory dorsal horn neurons and inhibits that to inhibitory neurons by both presynaptic and postsynaptic mechanisms. ^{Reference Boakye, Rancic and Whitlock87,Reference Lu, Ballanyi K.Colmers and Smith93,Reference Lu, Biggs and Stebbing94} This altered synaptic activity is capable of increasing spontaneous action potential discharge in excitatory neurons while reducing it in inhibitory neurons. ^{Reference Lu, Ballanyi K.Colmers and Smith93}

BDNF also enhances excitatory responses to N-methyl-d aspartate (NMDA) in rat spinal cord in vitro. ^{Reference Kerr, Bradbury and Bennett101} This may involve potentiation of the function of presynaptic NMDA receptors on primary afferent terminals ^{Reference Chen, Walwyn and Ennes102} with a resultant increase in excitatory glutamatergic transmission. This may contribute to the effectiveness of the NMDA blocker, ketamine in some patients. ^{Reference Dworkin, O’Connor and Backonja54}

Peripheral nerve injury reduces expression of the potassium-chloride exporter (KCC2) selectively in nociceptive dorsal horn neurons. ^{Reference Coull, Boudreau and Bachand90,Reference Ferrini, Perez-Sanchez and Ferland103} The resulting accumulation of intracellular Cl⁻ normally causes outward, inhibitory GABAergic synaptic currents mediated by Cl⁻ influx to become inward excitatory currents mediated by Cl⁻ efflux. ^{Reference Coull, Boudreau and Bachand90} In male rats, this downregulation of KCC2 is mediated by BDNF. ^{Reference Ferrini and De Koninck104} Since the loss of GABAergic inhibition enables non-noxious Aβ fiber-mediated excitatory transmission to access the superficial spinal dorsal horn, this process contributes to the establishment of allodynia. ^{Reference Baba, Ji and Kohno99}

Long-term potentiation (LTP) of synaptic transmission, sometimes known as “wind-up”, contributes to central sensitization in the dorsal horn. ^{Reference Sandkuhler, Benrath, Brechtel, Ruscheweyh and Heinke105,Reference Sandkuhler106} LTP of C-fibre responses is augmented by BDNF ^{Reference Li and Cai107} and LTP induced by nerve stimulation is occluded by BDNF pretreatment. ^{Reference Ding, Cia and li108} The importance of these effects was recently underlined by the observation that spinal LTP as well as microglial activation and upregulation of BDNF are inhibited by antibodies to the secondary mediator CSF-1. This strongly implicates the CSF-1-microglia-BDNF axis ^{Reference Boakye, Tang and Smith18} in the generation of spinal LTP. ^{Reference Zhou, Peng and Xu109}

As already mentioned, in females, changes in sensory processing in the dorsal horn involve the invasion of macrophages and T-lymphocytes. ^{Reference Mapplebeck, Beggs and Salter80,Reference Sorge and Mapplebeck81} Yet as in males, this leads to attenuation of inhibition following the collapse of the Cl⁻ gradient. ^{Reference Mapplebeck, Lorenzo and Lee110} In females, collapse of the Cl⁻ gradient is also brought about by the neuropeptide, CGRP ^{Reference Paige, Plasencia-Fernandez and Kume111} which is released from primary afferent terminals. ^{Reference Gardell, Vanderah and Gardell112}

IL-1β from microglia stimulates astrocytic production of both TNF-α and IL-1β itself ^{Reference Gajtko, Bakk, Hegedus, Ducza and Hollo113} thereby amplifying the initial IL-1β signal. Spinal actions of IL-1β involve increases in excitatory synaptic transmission. ^{Reference Gustafson-Vickers, Lu, Lai, Todd, Ballanyi and Smith65,Reference Kawasaki, Zhang, Cheng and Ji66} This may involve a reduction in the ability of astrocytes to take up glutamate as a result of internalization of the astrocytic glutamate transporter (EAAT2). ^{Reference Yan, Maixner and etal114}

TNF-α also augments excitatory transmission in the dorsal horn ^{Reference Boakye, Tang and Smith18,Reference Kawasaki, Zhang, Cheng and Ji66} as well as LTP by an action on glial cells. ^{Reference Gruber-Schoffnegger, Drdla-Schutting, Honigsperger, Wunderbaldinger, Gassner and Sandkuhler115} Blockade of TNF-1 receptors attenuates neuropathic pain in male rodents but not in females. ^{Reference del Rivero, Fischer, Yang, Swanson and Bethea116} Although anti-TNF antibodies and anti-TNF drugs such as thalidomide are available, none seem particularly useful in pain management. ^{Reference Goncalves Dos, Delay, Yaksh and Corr117}

IFN-γ from invading T-lymphocytes induces both tactile allodynia and altered microglia function. Genetic ablation of the interferon receptor (IFN-γR) impairs nerve injury-evoked activation of ipsilateral microglia and tactile allodynia. ^{Reference Tsuda, Masuda, Kitano, Shimoyama, Tozaki-Saitoh and Inoue118} IFN-γ also increases dorsal horn excitability ^{Reference Vikman, Duggan and Siddall119} and facilitates synaptic transmission between primary afferent C-fibres and Lamina 1 neurons via a microglial dependent mechanism. ^{Reference Reischer, Heinke and Sandkuhler120}

Failure to Resolve Chronic Neuroinflammation

All types of injury are capable of promoting inflammation and pain ^{Reference Ji, Xu, Strichartz and Serhan121} and the interactions of inflammatory mediators with neurons, glia, immunocompetent leucocytes and lymphocytes, and macrophages ^{Reference Scholz and Woolf14} promote neuroinflammation. Since identified “off signals” actively suppress the classical signs of inflammation, ^{Reference Ji, Xu, Strichartz and Serhan121,Reference Ji122} pain is usually short lasting or acute. The signals that actively resolve inflammation and pain include anti-inflammatory cytokines such as IL-10 and lipid-derived specialized pro-resolving mediators (SPMs). ^{Reference Buckley, Gilroy and Serhan123,Reference Buckley, Gilroy, Serhan, Stockinger and Tak124} Despite this, the neuroinflammation associated with neuropathic pain may not resolve, thereby promoting the transition from acute pain to chronic pain. ^{Reference Ji, Xu, Strichartz and Serhan121} As already mentioned, spontaneous and ectopic activity in primary afferent fibers is crucial for the maintenance and persistence of signs of neuropathic pain. ^{Reference Pitcher and Henry50–Reference Yatziv and Devor53,Reference Haroutounian, Ford and Frey56} Excessive neuronal activity releases glutamate and neuropeptides which interact with glia and immune cells to provoke the generation of inflammatory mediators. ^{Reference Xanthos and Sandkuhler73} It is possible that this incessant neurogenic neuroinflammation overcomes the resolution processes that normally terminate inflammation thereby contributing to the indefinite persistence of neuropathic pain.

In addition, the injury-induced structural changes in peripheral afferent ^{Reference Gangadharan, Zheng and Tabener29} and sympathetic nerves ^{Reference McLachlan, Janig and Michalis30,Reference Yen, Bennett and Ribeiro-da-Silva31} and in higher brain structures are almost certainly irreversible. ^{Reference Price, Basbaum and Bresnahan12} These enduring changes also contribute to the chronic nature of neuropathic pain.

Changes in Central Sensory Pathways in Higher Brain Regions

Cytokine/chemokine/growth factor/glial cell interactions are also involved in modulation of sensory information in the mesolimbic system, ^{Reference Taylor, Castonguay and Taylor125} thalamus, sensory cortex, nucleus accumbens, and amygdala. ^{Reference Taylor, Castonguay and Taylor125–Reference Wu and Zhu127} Peripheral nerve injury promotes microglial activation in the contralateral thalamus, sensory cortex, and amygdala as would be expected from the anatomical projections of ascending sensory fibers. Brain regions not directly involved in either sensory or affective aspects of pain, such as the motor cortex, do not display microglial activation. ^{Reference Taylor, Mehrabani, Liu, Taylor and Cahill128} Hyperactivity in parts of the anterior cingulate cortex and other limbic structures drives the anxiety and depression that represent a co-morbidity of chronic and neuropathic pain. ^{Reference Bannister, Sachau, Baron and Dickenson7,Reference Sellmeijer, Mathis and Hugel129}

Blood-borne inflammatory mediators ^{Reference Sandy-Hindmarch, Bennett, Wiberg, Furniss, Baskozos and Schmid130} from the site of peripheral injury increase the permeability of the blood–brain barrier. ^{Reference Xanthos, Pungel, Wunderbaldinger and Sandkuhler33} This allows CNS neurons to access blood cells and the cytokines and chemokines they produce. ^{Reference Greenhalgh, David and Bennett131} In addition, the selective activation of glia and immune cells in nociceptive pathways ^{Reference Taylor, Castonguay and Taylor125} likely reflects localized neurogenic neuroinflammation in response to enduring intense activity. ^{Reference Xanthos and Sandkuhler73}

Alterations in Descending Control of Spinal Processing

Spinal nociceptive processing is subject to modulation by descending serotonergic and noradrenergic pathways. ^{Reference Finnerup, Kuner and Jensen6,Reference Bannister and Dickenson132} Descending inhibition is mediated via α₂-adrenoceptors and 5HT₇ receptors whereas serotonergic activation of metabotropic 5HT₂ receptors and ionotropic 5HT₃ receptors facilitates transmission ^{Reference Bannister, Sachau, Baron and Dickenson7} . Brainstem excitatory pathways are more important in the maintenance than in the induction of pain and under these conditions, α2-noradrenergic inhibition is attenuated whilst facilitation through 5HT₂ and 5HT₃ receptors is enhanced. ^{Reference Bannister, Sachau, Baron and Dickenson7,Reference Bannister and Dickenson132–Reference Bannister, Lockwood, Goncalves, Patel and Dickenson134} Actions on these descending controls are thus likely to underlie the efficacy of tricyclic antidepressants and serotonin-noradrenaline reuptake inhibitors in pain management. ^{Reference Bannister, Sachau, Baron and Dickenson7,Reference Finnerup, Attal and Haroutounian10}

Different Injuries and Different Etiologies

As already stated, different types of nerve injury provoke different types of behavioral or physiological response in both humans and animals. ^{Reference Mogil1–Reference Fitzgerald and McKelvey4} Thus while mechanical allodynia produced in animals by SNI ^{Reference Decosterd and Woolf13} persists for many weeks, that produced by CCI is short-lived and recovery is seen in about 4 weeks. ^{Reference Decosterd and Woolf13,Reference Noh, Mikler, Joy and Smith37} Similarly, changes in synaptic transmission in the superficial dorsal horn are more robust after sciatic CCI than after complete sciatic nerve section (axotomy). ^{Reference Chen, Balasubramanyan, Lai, Todd and Smith92} These findings are consistent with the observation that CCI promotes stronger and more long-lasting upregulation of TNF-α, IL-1β, and CCL-2 than axotomy by nerve crush. ^{Reference Kleinschnit, Brinkhoff, Zelenka, Sommer and Stoll135} It has also been shown that the neuronal subtypes in the dorsal horn that are involved in generation of mechanical allodynia is defined by the nature of peripheral nerve injury. ^{Reference Peirs, Williams and Zhao136}

More clinically relevant observations include reports that neuropathic pain associated with multiple sclerosis is characterized by loss of spinal neurons ^{Reference Gushchina, Pryce and Yip137} but this effect is not seen with CCI. ^{Reference Polgar, Hughes, Riddell, Maxwell, Puskár and Todd138,Reference Polgar, Gray, Riddell and Todd139} The above findings imply that different types of injury provoke the generation of different sets of mediators ^{Reference Boakye, Tang and Smith18,Reference DeLeo, Colburn and Rickman140} and thus present different drug targets.

The Way Forward? Bridging the Gap between Basic Science and Clinical Practice

Given that patients with neuropathic pain are heterogeneous in pathophysiology, etiology, and clinical presentation ^{Reference Mogil1,Reference Baron, Maier and Attal5} it is hardly surprising that injury-specific pathologies are found in animal models. As in the clinic, there is the added complication that signs of pain and response to medication of each experimental animal is determined by factors such as their sex, prior exposure to neonatal injury, age, intestinal microbiome, and environmental factors. ^{Reference Mogil1–Reference Fitzgerald and McKelvey4,Reference Brewer and Bacceic141,Reference Gaudet, Fonken, Ayala, Maier and Watkins142}

Quantitative sensory testing (QST) may help to bridge the knowledge gap between clinical and laboratory findings. This involves formalization and quantification of a battery of neurological tests, such as response to von Frey filaments, vibration, heat, pressure, and cold as well as dynamic allodynia and wind-up ratio. ^{Reference Baron, Maier and Attal5} Findings are compared with datasets that represent normal responses to sensory tests. Neuropathic pain patients can then be grouped into clusters based on their sensory profiles and this may have a role in determining treatment. ^{Reference Vollert, Maier and Attal143} Technological improvements in microneurography have shown that the specific C-fibre subpopulation affected (mechanoinsensitive versus non-mechanoceptive) depends on the source of neuropathic pain and the type of neuropathy. ^{Reference Serra, Bostock and Sola144,Reference Middleton, Barry and Comini145} Modern microneurography approaches will thus play a role in future refinement of QST. The validity of QST is supported by the observation that post hoc analysis of responders to treatments in clinical trials suggest that clinical effectiveness may cluster according to pain phenotype. ^{Reference Vollert, Maier and Attal143} Beyond this, it may also be possible to subcategorize patients according to their cytokine profile. It then may be possible to correlate precisely quantified signs and symptoms in each individual patient to pathophysiology at the cellular and molecular level.

Recent improvements in basic science approaches also seek to bridge the gap between the “bench and bedside”. For example, improved methodologies are starting to differentiate probable pain in animal models from nociception or simple withdrawal reflexes. ^{Reference Alles and Smith57,Reference Mogil146} Also more attention is now paid to the genetics, environment, and sex of experimental animals ^{Reference Mogil1,Reference Mapplebeck, Beggs and Salter80} and improved methodologies are now available for bringing human tissue to the laboratory. These include the culture of human nociceptors either from surgical or post-mortem tissue or using human-induced pluripotent stem cell-derived nociceptors. ^{Reference Middleton, Barry and Comini145,Reference Alsaloum and Waxman147}

Taken together, these approaches will permit a rational and highly personalized medicine approach that will dictate the most appropriate therapeutic approach for each individual patient. ^{Reference Bannister, Sachau, Baron and Dickenson7,Reference Renthal, Chamessian and Curatolo148,Reference Bouali-Benazzouz, Landry, Benazzouz and Fossat149}