Neuroma Model

One of the earliest explicit models of neuropathic pain, nerve axotomy, was developed from the observation that transection of the sciatic nerve results in self-mutilation, or autotomy, of the denervated paw (Wall et al 1979). Autotomy was originally thought to occur because the animal perceived the denervated limb as a foreign object and not as a part of its own body (Wall et al 1979). However, Wall and colleagues surmised that the neuroma formed at the site of nerve section may be the source of a noradrenergic-sensitive afferent discharge that might be perceived as pain referred to the anesthetized, denervated limb (Wall and Gutnick 1974, Wall et al 1979). Based on these suppositions, the transection or neuroma model was developed to study anesthesia dolorosa and phantom limb pain. Nerve transection in mice and rats was performed by removing approximately 5 mm of the sciatic and saphenous nerves at the level of the mid-thigh (Wall et al 1979). Denervation, indicated by complete anesthesia, was confirmed by the complete absence of a nocifensive response to a strong pinch. The degree of autotomy was quantified by applying a scoring method reflecting the severity of damage to the paw (Wall et al 1979). Animals showed time-dependent damage to the paw during the first 5 weeks with no increased damage afterward (Wall et al 1979). Normal weight gain and behavioral activity were otherwise observed (Wall et al 1979). Interestingly, injury to the saphenous nerve (a purely sensory nerve), a crush injury of the sciatic nerve, or a combination of both manipulations produced little or no autotomy over a 9-week observation period (Wall et al 1979). Although crush produces wallerian degeneration, nerve regeneration and reinnervation of the hindpaw can occur to at least some degree and in this manner possibly attenuate the expression of autotomy (Devor and Govrin-Lippmann 1979). In contrast, sciatic nerve ligation and transection produced time-dependent autotomy that was observed even when saphenous nerve section was performed more than 100 days after sciatic nerve section (Wall et al 1979). Although autotomy can also occur after dorsal root avulsion, the driving mechanism may differ since the spinal horn neurons lose afferent input, thereby possibly resulting in hyperactivity of these transmission pathways so critical to pain processing, and it may thus represent deafferentation pain (Lombard et al 1979). After axotomy distal to the dorsal root ganglion (DRG), however, the proximal portions of the sectioned nerves continue to generate input to the spinal cord (Lombard et al 1979).

Although this model is one of the first reported attempts to model clinical neuropathic pain states, specifically, anesthesia dolorosa and phantom limb pain, autotomy behavior has not been widely adopted. One reason may be the uncertainty whether autotomy behavior reflects pain (but see Qu et al 2011). It has been suggested that autotomy is related to persistent, spontaneous ectopic discharges from the neuromas that result in ongoing pain (Wall et al 1979, Devor 1991). Other suggestions, however, are that autotomy may be an attempt to remove an insensate appendage or that it occurs as a result of excessive grooming because of the absence of sensory feedback (Rodin and Kruger 1984, Kruger 1992). Objections have also been raised that injury-induced autotomy as a model of neuropathic pain should be discarded for several reasons, including the fact that it is “esthetically repugnant” and that other models should be considered (Rodin and Kruger 1984, Kruger 1992).

Partial Sciatic Nerve Section

A variant of the neuroma model was designed to avoid the complications inherent with complete denervation of the hindpaw and to more closely resemble trauma-induced nerve injury by tightly ligating approximately one-third to one-half of the sciatic nerve fascicle and sparing the remaining fibers from injury (Seltzer et al 1990). The rats displayed licking and guarding behavior of the hindpaw, suggestive of spontaneous pain, but no autotomy was observed (Seltzer et al 1990). Tactile allodynia, suggested by exaggerated withdrawal responses to light touch, was observed bilaterally (Seltzer et al 1990). Mechanical and thermal hyperalgesia was also observed (Seltzer et al 1990). Chemical sympathectomy with guanethidine abolished these behavioral responses, thus suggesting that these signs of neuropathic pain are probably sympathetically maintained (Seltzer et al 1990, Seltzer and Shir 1991). The combination of an almost immediate onset and long duration of allodynia and hyperalgesia, the presence of mirror-image pain, and the dependence on sympathetic activity led to suggestions that this preparation may serve as a model for causalgia elicited by partial nerve injury (Seltzer et al 1990, Seltzer and Shir 1991). Neonatal rats were treated with capsaicin to selectively obliterate capsaicin-sensitive C-fiber nociceptors in an attempt to differentiate the fiber types mediating these behavioral responses (Seltzer and Shir 1991). Tactile hyperesthesia and touch-evoked tactile allodynia developed in the capsaicin-treated rats after partial sciatic nerve ligation, but not thermal hyperalgesia. These results indicated that tactile hyperesthesia is probably mediated through large-diameter myelinated fibers whereas thermal hyperalgesia is mediated through thermal nociceptive C fibers as noted previously (Seltzer and Shir 1991). One shortcoming of the model is that it is difficult to accurately reproduce the same injury repeatedly. Consequently, the degree of neuropathic pain behavior might be affected by the degree of the injury. Moreover, there is considerable mixing of injured with uninjured fibers, and the uninjured fibers may express enhanced discharges through ephaptic transmission.

Chronic Constriction Injury

The chronic constriction injury (CCI) model was developed to inflict reproducible nerve injury without complete denervation (Bennett and Xie 1988). A set of four chromic gut sutures were placed loosely around the common sciatic nerve at intervals of 1–2 mm so that they did not completely impede circulation through the epineurium (Bennett and Xie 1988). During the first week after surgery, gait and posture were highly variable among the animals and stabilized for up to 2 months thereafter. The rats walked with a definitive limp; exhibited ventriflexion of the toes, which were held tightly together; and avoided placing weight on the affected hindpaw (Bennett and Xie 1988). Guarding behavior, consisting of raising the limb and keeping it close to the flank, was observed. A striking feature of rats with CCI is thickening and elongation of the claws, to the point that in extreme cases they would curve back to the toe pads because of avoidance of grooming the injured hindlimb. Rats may show mutilation of the claw tips and roots but, unlike autotomy, no mutilation of the digits (Bennett and Xie 1988). Thermal hyperalgesia may be evident within 2 days and last 2 months after CCI. These changes in thresholds appear much sooner than autotomy behavior following complete nerve transection. Compound electromyographic activity of the biceps femoris and semitendinosus muscles in response to a heated probe applied to the hindlimb was markedly greater and showed long-lasting afterdischarge activity in rats with CCI when compared with sham-operated animals (Bennett and Xie 1988). Hypersensitivity to cold (4°C) and to light tactile stimuli, but not to noxious mechanical stimuli, was observed after CCI (Bennett and Xie 1988). Gentle stroking of the affected hindlimb resulted in a marked increase in Fos expression in the superficial laminae of the spinal dorsal horn (Catheline et al 1999). These behavioral observations may indicate that the CCI model might suggest spontaneous pain, as revealed by abnormal postures, guarding, and coincident grooming behavior, actions mimicking the signs of complex regional pain syndrome (CRPS). For example, some CRPS patients do not trim their nails because of the pain experienced (Bennett and Xie 1988).

The CCI model may elicit both spontaneous and evoked neuropathic pain because some afferent fibers of the sciatic nerve survive after surgery. Within 1 day of CCI, intraneural edema caused by partial constriction of the vasculature of the epineurium results in translucent, demyelinated constrictions 25–75% of the original diameter of the sciatic trunk; the constrictions merge into a single area of uniform thinning with swelling proximal to the constricted area (Bennett and Xie 1988). This is reminiscent of entrapment neuropathies (e.g., carpal tunnel syndrome) (Bennett and Xie 1988). The proximal sciatic nerve appeared normal to within 0.5 cm of the constriction, where marked degeneration and endoneurial edema became apparent along with massive demyelination and nearly total loss of large-diameter Aα and Aβ fibers and a lesser loss of small-diameter myelinated Aδ fibers (Munger et al 1992). Within 3 days of CCI, 89% of Aβ, 87% of Aδ, and 32% of C fibers were lost, with progression to loss of 94% of myelinated fibers and 73% of unmyelinated fibers within 14 days (Munger et al 1992, Coggeshall et al 1993). Subsequently, there was regeneration of unmyelinated and small-diameter myelinated but not large-diameter myelinated fibers to nearly normal levels (Munger et al 1992, Coggeshall et al 1993). The hyperalgesia observed after CCI is probably not due to the loss of large-diameter mechanosensitive fibers since it was resolved 56 days after CCI whereas the large-diameter fibers remained absent (Coggeshall et al 1993). It is possible that hyperalgesia may be mediated through sensitized Aδ and C fibers, some of which normally act as low-threshold mechanoreceptors (Munger et al 1992). Electrophysiological studies have shown that primary afferents, including large-diameter myelinated fibers, spontaneously discharge at ectopic foci proximal to the injury and that these abnormal discharges might play a role in spontaneous and evoked manifestations of neuropathic pain (Munger et al 1992, Coggeshall et al 1993).

An extension of the CCI model is CCI of the infraorbital nerve, which has been used as a model of trigeminal neuropathic pain (Vos et al 1994). The edge of the orbit of anesthetized rats was exposed and the contents of the orbit gently pushed aside with a cotton-tipped swab to expose the infraorbital nerve just caudal to the infraorbital foramen (Vos et al 1994). A pair of chromic gut sutures were placed around the nerve without occluding circulation in the nerve sheath. Rats with constriction injury of the infraorbital nerve demonstrated a significant increase in facial grooming along with decreased exploratory behavior (Vos et al 1994). They also spent increased periods immobile in a “frozen” posture (Vos et al 1994). The rats expressed touch-evoked agitation and vigorous nocifensive responses to probing the periorbital area with von Frey filaments in the later (15–130 days) postoperative period but were hypoesthetic during the immediate postoperative period (Vos et al 1994). Moreover, the constriction injury resulted in increased expression of Fos in the medullary dorsal horn corresponding to the intensity of the touch stimulus (Vos and Strassman 1995). Tactile allodynia was blocked by injections of triptans (Kayser et al 2002), endothelin ET_B receptor antagonists (Chichorro et al 2006), and the antiepileptic lacosamide (Hao et al 2006). This model may lead to better understanding of trigeminal neuropathic pain states and to enhanced therapeutic regimens for this condition.

Spinal Nerve Ligation

The spinal nerve ligation (SNL) model of traumatic nerve injury has become one of the most commonly used and studied models (Kim and Chung 1992). The primary impetus for development of the SNL model derived from the belief that it was not possible to adequately control the numbers and types of primary afferent fibers that were injured with the previous models (Kim and Chung 1992). The L5 and L6 spinal nerve branches of the sciatic nerve are carefully identified, isolated, and tightly ligated with 3–0 silk suture between the trifurcation of the sciatic nerve and distal to the DRGs. Removal of the L6 spinal transverse process is required for unobstructed contact with this spinal nerve (Kim and Chung 1992). The hindpaws of rats with L5/L6 SNL are slightly everted and the toes held together, and the rats shift weight away from the injured hindpaw and limp with an uncoordinated gait (Kim and Chung 1992). After SNL, rats would frequently suddenly withdraw the injured hindpaw and lick or gently bite the claws of the hindlimb (Kim and Chung 1992). Most importantly, no signs of autotomy were observed in these rats (Kim and Chung 1992). Critically, ligation of only the L4 spinal nerve caused extreme motor deficits and dragging of the hindlimb because of denervation of the proximal muscles of the leg (Kim and Chung 1992). The many subsequent experiments performed with the SNL model indicate that limited motor deficits are seen with SNL.

Robust enhanced responses to gentle tactile and noxious thermal stimuli were present within 1–2 days after SNL and persisted throughout the observation period; they were interpreted as evidence of tactile allodynia and thermal hyperalgesia lasting longer than 10 weeks (Kim and Chung 1992). Other studies using the paw withdrawal threshold, which involved the application of a series of von Frey filaments of increasing and decreasing strength, demonstrated that tactile allodynia developed within 1–2 days after SNL and remained throughout the observation period of more than 50 days (Chaplan et al 1994, Ossipov et al 1999). Hyperalgesia in response to noxious radiant heat had a similar time course as tactile allodynia did (Chaplan et al 1994, Ossipov et al 1999). The cold allodynia after SNL is much less robust than that seen after CCI (Kim et al 1997). Application of acetone drops to the hindpaw elicits brisk withdrawal responses or increased dorsal horn unit activity (Kim et al 1997). However, interpretation of such cold allodynia may be adulterated by the temperature of the hindpaw, the acetone, the rate of evaporation, and the tactile stimulation produced by the drop itself (Jasmin et al 1998). Behavioral responses to a 5°C cold plate were significantly less pronounced in SNL rats than in CCI rats (Kim et al 1997). Electrophysiological studies of convergent dorsal horn units showed an increase in the number of responsive neurons, an increase in response frequencies, and an increase in the slope of the stimulus–response function of the dorsal horn units in response to light touch, brush, or heat (Chapman et al 1998, Suzuki et al 1999). A presumptive cooling sensation after a drop of acetone onto the hindpaw also produced enhanced responses of spinal dorsal horn units after SNL (Chapman et al 1998, Suzuki et al 1999). Interestingly, the response thresholds or response frequencies of spinal dorsal horn neurons to electrical stimulation of Aβ or C fibers applied to their receptive fields did not differ significantly among naïve, sham-operated, or SNL rats (Suzuki et al 1999).

Whether expression of the behavioral signs of neuropathic pain requires input from the injured nerves has been controversial. Interruption of neuronal communication between the DRG of the ligated nerves and the spinal cord through either dorsal rhizotomy or the local application of bupivacaine reversed the hyperesthetic responses to thermal, tactile, and cool stimuli (Sheen and Chung 1993, Yoon et al 1996), thus suggesting the importance of the injured nerves. Rhizotomy of the L5/L6 spinal nerve roots did not produce any behavioral signs of neuropathic pain, and dorsal rhizotomy of the L3 and L4 spinal nerve roots, in addition to L5/L6 SNL, produced complete denervation of the hindlimb along with loss of motor activity and autotomy, in effect mimicking sciatic nerve section (Sheen and Chung 1993, Yoon et al 1996). Bupivacaine applied onto the L3 and L4 spinal nerve roots of rats with L5/L6 SNL blocked tactile but not thermal or cold hyperesthetic responses (Sheen and Chung 1993). It was proposed that evoked pain requires input from both injured and uninjured afferent fibers whereas spontaneous pain may be mediated through injured afferent input (Sheen and Chung 1993, Yoon et al 1996). However, other studies have questioned this conclusion. For example, Li and colleagues (2000) reported that section of the L5 spinal nerve followed by L5 dorsal rhizotomy did not block enhanced evoked responses. In contrast, L5 spinal nerve section followed by L4 dorsal rhizotomy blocked evoked input, which suggests that the hypersensitivity was mediated via uninjured fibers (Li et al 2000), although this conclusion may have been confounded by significant denervation of the hindpaw under these conditions. Recent studies evaluating non-evoked pain (see later) have supported a role for injured fibers (Qu et al 2011), consistent with the clinical observations (Gracely et al 1992). However, this study could not exclude an additional role for uninjured fibers. Notably, a recent microneurographic study found that individuals with small-fiber neuropathic pain, as well as animals with five different types of experimental nerve pathologies, demonstrated markedly increased spontaneous activity of intact C-fiber nociceptors, thus providing evidence that the spontaneous activity of nociceptors may additionally contribute to spontaneous pain (Serra et al 2012).

A variation of the L5/L6 SNL model is to ligate or cut the L5 spinal nerve only. This procedure produced a similar behavioral profile, but of lesser magnitude, perhaps because of the involvement of fewer nerve fibers (Kim and Chung 1992). Another adaptation that has been reported, though not widely used, called for ligation of the sacral rather than the lumbar spinal nerves to induce tactile and thermal hyperesthesia of the rat tail (Sung et al 1998). An interesting feature of ligation of the sacral afferents is the production of bilateral tactile allodynia and thermal hyperalgesia of the hindpaws (Sung et al 1998). This model allows injury at a given spinal segment but hyperalgesia revealed through evoked input at a different level. Moreover, clear, robust signs of tactile allodynia and thermal hyperalgesia are produced within a few days and last for months, which allows extensive examination of the progression of changes after nerve injury. A unique advantage of ligation or section of the spinal nerve is that this process allows independent examination of damaged and spared nerves and their associated DRG neurons. This permits examination of the changes in expression of multiple potential mediators and neuromarkers in injured and adjacent, uninjured primary afferent nerves.

Spared Nerve Injury

The spared nerve injury (SNI) model is created by tight ligation and subsequent resection of 2–4 mm of the common peroneal and tibial nerves while leaving the sural nerve intact (Decosterd and Woolf 2000). These nerves represent the three distal branches of the sciatic nerve. Behavioral signs of neuropathic pain are evident within 1 day after SNI and are maintained for more than 9 weeks (Decosterd and Woolf 2000). Spontaneous pain is suggested by avoidance of weight bearing on the injured hindpaw and eversion of the paw and rapid hindpaw flexion on contact (Decosterd and Woolf 2000). Animals with SNI demonstrated enhanced responses to light and noxious tactile stimuli and to acetone drops, thus suggesting tactile, thermal, and cold hyperesthesia (Decosterd and Woolf 2000). Notably, thermal hyperalgesia was not demonstrated by decreased latency to noxious heat; rather, the duration of the withdrawal response was increased. Evidence of neuropathic pain behavior occurred in response to stimuli applied to regions innervated by the sural nerve and the uninjured saphenous territories. The level of responses appeared to be greater when the stimuli were applied to the receptive field of the sural nerve than when applied to the saphenous nerve (Decosterd and Woolf 2000).

With this model, the territories of injured and uninjured nerves can be examined independently (Decosterd and Woolf 2000). Furthermore, the effects of nerve injury on an uninjured nerve (sural nerve) that shares a common nerve trunk with the injured nerve may be compared with an adjacent uninjured nerve that is anatomically isolated (saphenous nerve) (Decosterd and Woolf 2000). Therefore, it is possible to study changes that occur in DRG neurons whose axons have minimal commingling with injured fibers but whose terminals overlap the territory of injured nerves (Decosterd and Woolf 2000).

Distal Nerve Injury

CRPS type 1 was thought to occur in the absence of verifiable nerve injury (Boas et al 1996, Scadding et al 1999, Birklein 2005). This condition may be precipitated by a “noxious event” that might include fractures, joint sprains, strains, thoracic surgery, soft tissue injury, and cardiac ischemia and can be of short duration or continue long after the original injury has healed (Dijkstra et al 2003, Baron et al 2005). Idiopathic CRPS-1 occurs after noxious events that are so trivial that they may not even be recalled by the patient (Bonica et al 1990; Baron et al 2005; Oaklander 2008, 2010; Oaklander and Fields 2009). Such injuries would include venipuncture, lacerations, and other types of minor trauma (Bonica et al 1990, Baron et al 2005, Oaklander 2010). Because of difficulty determining the presence of verified nerve injury, inclusion of CPRS-1 as a neuropathic pain state has been challenged (Max 2002). However, evidence is emerging that CRPS-1 is associated with nerve injury but that technologies have been unable to detect such injury until recently (Oaklander 2010).

Minimally invasive techniques to visualize intracutaneous axons in skin biopsy specimens now exist (Oaklander 2010). Rice and colleagues (Albrecht et al 2006) found reduced innervation of the epidermis, sweat glands, and vasculature by small fibers in skin sections obtained from the amputated legs of patients with CRPS-1. Oaklander and co-workers found a 29% reduction in intraepidermal neurites in biopsy samples taken from an affected site when compared with an unaffected site (Oaklander et al 2006). Since venipuncture is a cause of CRPS-1 in susceptible individuals, needlestick of the left tibial nerve of rats (i.e., distal nerve injury [DNI]) was used as a model of this condition (Siegel et al 2007). The left tibial nerve of anesthetized Sprague-Dawley rats was exposed and a flat wooden platform inserted under the nerve. The nerve was pierced through with either a 30-, 22-, or 18-gauge needle and the wound closed (Siegel et al 2007). Paw withdrawal responses to probing with von Frey filaments or a pinprick along with hindpaw position, color, and edema were measured at various times after the injury. Seven days after DNI, 67, 88, and 89% of rats with tibial nerves pierced by the 18-, 22-, and 30-gauge needles, respectively, showed a 50% or greater reduction in the paw withdrawal threshold (Siegel et al 2007). Importantly, the development of mechanical hypersensitivity did not correlate with the size of the lesion. Moreover, 57% of the rats also showed sensitivity of the contralateral hindpaw, a model of the “mirror pain” seen in CRPS patients (Siegel et al 2007). The prevalence of hypersensitivity to pinprick or cold was very low, but 14% of the rats showed abnormal posture as indicated by elevation of the lateral hindpaw along with paw eversion or plantar flexion of all digits (Siegel et al 2007). Evidence of wallerian degeneration was seen in tibial nerve stumps taken 14 days after DNI (Siegel et al 2007).

Ischemic Peripheral Nerve Injury

Injury to the sciatic nerve of mice or rats has been produced by photochemically induced ischemia. The photosensitive dye erythrosin B is injected intravenously, and the exposed sciatic nerve is irradiated with an argon laser with emission at a wavelength of 514 nm (Yu et al 2000). It was found that exposure lasting 30 seconds would selectively injure myelinated fibers and an exposure duration of 2 minutes would cause injury to both myelinated and unmyelinated fibers (Yu et al 2000). Within 1 day of irradiation, the blood vessels of the epineurium and within the fascicles were occluded. Axons demonstrated signs of initiation of degeneration at the site of irradiation (Yu et al 2000). Inflammatory and fibrotic tissue, wallerian degeneration, edema, and demyelination were evident within 7 days. Although the nerves remained thinner than normal, there was evidence of reinnervation after 3 months. The unmyelinated axons appeared to have normal morphology, whereas the myelinated axons were smaller with a thin myelin sheath (Yu et al 2000). Hyperesthesia to light touch and to cold was maximal 7 days after injury and resolved within 3 months. Interestingly, tactile and cold hyperesthesia developed only when both myelinated and unmyelinated fibers were injured. Damage to myelinated axons only was insufficient to produce signs of neuropathic pain (Yu et al 2000). Behavioral signs of spontaneous pain were not evident in this model.

Postischemic Pain

In addition to injury, CRPS-1 may depend partly on localized tissue ischemia (Koban et al 2003, Coderre et al 2004). Studies have demonstrated diminished blood oxygenation in skin capillaries (Koban et al 2003), along with biochemical evidence of anaerobic metabolism (Birklein et al 2000a, 2000b). Tissue obtained from the amputated limbs of patients with CRPS-1 showed signs of oxidative stress and ischemic conditions (van der Laan et al 1998). These observations led to the development of an animal model of CRPS-1 based on ischemia and reperfusion (Coderre et al 2004). Male Long-Evans rats were maintained under pentobarbital anesthesia for a period of 3 hours. A nitrile O-ring, 5.5 mm in diameter, was placed on the left hindlimb proximal to the ankle joint and remained there for 3 hours. This produced the equivalent of a tourniquet inflated to a pressure of 350 mm Hg (Coderre et al 2004). The ring was removed, anesthesia was terminated, and the rats recovered during reperfusion (Coderre et al 2004). Sham rats were prepared in an identical fashion but with a loosely fitting O-ring. Rats showed elevated paw temperature 5 minutes after reperfusion that lasted 2 hours and extravasation lasting from 2–12 hours after reperfusion (Coderre et al 2004). Rats with postischemic pain demonstrated behavioral hypersensitivity to light touch with von Frey filaments, to pinprick, and to cold stimuli in the form of an acetone drop that lasted up to 4 weeks after the procedure (Coderre et al 2004). Thermal hyperalgesia was not observed. Administration of the free radical scavengers N-acetylcysteine and Tempol blocked the behavioral signs of enhanced pain, which is consistent with the role of free oxygen radicals in reperfusion injury and suggests a possible role for these radicals in CRPS-1 (Coderre et al 2004).

Vasculitic Neuropathy

A more widespread ischemia and reperfusion protocol was developed as a model of vasculitic neuropathy (Muthuraman et al 2010). Wistar rats were anesthetized and the femoral artery and nerve isolated. A slip knot formed from 6–0 silk suture was used to constrict the femoral artery near the trifurcation of the sciatic nerve. Reperfusion was achieved by removing the knot after 2, 4, or 6 hours of ischemia. The wound was closed and the animals observed for up to 16 days (Muthuraman et al 2010). The ischemia and reperfusion caused behavioral hypersensitivity to noxious radiant heat or pinprick applied to the hindpaw, as well as to noxious thermal and mechanical stimuli applied to the tail (Muthuraman et al 2010). Moreover, this procedure produced increased blood levels of lipid peroxidation, serum nitrate, and tumor necrosis factor-α (TNF-α) and decreased levels of interleukin-10 (Muthuraman et al 2010). These changes correlated with the duration of the ischemia. Moreover, the ischemia-reperfusion protocol resulted in pathological changes in the sciatic nerve as reflected by a decrease in nerve fiber density, axonal degeneration, and reductions in sensory and motor nerve conduction velocity (Muthuraman et al 2010). The behavioral and neurophysiological evidence suggests that ischemia and reperfusion produce vasculitic neuropathy that mimics the clinical manifestations of CRPS (Muthuraman et al 2010).

Streptozotocin-Induced Diabetes

Insulin-dependent diabetes is thought to cause one of the most prevalent forms of peripheral neuropathy in the developed world (Horowitz 1993). Diabetes-induced degeneration of the microvasculature begins at the most distal regions of the limbs and progresses proximally; it results in progressive localized ischemia and degeneration of neuronal processes and leads to the eventual loss of myelinated and unmyelinated axons (Horowitz 1993). Neuropathic pain secondary to diabetes is difficult to treat and is generally unresponsive to current therapies. A model of diabetes has been developed whereby a single systemic injection of streptozotocin (also referred to as streptozocin) produces progressive and permanent degeneration of the beta cells of the pancreatic islets of Langerhans along with hyperglycemia and loss of serum insulin (Katsilambros et al 1970). Such treatment causes pathological changes in peripheral axons, including large vacuoles, accumulation of neurofilaments, thinning of myelin sheaths, and degeneration of Schwann cells (Katsilambros et al 1970).

There had been conflicting reports regarding the effect of streptozotocin-induced diabetes on nociceptive responses until the model was characterized and standardized by Courteix and colleagues (1993). A single intraperitoneal injection of 75 mg/kg of streptozotocin produced progressively increasing signs of diabetes mellitus, including hyperglycemia, polyuria, polydipsia, and weight loss (Courteix et al 1993). By the fourth week after injection of streptozotocin, the measured blood glucose level was higher than 34 mM, whereas that of control rats was approximately 6.5 mM, and the animals also show signs of hypothermia and decreased motor activity (Courteix et al 1993). Streptozotocin-induced diabetes produces consistent, reliable signs of mechanical hyperalgesia to noxious paw pinch and tactile allodynia to probing with light tactile (von Frey filaments) stimuli (Courteix et al 1993, Porreca et al 2000). Thermal hyperalgesia was demonstrated by significantly decreased tail flick latencies from water baths maintained at normally non-noxious temperatures (i.e., 42–46°C) and by decreased paw withdrawal latencies to radiant heat (Katsilambros et al 1970, Courteix et al 1993). Cold allodynia was demonstrated by significant decreases in tail flick latencies in response to a water bath maintained at 10°C (Courteix et al 1993). Diabetic rats also showed a marked increase in pain responses to injection of formalin into the hindpaw (Courteix et al 1993).

Streptozotocin-induced diabetes is associated with hyperexcitability of nociceptive C fibers in response to sustained noxious mechanical stimuli, although the activation threshold is not changed (Ahlgren and Levine 1994). Approximately one-third of C fibers demonstrated marked hyper-responsiveness to suprathreshold mechanical stimuli that resulted in a three-fold increase in firing rate and faster conduction velocity (Chen and Levine 2001). Electrophysiological studies performed with a skin–nerve preparation reported enhanced frequency and intensity of responses of polymodal C-fiber nociceptors to light tactile stimuli (Suzuki et al 2002). Both basal and capsaicin-evoked release of calcitonin gene–related peptide (CGRP) was doubled in spinal tissue obtained from rats with streptozotocin-induced diabetic neuropathy (Ellington et al 2002). Diabetic rats that were also treated with resiniferatoxin, an ultrapotent analogue of capsaicin that results in desensitization of transient receptor potential vanilloid 1 (TRPV1)-positive small afferents, lost thermal hyperalgesia but maintained tactile allodynia, thus indicating a significant contribution of myelinated fibers to tactile allodynia (Khan et al 2002). Moreover, Aβ and Aδ fibers exhibited ectopic discharges and increased spontaneous activity along with a lower activation threshold and augmented responses to mechanical stimuli (Khan et al 2002).

Importantly, streptozotocin injection elicited a time-dependent expression of behavior together with a progressive increase in the number of animals responding with hyperalgesia, as well as an increase in the intensity of hyperalgesia, over the 4-week observation period (Courteix et al 1996). This time course was said to mimic the slow progression of painful neuropathy in individuals with diabetes (Courteix et al 1996). However, 1 month after the streptozotocin injection, animals would show significant hypoalgesia (Calcutt 2004). Moreover, the thermal hypoalgesia would appear before detectable loss of intraepidermal nerve fibers and may reflect dysfunction of the fibers before complete loss (Beiswenger et al 2008). Thus, the streptozotocin model of diabetic neuropathy may model clinical progression of the disease from the painful stage to the degenerative and painless stage (Calcutt 2004, Beiswenger et al 2008). However, the mechanisms that produce neuropathic pain in this model are still not well understood. The time course of disease progression with periods of normal responses followed by hyperalgesia and then hypoalgesia can produce confusing results. Critically, animals with streptozotocin-induced diabetes also have severe weight loss and overall poor health, which further complicates the interpretation of behavioral end points.

Chemotherapeutic-Induced Neuropathies

Varicella-Zoster Virus

Post-herpetic neuralgia is a common complaint after an outbreak of herpes zoster (Fleetwood-Walker et al 1999, Dalziel et al 2004, Garry et al 2005). After primary infection with varicella-zoster virus (VZV; “chickenpox”), the virus establishes a latent infection of the DRG. Reactivation of the virus results in the production of painful lesions along the infected dermatomes (i.e., herpes zoster or “shingles”). After the lesions have healed and the virus returns to a dormant state, post-herpetic neuralgia, which is characterized by spontaneous burning or aching pain, along with mechanical allodynia and hyperalgesia (Rowbotham and Fields 1996), develops in some (but not all) individuals. The pain of post-herpetic neuralgia is refractory to treatment with many analgesics (Rowbotham and Fields 1996).

An animal model of post-herpetic neuralgia is based on latent VZV infection in rats, where the footpad is injected with approximately 4 × 10⁶ cultured CV-1 cells infected with VZV (Fleetwood-Walker et al 1999). Time-dependent behavioral signs of tactile allodynia and thermal hyperalgesia developed in the rats (Fleetwood-Walker et al 1999). The VZV protein was expressed in DRGs in the absence of neuronal cell death or inflammation, thus suggesting that the neuropathic pain state was caused by the virus (Fleetwood-Walker et al 1999). Moreover, injection of killed virus did not produce neuropathic signs, and the antiviral drug valacyclovir blocked the development of allodynia in infected rats (Dalziel et al 2004). Infection with VZV also resulted in expression of the VZV immediate–early protein IE62 in myelinated and unmyelinated sensory DRG neurons ipsilateral but not contralateral to the injection (Garry et al 2005). Moreover, VZV also elicited up-regulation of neuropeptide Y (NPY) in myelinated DRG neurons (Garry et al 2005), which is also seen after traumatic injury and may mediate tactile hypersensitivity (Ossipov et al 2002, Garry et al 2005). These observations reveal a similarity with models of peripheral nerve injury as a result of trauma and are consistent with VZV infection producing a neuropathic pain state (Garry et al 2005).

gp120-Induced Model of HIV Neuropathy

Infection with human immunodeficiency virus (HIV) can cause neuropathic pain. The HIV envelope protein gp120 binds to epitopes and activates microglia and astrocytes, thereby evoking behavioral signs of neuropathic pain, in part by the abundant production of cytokines, TNF-α, eicosanoids, nitric oxide, excitatory amino acids, and other pro-nociceptive mediators (Milligan et al 2000, Herzberg and Sagen 2001). Spinal administration of recombinant gp120 produced enhanced behavioral responses to thermal stimuli and to light touch within 30 minutes of injection (Milligan et al 2000). It also elicited touch-evoked agitation, as indicated by aggression, biting, and vocalization in response to light brushing of the hindquarters (Milligan et al 2000). Spinal injections of heat-denatured gp120 were inactive. The glial inhibitors fluorocitrate and CNI-1493 blocked the behavioral signs of neuropathic pain elicited by gp120 (Milligan et al 2000). Application of gp120 in bands of oxidized cellulose containing 40 or 400 ng of gp120 loosely wrapped around the sciatic nerve produced swelling, edema, and muscular weakness for 5–7 days after application (Herzberg and Sagen 2001). Within 2–4 days of gp120 exposure, the rats expressed behavioral signs of tactile allodynia, mechanical and thermal hyperalgesia, and cold allodynia (Herzberg and Sagen 2001). These behavioral signs of neuropathic pain were evident for 3–4 weeks (Herzberg and Sagen 2001). Edema, axonal swelling, and immunostaining for TNF-α were evident within the sciatic nerve trunk within 5 days of gp120 application but resolved within 3 weeks and were suggestive of neuritis (Herzberg and Sagen 2001). Activation of astrocytes and microglia in the L5 and L6 spinal dorsal horn was evident between 5 and 30 days after gp120 application. These studies indicate that peripheral application of gp120 may elicit a neuropathic pain state that is independent of direct neurogenic inflammation (Milligan et al 2000, Herzberg and Sagen 2001). It is possible that gp120 or inflammatory mediators released by exposure to gp120 are transported to the spinal cord and initiate a pro-nociceptive cascade, possibly through spinal glial activation (Milligan et al 2000, Herzberg and Sagen 2001). Binding of gp120 to the CXCR4 chemokine receptor of Schwann cells causes release of regulated upon activation, normal T-cell expressed and secreted (RANTES), which induces TNF-α production in DRG neurons and thereby leads to neurotoxicity (Keswani et al 2003). Addition of gp120 to DRG cultures resulted in reduced neurite growth and evidence of apoptosis (Melli et al 2006, Hoke et al 2009). The neurotoxicity induced by gp120 may be mediated by the release of Ca²⁺ from storage sites in the endoplasmic reticulum, possibly through activation of the CXCR4 chemokine receptor (Hoke et al 2009). More recent studies involving transgenic mice expressing the gp120 protein in astrocytes under the control of a modified GFAP (glial fibrillary acidic protein) gene showed that the retroviral drugs used to treat HIV promote HIV-induced neuropathy. Degeneration of dendrites, loss of presynaptic terminals, and reactive astrocytosis develop in these transgenic mice (Keswani et al 2006). Daily oral administration of the dideoxynucleoside reverse transcriptase inhibitor didanosine produced significant axonal degeneration of unmyelinated peripheral neurons in these transgenic mice (Keswani et al 2006). This effect corresponds to the dying back of distal sensory axons of peripheral nerves seen in patients with HIV (Keswani et al 2006). Perineural delivery of gp120 to the sciatic nerve resulted in decreased intraepidermal nerve fiber density and provoked macrophage infiltration into the area of gp120 infiltration (Wallace et al 2007). Moreover, gp120 infiltration resulted in increased expression of activated transcription factor 3 (ATF3), galanin, and NPY in DRG neurons, but no change in TRPV1 and CGRP (Wallace et al 2007). Thermal hypersensitivity was blocked by systemic morphine and gabapentin, but not by amitriptyline (Wallace et al 2007). Spinal gliosis, indicated by activation of microglia and astrocytes in the spinal cord, represents a means through which HIV infection could promote central neuropathology even in the absence of direct infection of the central nervous system (Wallace et al 2007).

Spinal Cord Injury Models of Central Neuropathic Pain

In addition to peripheral nerve injury, behavioral signs of neuropathic pain may be evident after nerve damage that occurs within the central nervous system. Traumatic spinal cord injury may be produced by graded contusions of the cord. The sequelae of traumatic or ischemic spinal cord injury have been modeled by spinal injection of the excitatory amino acids quisqualic acid and kainic acid. These and related models have provided a significant contribution to our understanding of centrally mediated neuropathic pain states. Pain associated with spinal cord injury is discussed in Chapter 68.

Dynamic and Static Allodynia

Patients with neuropathic pain may have either static allodynia, which can be evoked by pressing the neuropathic region, or dynamic allodynia, which is evoked by lightly brushing the affected skin, or both forms of mechanical allodynia may be present (Koltzenburg et al 1992, Ochoa and Yarnitsky 1993). Studies performed with differential nerve blocks achieved through the application of a tourniquet or injections of local anesthetics revealed that dynamic allodynia is probably mediated through myelinated A fibers and static allodynia through unmyelinated C fibers (Koltzenburg et al 1992, Ochoa and Yarnitsky 1993). Animal models were used to explore the different responses of static and dynamic allodynia to potential therapeutic interventions. Animals with either CCI or SNL demonstrated dynamic allodynia in response to stroking the hindpaw and static allodynia with the application of von Frey filaments (Field et al 1999a). Systemic morphine blocked static allodynia but did not alter dynamic allodynia (Field et al 1999a). In contrast, both forms of mechanical allodynia were blocked by pregabalin (Field et al 1999a). Thermal hyperalgesia and static allodynia were blocked by capsaicin injected into the hindpaw, whereas dynamic allodynia was unaffected, thus leading to the conclusion that dynamic allodynia is mediated through Aβ and capsaicin-insensitive Aδ fibers (Field et al 1999a). Static and dynamic allodynia was also observed in rats with streptozotocin-induced diabetic neuropathy (Field et al 1999b). Systemic morphine and amitriptyline both produced a dose-dependent block of static, but not dynamic allodynia, whereas gabapentin and pregabalin blocked both forms of mechanical allodynia (Field et al 1999b). These observations indicate that patients with mechanical allodynia may respond differently to a given therapeutic regimen, depending on the form of mechanical allodynia present.

Assessment of Spontaneous or Ongoing Pain

An important criticism directed against animal models of neuropathic pain and its behavioral quantification is that such assessments depend on evoked responses to innocuous or noxious external stimuli. The principal complaint from patients with neuropathic pain, however, is ongoing pain that may be continuous or paroxysmal (i.e., pain that is independent of an external stimulus). The reliance of testing protocols on evoking a nocifensive response has been considered a significant impediment to the development of new therapies for treatment of pain (Campbell and Meyer 2006, Rice et al 2008, Vierck et al 2008). Consequently, effort has been directed toward the development of animal models designed to evaluate spontaneous, or stimulus-independent, pain in neuropathic models. Evidence confirming the presence of spontaneous pain in experimental models of neuropathic pain has been lacking until recently.

Place Escape Avoidance Paradigm

Pain is aversive and provides motivation to avoid stimuli that would produce or enhance pain (Price et al 1980). Fuchs and colleagues (LaBuda and Fuchs 2000) developed an important protocol to measure the aversive qualities of pain based on avoidance of a location that has been associated with the application of a stimulus to an inflamed or nerve-injured paw. This assay, the “place escape avoidance paradigm,” provided significant mechanistic insight into the aversive nature of experimental neuropathic pain (LaBuda and Fuchs 2000). Rats with ligation of the L5 spinal nerve or hindpaw injection of complete Freund’s adjuvant are placed in a two-chamber box, with one chamber being light and the other dark and the floor made of mesh to allow the application of von Frey filaments to the hindpaw. The animals are allowed unrestricted movement during the 30-minute testing paradigm. While the rats are in the dark chamber, mechanical stimuli (476-mN von Frey filament) are applied at 15-second intervals to the hindpaw ipsilateral to the injury or inflammation, and while the animal is in the light chamber, the stimulus is applied to the contralateral hindpaw (LaBuda and Fuchs 2000). The rats with either nerve injury or inflammation spent a significantly greater amount of time in the light chamber, thus suggesting avoidance of the chamber associated with hyperalgesia, whereas the control groups consisting of sham-operated or vehicle-injected rats spent an equivalent amount of time in each chamber (LaBuda and Fuchs 2000). Additionally, it was noted that although the rats would occasionally explore the dark chamber, they would leave before application of the next stimulus, which suggests that the rats would anticipate and avoid the stimulus applied to the hyperalgesic hindpaw (LaBuda and Fuchs 2000).

Additionally, lesions of the anterior cingulate cortex (ACC) (LaGraize et al 2004) or micro-injection of morphine therein (LaGraize et al 2006) blocked escape/avoidance without altering responses to evoked hyperalgesia, thereby providing additional support that this region modulates affective responses to pain and hyperalgesia and demonstrating the relevance of this assay (LaGraize et al 2004, 2006).

Intrathecal Self-Administration in Experimental Pain States

A novel methodology was developed in which intrathecal self-administration was used to determine the effectiveness of a treatment against spontaneous neuropathic pain. The α₂-adrenergic agonist clonidine has been used successfully for the treatment of neuropathic pain clinically and has been effective against evoked measures of neuropathic pain in animal models (Xu et al 1992). Because clonidine does not have reinforcing properties, it is not expected that rats would self-administer this drug unless the animal received some benefit, presumably pain relief (Martin et al 2006). Rats with SNL and with tactile hypersensitivity were placed in operant conditioning chambers and trained to press a lever to administer drug or saline. Rats received 10 μg of clonidine intrathecally when one lever was pressed. Even though uninjured rats and rats with SNL showed similar rates of lever pressing during the first day, the response of uninjured animals decreased over the following 4 days. In contrast, the rats with SNL showed significant increases in the number of clonidine infusions (Martin et al 2006). Changing the infusion to deliver 3 or 30 μg of clonidine resulted in a corresponding change in the number of lever presses required, and a dose-dependent effect was demonstrated (Martin et al 2006). The self-administration behavior was extinguished by substituting saline for clonidine and was abolished by the α₂-adrenergic antagonist idazoxan (Martin et al 2006). Since uninjured rats self-administered clonidine at a similar rate as saline whereas rats with SNL showed increased clonidine self-administration, it was suggested that the pain relief obtained provided the rewarding cue that reinforced the self-administration behavior (Martin et al 2006). Thus, this method probably tests motivational aspects related to neuropathic pain. This premise was emphasized with the self-administration of opioids. It was found that rats with SNL would self-administer opioids less frequently and maintain lower drug intake than sham-operated animals would (Martin et al 2006). Moreover, the maximum doses of opioids that the rats with SNL would self-administer correlated with loss of the behavioral signs of tactile allodynia. It was suggested that the reward mechanisms influencing opioid consumption are altered with nerve injury and that the pain relief itself might be rewarding. This is further supported by observations that intrathecal clonidine reduced opioid self-administration in rats with SNL relative to sham-operated rats (Martin et al 2006, 2007; Martin and Ewan 2008).

Negative Reinforcement with Conditioned Place Preference

The development of this approach (King et al 2009) was based on the knowledge that relief of pain is rewarding in humans (Seymour et al 2005). Pain has a strong emotional component as exemplified by its unpleasantness, and chronic pain produces an aversive state (Johansen et al 2001, Vierck et al 2008). The unpleasantness of pain serves as the “teaching signal” that forces avoidance of stimuli that can potentially produce damage to tissues (Price 2000, Johansen et al 2001, King et al 2009). Negative reinforcement denotes the motivation to escape an aversive stimulus (i.e., pain). For this reason, pairing pain relief with a context resulted in negative enforcement (King et al 2009) and led to the demonstration of “unmasking” of spontaneous experimental neuropathic pain. In this way, drugs that do not normally elicit reward do so in the presence of chronic pain. Validation of this model for detecting ongoing, or non-evoked, pain was performed in rats with SNL or SNI placed in conditioned place preference (CPP) boxes consisting of three chambers. Two chambers separated by a neutral chamber had different visual and textural characteristics. After a period of preconditioning, a non-active control treatment is paired with one chamber and a treatment demonstrated to be effective for human neuropathic pain with the other chamber. Clonidine or ω-conotoxin delivered spinally to rats with nerve injury blocked the behavioral signs of tactile allodynia. Critically, these treatments produced place preference selectively in animals with nerve injury, thus indicating that the animals preferred the chamber where pain relief occurred (King et al 2009). However, spinally injected adenosine, which abolished the behavioral signs of tactile allodynia in the rats with SNL, did not produce any evidence of CPP (King et al 2009). Clinically, spinal clonidine (Uhle et al 2000, Krames 2002, Ackerman et al 2003) and ω-conotoxin (ziconotide, generic for PRIALT) (Rauck et al 2009) are both effective against ongoing neuropathic pain, whereas spinal adenosine was reported to block hyperalgesia, but not ongoing pain, in a small clinical study (Eisenach et al 2003). Moreover, CPP was observed selectively in rats with nerve injury following micro-injection of lidocaine into the rostral ventromedial (RVM) medulla, thus demonstrating the relevance of descending pain modulation to spontaneous neuropathic pain. Animals with either SNL or SNI showed CPP, a finding indicative of independence of the injury model. These effects were demonstrated by interventions that are made outside the reward pathway (i.e., the spinal cord or RVM medulla) and with drugs that do not directly activate the reward pathway (i.e., ω-conotoxin, clonidine, lidocaine). Finally, place preference could be demonstrated in animals after sciatic axotomy or sciatic/saphenous axotomy with either RVM lidocaine or spinal clonidine (Qu et al 2011). The demonstration of CPP in animals with complete denervation of the hindpaw strongly supported the fact that the approach reflected the presence of spontaneous pain rather than any tactile stimulation that might be associated with ambulation within the testing chamber. Additionally, this result suggested that injured nerve fibers can mediate pain in animals, consistent with observations in humans that aggravating a neuroma would produce pain whereas local anesthesia near the neuroma would reduce pain (Gracely et al 1992). The place preference associated with pain relief in rats with SNL was prevented by a lesion of the rostral ACC (Qu et al 2011), thus suggesting that the procedure was evaluating the aversive state produced by peripheral nerve injury. Place preference was produced with a single pairing, a result suggesting that the peripheral nerve injury produces significant spontaneous pain. Adoption of the CPP method for evaluation of relief of neuropathic pain has the potential to allow investigation of the mechanism and offers the possibility of improving translation to human therapeutics.

Pain-Induced Aversion

A conditioned place aversion paradigm was used to assess the affective/motivational and the sensory aspects of neuropathic pain (Hummel et al 2008). Rats with nerve injury and tactile allodynia were placed in a two-chamber conditioning box 3 weeks after surgery and allowed to roam freely for 30 minutes. Conditioning sessions were performed by randomly assigning one-half of the sham-operated and the SNL rats to a black chamber paired with a noxious stimulus (15-g von Frey filament) or innocuous stimulus (1-g von Frey filament) and a novel item (Hummel et al 2008). The remaining rats were placed in a striped chamber paired with the same stimuli. Following a 5-minute adaptation period, the left hindpaw of the rats was stimulated with the filament every minute for 15 minutes. For 5 consecutive days the rats would receive one stimulus in one chamber in the morning and the opposite stimulus in the other chamber 4 hours later (Hummel et al 2008). The sequence of innocuous and noxious exposure alternated daily and the novel objects were changed daily (Hummel et al 2008). Morphine-treated rats received the drug 45 minutes before the pain-pairing session. The day after the last conditioning session, the rats were allowed free access to both chambers, and time spent in each one was determined by an unbiased observer who reviewed video recordings of the sessions (Hummel et al 2008). The rats with SNL developed a strong, significant aversion to the pain-paired chamber, as indicated by a reduction in time spent in this chamber (Hummel et al 2008). Importantly, the aversion was still present 1 month after the last conditioning trial. In contrast, the sham-operated control animals did not show a preference (Hummel et al 2008). Pretreatment with doses of morphine that are not analgesic or rewarding attenuated the pain-induced aversion in rats with SNL. This method provides a means to study the negative affect associated with painful conditions and allows exploration of how the memory of pain may have an impact on motivation and affect (Hummel et al 2008).

Ultrasonic Vocalization

Measurement of ultrasonic vocalizations in rodents as an indicator of pain has been attempted numerous times with contradictory findings (Jourdan et al 2002, Han et al 2005, Wallace et al 2005, Williams et al 2008). Possible confounding factors included vocalizations as a result of stress, a novel environment, or immobilization, which prevented accurate quantification of spontaneous pain (Kurejova et al 2010). These factors were minimized in mice via acclimatization to the testing chamber by permitting free roaming and isolation from background noise (Kurejova et al 2010). Additionally, recordings were performed at 37 and 50 kHz, with avoidance of the 22-kHz signal, which is associated with stress or alarm (Kurejova et al 2010). Increased ultrasonic vocalizations were observed in mice with SNI 2 to 4 weeks after the injury, and vocalizations returned to baseline levels by the sixth week (Kurejova et al 2010). In contrast, vocalizations were evoked by probing with von Frey filaments up to 8 weeks after SNI, thus suggesting that the ongoing pain was shorter in duration relative to the evoked pain (Kurejova et al 2010). Gabapentin, which is effective clinically against neuropathic pain and shows efficacy in animals models of neuropathy, also blocked ultrasonic vocalizations in mice with SNI (Kurejova et al 2010).

Flinching and Guarding Behavior in Bone Cancer

Injection of NCTC 2472 osteolytic tumor cells into the femur of mice produces tumor growth in bone along with sensitization and damage to the sensory fibers innervating bone, thus introducing a neuropathic component to bone cancer pain (Peters et al 2005). Mice with experimental bone cancer show behavioral signs of spontaneous pain, as measured by scoring flinching behavior, limb use, and locomotion (Luger et al 2001). Spontaneous and touch-evoked flinches, defined as raising the hindlimb and holding it up while not ambulatory, are counted (Luger et al 2001). Limb use is scored from 0–4, with 0 indicating complete lack of limb use and 4 indicating normal activity (Luger et al 2001). Forced ambulatory guarding is determined on a Rotorod and scored such that 0 indicates normal limb use and 5 indicates complete lack of use of the limb (Luger et al 2001). Gabapentin blocked these behavioral signs of spontaneous pain in this model (Peters et al 2005). Gabapentin did not alter progression of the disease or reduce activity, thus suggesting that these behavioral measures were indicative of a neuropathic component of ongoing pain (Peters et al 2005).

Spontaneous Foot Lift

Although “spontaneous foot lifting” (SFL) following injury to peripheral nerves was suggested as an indicator of spontaneous neuropathic pain (Bennett and Xie 1988, Choi et al 1994), it has not been validated as such, in part because foot lifting occurs in the absence of injury and in other pain states as well. Furthermore, the presence of SFL behavior is inconsistent in models of neuropathic pain. Although SFL was observed in the CCI model (Bennett and Xie, 1988), it is rarely observed after SNL (Djouhri et al 2006). No correlation was found between SFL and afferent activity (Djouhri et al 2006). Loose ligation of the L4 spinal nerve produced SFL only when the potential inflammatory chromic gut suture was used (Djouhri et al 2006). It was concluded that inflammation elicits spontaneous activity in C-fiber nociceptors and is the critical drive for SFL behavior (Djouhri et al 2006).

Mouse Grimace Scale

Development of a method of coding the facial expressions of mice was based on the premise that non-primate animals show facial expressions associated with their emotional state (Langford et al 2010). Five facial features observed in several presumably painful conditions were designated as “action units” (AUs) and were scored by observers monitoring videotape of the mice. A score of 0 indicated a high degree of confidence by the observer that the AU was not present, 1 indicated equivocation, and 2 indicated a high degree of confidence that the AU was present (Langford et al 2010). The AUs were orbital tightening, indicated by tightening of the orbital area or a tightly closed eyelid; nose bulge, indicated by rounded skin on the bridge of the nose; cheek bulge; pulling apart of the ears from a baseline or normal position; and movement of whiskers away from the normal or baseline position. Grimace was evaluated following acute nociception, such as noxious radiant heat or warm-water immersion, as well as longer-lasting conditions induced by formalin injection, paw incision, mustard oil application, and intraplantar zymosan. Stimuli lasting from 10 minutes to 4 hours were most likely associated with evidence of pain according to the mouse grimace scale (MGS). The MSG, however, failed to detect spontaneous pain in the CCI and SNI nerve injury models, thus preventing this assay from being used for neuropathic pain (Langford et al 2010). Additionally, lesion of the rostral ACC or the amygdala with ibotenic acid did not block the facial grimace produced by intraperitoneal acetic acid (Langford et al 2010). This assay is acknowledged to be extraordinarily labor-intensive and has yet to be validated with therapies that are clinically effective or ineffective.

Changes in Natural Behavior as Outcomes

Burrowing is a highly conserved natural behavior of laboratory rats, and disruption of burrowing is indicative of behavioral dysfunction (Deacon 2006). It has been suggested that ongoing pain behavior could be estimated by placing a tube filled with a weighed amount of gravel in the animal’s cage. The amount of gravel removed because of burrowing is determined by weighing the amount remaining and subtracting from the total (Andrews et al 2011). Rats with L5 spinal nerve transection, tibial nerve transection, or partial sciatic nerve ligation showed a significant reduction in burrowing behavior that was reversed by a non-sedating dose of gabapentin (Andrews et al 2011). Importantly, reduction in burrowing did not correlate with evoked indices of neuropathic pain, thus suggesting that different mechanisms are being examined (Andrews et al 2011). Since burrowing behavior is indicative of the animal’s state of “well-being” (Deacon 2006, Cunningham et al 2007), it is suggested that this behavior may be useful in examining ongoing pain from different manipulations (Andrews et al 2011). However, interpretation of reduced burrowing activity is also influenced by other behavioral factors. For example, the sedation induced by high doses of gabapentin also reduced burrowing activity in animals with nerve injury (Andrews et al 2011).

The behavior of rodents placed in an open-field paradigm is a classic protocol for determining the “emotionality” of rodents and is often used in experimental psychology, especially for measuring stress and anxiety (Ivinskis 1970, Geerse et al 2006). Animals showing increased defecation rates and/or reduced locomotor activity are regarded to be in a state of high emotional reactivity in this paradigm (Ivinskis 1970, Geerse et al 2006). Geerse and colleagues (2006) suggested a correlation between prior stressful experience and hyperalgesia that was uncovered by application of the open-field paradigm. Seltzer and colleagues found that animals showing high emotionality on the open-field test exhibited greater rates of autotomy after peripheral nerve injury (Vatine et al 2000). Although the open-field paradigm does not measure pain directly, it appears to predict animals that may show greater sensitivity to pain after nerve injury or inflammation and may have important implications in work aimed at conditions such as post-traumatic stress syndrome (Vatine et al 2000, Geerse et al 2006).

Ackerman L.L., Follett K.A., Rosenquist R.W. Long-term outcomes during treatment of chronic pain with intrathecal clonidine or clonidine/opioid combinations. Journal of Pain and Symptom Management. 2003;26:668–677.

Ahlgren S.C., Levine J.D. Protein kinase C inhibitors decrease hyperalgesia and C-fiber hyperexcitability in the streptozotocin-diabetic rat. Journal of Neurophysiology. 1994;72:684–692.

Albrecht P.J., Hines S., Eisenberg E., et al. Pathologic alterations of cutaneous innervation and vasculature in affected limbs from patients with complex regional pain syndrome. Pain. 2006;120:244–266.

Aley K.O., Reichling D.B., Levine J.D. Vincristine hyperalgesia in the rat: a model of painful vincristine neuropathy in humans. Neuroscience. 1996;73:259–265.

Andrews N., Legg E., Lisak D., et al. Spontaneous burrowing behaviour in the rat is reduced by peripheral nerve injury or inflammation associated pain. European Journal of Pain. 2012;16(4):485–495.

Authier N., Balayssac D., Marchand F., et al. Animal models of chemotherapy-evoked painful peripheral neuropathies. Neurotherapeutics. 2009;6:620–629.

Authier N., Gillet J.P., Fialip J., et al. Description of a short-term Taxol-induced nociceptive neuropathy in rats. Brain Research. 2000;887:239–249.

Baron R., Binder A., Pappagallo M. Complex regional pain syndromes. In: Pappagallo M., ed. The neurological basis of pain. New York: McGraw-Hill; 2005:359–378.

Beiswenger K.K., Calcutt N.A., Mizisin A.P. Dissociation of thermal hypoalgesia and epidermal denervation in streptozotocin-diabetic mice. Neuroscience Letters. 2008;442:267–272.

Bennett G.J. Pathophysiology and animal models of cancer-related painful peripheral neuropathy. Oncologist. 2010;15(Suppl 2):9–12.

Bennett G.J., Xie Y.K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107.

Birklein F. Complex regional pain syndrome. Journal of Neurology. 2005;252:131–138.

Birklein F., Riedl B., Sieweke N., et al. Neurological findings in complex regional pain syndromes—analysis of 145 cases. Acta Neurologica Scandinavica. 2000;101:262–269.

Birklein F., Weber M., Ernst M., et al. Experimental tissue acidosis leads to increased pain in complex regional pain syndrome (CRPS). Pain. 2000;87:227–234.

Boas R.A., Jänig W., Stanton-Hicks M. Complex regional pain syndromes: symptoms, signs and differential diagnosis. In: Jänig W., Stanton-Hicks M., eds. Progress in pain research and management. Seattle: IASP Press; 1996:79–92.

Bonica J.J., Loeser J.D., Chapman C.R., et al. Causalgia and other reflex sympathetic dystrophies. In: Bonica J.J., ed. The management of pain. Philadelphia: Lea & Febiger; 1990:220–243.

Calcutt N.A. Experimental models of painful diabetic neuropathy. Journal of the Neurological Sciences. 2004;220:137–139.

Campbell J.N., Meyer R.A. Mechanisms of neuropathic pain. Neuron. 2006;52:77–92.

Catheline G., Le Guen S., Honore P., et al. Are there long-term changes in the basal or evoked Fos expression in the dorsal horn of the spinal cord of the mononeuropathic rat? Pain. 1999;80:347–357.

Chaplan S.R., Bach F.W., Pogrel J.W., et al. Quantitative assessment of tactile allodynia in the rat paw. Journal of Neuroscience Methods. 1994;53:55–63.

Chapman V., Suzuki R., Dickenson A.H. Electrophysiological characterization of spinal neuronal response properties in anaesthetized rats after ligation of spinal nerves L5-L6. Journal of Physiology. 1998;507:881–894.

Chen X., Levine J.D. Hyper-responsivity in a subset of C-fiber nociceptors in a model of painful diabetic neuropathy in the rat. Neuroscience. 2001;102:185–192.

Chichorro J.G., Zampronio A.R., Rae G.A. Endothelin ET(B) receptor antagonist reduces mechanical allodynia in rats with trigeminal neuropathic pain. Experimental Biology and Medicine. 2006;231:1136–1140.

Choi Y., Yoon Y.W., Na H.S., et al. Behavioral signs of ongoing pain and cold allodynia in a rat model of neuropathic pain. Pain. 1994;59:369–376.

Cliffer K.D., Siuciak J.A., Carson S.R., et al. Physiological characterization of Taxol-induced large-fiber sensory neuropathy in the rat. Annals of Neurology. 1998;43:46–55.

Coderre T.J., Xanthos D.N., Francis L., et al. Chronic post-ischemia pain (CPIP): a novel animal model of complex regional pain syndrome-type I (CRPS-I; reflex sympathetic dystrophy) produced by prolonged hindpaw ischemia and reperfusion in the rat. Pain. 2004;112:94–105.

Coggeshall R.E., Dougherty P.M., Pover C.M., et al. Is large myelinated fiber loss associated with hyperalgesia in a model of experimental peripheral neuropathy in the rat? Pain. 1993;52:233–242.

Costigan M., Scholz J., Woolf C.J. Neuropathic pain: a maladaptive response of the nervous system to damage. Annual Review of Neuroscience. 2009;32:1–32.

Courteix C., Bardin M., Massol J., et al. Daily insulin treatment relieves long-term hyperalgesia in streptozocin diabetic rats. Neuroreport. 1996;7:1922–1924.

Courteix C., Eschalier A., Lavarenne J. Streptozocin-induced diabetic rats: behavioural evidence for a model of chronic pain. Pain. 1993;53:81–88.

Cunningham C., Campion S., Teeling J., et al. The sickness behaviour and CNS inflammatory mediator profile induced by systemic challenge of mice with synthetic double-stranded RNA (poly I: C). Brain, Behavior, and Immunity. 2007;21:490–502.

Dalziel R.G., Bingham S., Sutton D., et al. Allodynia in rats infected with varicella zoster virus—a small animal model for post-herpetic neuralgia. Brain Research. Brain Research Reviews. 2004;46:234–242.

Deacon R.M. Burrowing in rodents: a sensitive method for detecting behavioral dysfunction. Nature Protocols. 2006;1:118–121.

Decosterd I., Woolf C.J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain. 2000;87:149–158.

De Felice M., Sanoja R., Wang R., et al. Engagement of descending inhibition from the rostral ventromedial medulla protects against chronic neuropathic pain. Pain. 2011;152:2701–2709.

Devor M. Sensory basis of autotomy in rats. Pain. 1991;45:109–110.

Devor M., Govrin-Lippmann R. Selective regeneration of sensory fibers following nerve crush injury. Experimental Neurology. 1979;65:243–254.

Dijkstra P.U., Groothoff J.W., Jan ten Duis H., et al. Incidence of complex regional pain syndrome type I after fractures of the distal radius. European Journal of Pain. 2003;7:457–462.

Dina O.A., Chen X., Reichling D., et al. Role of protein kinase C epsilon and protein kinase A in a model of paclitaxel-induced painful peripheral neuropathy in the rat. Neuroscience. 2001;108:507–515.

Djouhri L., Koutsikou S., Fang X., et al. Spontaneous pain, both neuropathic and inflammatory, is related to frequency of spontaneous firing in intact C-fiber nociceptors. Journal of Neuroscience. 2006;26:1281–1292.

Eisenach J.C., Rauck R.L., Curry R. Intrathecal, but not intravenous adenosine reduces allodynia in patients with neuropathic pain. Pain. 2003;105:65–70.

Ellington H.C., Cotter M.A., Cameron N.E., et al. The effect of cannabinoids on capsaicin-evoked calcitonin gene–related peptide (CGRP) release from the isolated paw skin of diabetic and non-diabetic rats. Neuropharmacology. 2002;42:966–975.

Field M.J., Bramwell S., Hughes J., et al. Detection of static and dynamic components of mechanical allodynia in rat models of neuropathic pain: are they signalled by distinct primary sensory neurones? Pain. 1999;83:303–311.

Field M.J., McCleary S., Hughes J., et al. Gabapentin and pregabalin, but not morphine and amitriptyline, block both static and dynamic components of mechanical allodynia induced by streptozocin in the rat. Pain. 1999;80:391–398.

Flatters S.J., Bennett G.J. Studies of peripheral sensory nerves in paclitaxel-induced painful peripheral neuropathy: evidence for mitochondrial dysfunction. Pain. 2006;122:245–257.

Fleetwood-Walker S.M., Quinn J.P., Wallace C., et al. Behavioural changes in the rat following infection with varicella-zoster virus. Journal of General Virology. 1999;80:2433–2436.

Garry E.M., Delaney A., Anderson H.A., et al. Varicella zoster virus induces neuropathic changes in rat dorsal root ganglia and behavioral reflex sensitisation that is attenuated by gabapentin or sodium channel blocking drugs. Pain. 2005;118:97–111.

Geerse G.-J., van Gurp L.C.A., Wiegant V.M., et al. Individual reactivity to the open-field predicts the expression of stress-induced behavioural and somatic pain sensitisation. Behavioural Brain Research. 2006;174:112–118.

Gracely R.H., Lynch S.A., Bennett G.J. Painful neuropathy: altered central processing maintained dynamically by peripheral input. Pain. 1992;51:175–194.

Han J.S., Bird G.C., Li W., et al. Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior. Journal of Neuroscience Methods. 2005;141:261–269.

Hao J.X., Stohr T., Selve N., et al. Lacosamide, a new anti-epileptic, alleviates neuropathic pain–like behaviors in rat models of spinal cord or trigeminal nerve injury. European Journal of Pharmacology. 2006;553:135–140.

Herzberg U., Sagen J. Peripheral nerve exposure to HIV viral envelope protein gp120 induces neuropathic pain and spinal gliosis. Journal of Neuroimmunology. 2001;116:29–39.

Hoke A., Morris M., Haughey N.J. GPI-1046 protects dorsal root ganglia from gp120-induced axonal injury by modulating store-operated calcium entry. Journal of the Peripheral Nervous System. 2009;14:27–35.

Horowitz S.H. Diabetic neuropathy. Clinical Orthopaedics and Related Research. 1993;296:78–85.

Hummel M., Lu P., Cummons T.A., et al. The persistence of a long-term negative affective state following the induction of either acute or chronic pain. Pain. 2008;140:436–445.

Ivinskis A. A study of validity of open-field measures. Australian Journal of Psychology. 1970;22:175–183.

Jasmin L., Kohan L., Franssen M., et al. The cold plate as a test of nociceptive behaviors: description and application to the study of chronic neuropathic and inflammatory pain models. Pain. 1998;75:367–382.

Johansen J.P., Fields H.L., Manning B.H. The affective component of pain in rodents: direct evidence for a contribution of the anterior cingulate cortex. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:8077–8082.

Jourdan D., Ardid D., Eschalier A. Analysis of ultrasonic vocalisation does not allow chronic pain to be evaluated in rats. Pain. 2002;95:165–173.

Katsilambros N., Rahman Y.A., Hinz M., et al. Action of streptozotocin on insulin and glucagon responses of rat islets. Hormone and Metabolic Research. 1970;2:268–270.

Kayser V., Aubel B., Hamon M., et al. The antimigraine 5-HT 1B/1D receptor agonists, sumatriptan, zolmitriptan and dihydroergotamine, attenuate pain-related behaviour in a rat model of trigeminal neuropathic pain. British Journal of Pharmacology. 2002;137:1287–1297.

Kehlet H., Jensen T.S., Woolf C.J. Persistent postsurgical pain: risk factors and prevention. Lancet. 2006;367:1618–1625.

Keswani S.C., Jack C., Zhou C., et al. Establishment of a rodent model of HIV-associated sensory neuropathy. Journal of Neuroscience. 2006;26:10299–10304.

Keswani S.C., Polley M., Pardo C.A., et al. Schwann cell chemokine receptors mediate HIV-1 gp120 toxicity to sensory neurons. Annals of Neurology. 2003;54:287–296.

Khan G.M., Chen S.R., Pan H.L. Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats. Neuroscience. 2002;114:291–299.

Kilpatrick T.J., Phan S., Reardon K., et al. Leukaemia inhibitory factor abrogates paclitaxel-induced axonal atrophy in the Wistar rat. Brain Research. 2001;911:163–167.

Kim J.H., Suh S.I., Seol H.Y., et al. Regional grey matter changes in patients with migraine: a voxel-based morphometry study. Cephalalgia. 2008;28(6):598–604.

Kim K.J., Yoon Y.W., Chung J.M. Comparison of three rodent neuropathic pain models. Experimental Brain Research. 1997;113:200–206.

Kim S.H., Chung J.M. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain. 1992;50:355–363.

King T., Vera-Portocarrero L., Gutierrez T., et al. Unmasking the tonic-aversive state in neuropathic pain. Nature Neuroscience. 2009;12:1364–1366.

Koban M., Leis S., Schultze-Mosgau S., et al. Tissue hypoxia in complex regional pain syndrome. Pain. 2003;104:149–157.

Koltzenburg M., Lundberg L.E., Torebjork H.E. Dynamic and static components of mechanical hyperalgesia in human hairy skin. Pain. 1992;51:207–219.

Krames E. Implantable devices for pain control: spinal cord stimulation and intrathecal therapies. Best Practice & Research. Clinical Anaesthesiology. 2002;16:619–649.

Kruger L. The non-sensory basis of autotomy in rats: a reply to the editorial by Devor and the article by Blumenkopf and Lipman. Pain. 1992;49:153–156.

Kurejova M., Nattenmuller U., Hildebrandt U., et al. An improved behavioural assay demonstrates that ultrasound vocalizations constitute a reliable indicator of chronic cancer pain and neuropathic pain. Molecular Pain. 2010;6:18.

LaBuda C.J., Fuchs P.N. A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Experimental Neurology. 2000;163:490–494.

LaGraize S.C., Borzan J., Peng Y.B., et al. Selective regulation of pain affect following activation of the opioid anterior cingulate cortex system. Experimental Neurology. 2006;197:22–30.

LaGraize S.C., Labuda C.J., Rutledge M.A., et al. Differential effect of anterior cingulate cortex lesion on mechanical hypersensitivity and escape/avoidance behavior in an animal model of neuropathic pain. Experimental Neurology. 2004;188:139–148.

Langford D.J., Bailey A.L., Chanda M.L., et al. Coding of facial expressions of pain in the laboratory mouse. Nature Methods. 2010;7:447–449.

Li Y., Dorsi M.J., Meyer R.A., et al. Mechanical hyperalgesia after an L5 spinal nerve lesion in the rat is not dependent on input from injured nerve fibers. Pain. 2000;85:493–502.

Lombard M.C., Nashold B.S., Jr., Albe-Fessard D., et al. Deafferentation hypersensitivity in the rat after dorsal rhizotomy: a possible animal model of chronic pain. Pain. 1979;6:163–174.

Luger N.M., Honore P., Sabino M.A., et al. Osteoprotegerin diminishes advanced bone cancer pain. Cancer Research. 2001;61:4038–4047.

Martin T.J., Ewan E. Chronic pain alters drug self-administration: implications for addiction and pain mechanisms. Experimental and Clinical Psychopharmacology. 2008;16:357–366.

Martin T.J., Kim S.A., Buechler N.L., et al. Opioid self-administration in the nerve-injured rat: relevance of antiallodynic effects to drug consumption and effects of intrathecal analgesics. Anesthesiology. 2007;106:312–322.

Martin T.J., Kim S.A., Eisenach J.C. Clonidine maintains intrathecal self-administration in rats following spinal nerve ligation. Pain. 2006;125:257–263.

Max M.B. Clarifying the definition of neuropathic pain. Pain. 2002;96:406–407.

McCarberg B., Billington R. Consequences of neuropathic pain: quality-of-life issues and associated costs. American Journal of Managed Care. 2006;12:S263–S268.

Melli G., Keswani S.C., Fischer A., et al. Spatially distinct and functionally independent mechanisms of axonal degeneration in a model of HIV-associated sensory neuropathy. Brain. 2006;129:1330–1338.

Milligan E.D., Mehmert K.K., Hinde J.L., et al. Thermal hyperalgesia and mechanical allodynia produced by intrathecal administration of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp120. Brain Research. 2000;861:105–116.

Mosconi T., Kruger L. Fixed-diameter polyethylene cuffs applied to the rat sciatic nerve induce a painful neuropathy: ultrastructural morphometric analysis of axonal alterations. Pain. 1996;64:37–57.

Munger B.L., Bennett G.J., Kajander K.C. An experimental painful peripheral neuropathy due to nerve constriction. I. Axonal pathology in the sciatic nerve. Experimental Neurology. 1992;118:204–214.

Muthuraman A., Ramesh M., Sood S. Development of animal model for vasculitic neuropathy: induction by ischemic-reperfusion in the rat femoral artery. Journal of Neuroscience Methods. 2010;186:215–221.

Oaklander A.L. RSD/CRPS: the end of the beginning. Pain. 2008;139:239–240.

Oaklander A.L. Role of minimal distal nerve injury in complex regional pain syndrome-I. Pain Medicine. 2010;11:1251–1256.

Oaklander A.L., Fields H.L. Is reflex sympathetic dystrophy/complex regional pain syndrome type I a small-fiber neuropathy? Annals of Neurology. 2009;65:629–638.

Oaklander A.L., Rissmiller J.G., Gelman L.B., et al. Evidence of focal small-fiber axonal degeneration in complex regional pain syndrome-I (reflex sympathetic dystrophy). Pain. 2006;120:235–243.

Ochoa J.L., Yamitsky D. Mehanical hyperalgesias in neuropathic pain patients: dynamic and static subtypes. Annals of Neurology. 1993;33(5):465–472.

Ossipov M.H., Bian D., Malan T.P., Jr., et al. Lack of involvement of capsaicin-sensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats. Pain. 1999;79:127–133.

Ossipov M.H., Zhang E.T., Carvajal C., et al. Selective mediation of nerve injury–induced tactile hypersensitivity by neuropeptide Y. Journal of Neuroscience. 2002;22:9858–9867.

Peters C.M., Ghilardi J.R., Keyser C.P., et al. Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain. Experimental Neurology. 2005;193:85–100.

Polomano R.C., Mannes A.J., Clark U.S., et al. A painful peripheral neuropathy in the rat produced by the chemotherapeutic drug, paclitaxel. Pain. 2001;94:293–304.

Porreca F., Ossipov M.H., Lai J., et al. Blockade of diabetic, chemotherapeutic, and NGF-induced pain by antisense knockdown of PN3/SNS, a TTX-resistant sodium channel. In: Devor M., Rowbotham M.C., Wiesenfeld-Hallin Z., eds. Proceedings of the 9th World Congress on Pain. Seattle: IASP Press, 2000.

Price D.D. Psychological and neural mechanisms of the affective dimension of pain. Science. 2000;288:1769–1772.

Price D.D., Barrell J.J., Gracely R.H. A psychophysical analysis of experimental factors that selectively influence the affective dimension of pain. Pain. 1980;8:137–149.

Qu C., King T., Okun A., et al. Lesion of the rostral anterior cingulate cortex eliminates the aversiveness of spontaneous neuropathic pain following partial or complete axotomy. Pain. 2011;152:1641–1648.

Rauck R.L., Wallace M.S., Burton A.W., et al. Intrathecal ziconotide for neuropathic pain: a review. Pain Practice. 2009;9:327–337.

Rice A.S., Cimino-Brown D., Eisenach J.C., et al. Animal models and the prediction of efficacy in clinical trials of analgesic drugs: a critical appraisal and call for uniform reporting standards. Pain. 2008;139:243–247.

Rodin B.E., Kruger L. Deafferentation in animals as a model for the study of pain: an alternative hypothesis. Brain Research. 1984;319:213–228.

Rowbotham M.C., Fields H.L. The relationship of pain, allodynia and thermal sensation in post-herpetic neuralgia. Brain. 1996;119:347–354.

Roytta M., Raine C.S. Taxol-induced neuropathy: chronic effects of local injection. Journal of Neurocytology. 1986;15:483–496.

Sahenk Z., Brady S.T., Mendell J.R. Studies on the pathogenesis of vincristine-induced neuropathy. Muscle & Nerve. 1987;10:80–84.

Scadding J.W., Wall P.D., Melzack R. Complex regional pain syndromes. In: Wall P., Melzak R., eds. Textbook of Pain. 4th ed. Edinburgh: Churchill Livingstone; 1999:835–849.

Schmidt-Wilcke T., Leinisch E., Ganssbauer S., et al. Affective components and intensity of pain correlate with structural differences in gray matter in chronic back pain patients. Pain. 2006;125(1-2):89–97.

Schweinhardt P., Kuchinad A., Pukall C.F., et al. Increased gray matter density in young women with chronic vulvar pain. Pain. 2008;140(3):411–419.

Seltzer Z., Dubner R., Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain. 1990;43:205–218.

Seltzer Z., Shir Y. Sympathetically-maintained causalgiform disorders in a model for neuropathic pain: a review. Journal of Basic and Clinical Physiology and Pharmacology. 1991;2:17–61.

Serra J., Bostock H., Sola R., et al. Microneurographic identification of spontaneous activity in C-nociceptors in neuropathic pain states in humans and rats. Pain. 2012;153:42–55.

Seymour B., O’Doherty J.P., Koltzenburg M., et al. Opponent appetitive-aversive neural processes underlie predictive learning of pain relief. Nature Neuroscience. 2005;8:1234–1240.

Sheen K., Chung J.M. Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model. Brain Research. 1993;610:62–68.

Siau C., Bennett G.J. Dysregulation of cellular calcium homeostasis in chemotherapy-evoked painful peripheral neuropathy. Anesthesia and Analgesia. 2006;102:1485–1490.

Siegel S.M., Lee J.W., Oaklander A.L. Needlestick distal nerve injury in rats models symptoms of complex regional pain syndrome. Anesthesia and Analgesia. 2007;105:1820–1829.

Sung B., Na H.S., Kim Y.I., et al. Supraspinal involvement in the production of mechanical allodynia by spinal nerve injury in rats. Neuroscience Letters. 1998;246:117–119.

Suzuki R., Chapman V., Dickenson A.H. The effectiveness of spinal and systemic morphine on rat dorsal horn neuronal responses in the spinal nerve ligation model of neuropathic pain. Pain. 1999;80:215–228.

Suzuki Y., Sato J., Kawanishi M., et al. Lowered response threshold and increased responsiveness to mechanical stimulation of cutaneous nociceptive fibers in streptozotocin-diabetic rat skin in vitro—correlates of mechanical allodynia and hyperalgesia observed in the early stage of diabetes. Neuroscience Research. 2002;43:171–178.

Tanner K.D., Levine J.D., Topp K.S. Microtubule disorientation and axonal swelling in unmyelinated sensory axons during vincristine-induced painful neuropathy in rat. Journal of Comparative Neurology. 1998;395:481–492.

Tanner K.D., Reichling D.B., Gear R.W., et al. Altered temporal pattern of evoked afferent activity in a rat model of vincristine-induced painful peripheral neuropathy. Neuroscience. 2003;118:809–817.

Uhle E.I., Becker R., Gatscher S., et al. Continuous intrathecal clonidine administration for the treatment of neuropathic pain. Stereotactic and Functional Neurosurgery. 2000;75:167–175.

van der Laan L., ter Laak H.J., Gabreels-Festen A., et al. Complex regional pain syndrome type I (RSD): pathology of skeletal muscle and peripheral nerve. Neurology. 1998;51:20–25.

Vatine J.-J., Devor M., Belfer I., et al. Preoperative open field behavior predicts levels of neuropathic pain–related behavior in mice. Neuroscience Letters. 2000;279:141–144.

Vierck C.J., Hansson P.T., Yezierski R.P. Clinical and pre-clinical pain assessment: are we measuring the same thing? Pain. 2008;135:7–10.

Vos B.P., Strassman A.M. Fos expression in the medullary dorsal horn of the rat after chronic constriction injury to the infraorbital nerve. Journal of Comparative Neurology. 1995;357:362–375.

Vos B.P., Strassman A.M., Maciewicz R.J. Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat’s infraorbital nerve. Journal of Neuroscience. 1994;14:2708–2723.

Wall P.D., Gutnick M. Properties of afferent nerve impulses originating from a neuroma. Nature. 1974;248:740–743.

Wall P.D., Scadding J.W., Tomkiewicz M.M. The production and prevention of experimental anesthesia dolorosa. Pain. 1979;6:175–182.

Wallace V.C., Blackbeard J., Pheby T., et al. Pharmacological, behavioural and mechanistic analysis of HIV-1 gp120 induced painful neuropathy. Pain. 2007;133:47–63.

Wallace V.C., Norbury T.A., Rice A.S. Ultrasound vocalisation by rodents does not correlate with behavioural measures of persistent pain. European Journal of Pain. 2005;9:445–452.

Williams W.O., Riskin D.K., Mott A.K. Ultrasonic sound as an indicator of acute pain in laboratory mice. Journal of the American Association of Laboratory Animal Science. 2008;47:8–10.

Xu X.J., Puke M.J., Wiesenfeld-Hallin Z. The depressive effect of intrathecal clonidine on the spinal flexor reflex is enhanced after sciatic nerve section in rats. Pain. 1992;51:145–151.

Yoon Y.W., Na H.S., Chung J.M. Contributions of injured and intact afferents to neuropathic pain in an experimental rat model. Pain. 1996;64:27–36.

Yu W, Kauppila T, Hultenby K, et al. Photochemically-induced ischemic injury of the rat sciatic nerve: a light- and electron microscopic study. Journal of the Peripheral Nervous System. 2000;5:209–217.

Aley K.O., Reichling D.B., Levine J.D. Vincristine hyperalgesia in the rat: a model of painful vincristine neuropathy in humans. Neuroscience. 1996;73:259–265.

Apkarian A.V., Sosa Y., Sonty S., et al. Chronic back pain is associated with decreased prefrontal and thalamic gray matter density. Journal of Neuroscience. 2004;24:10410–10415.

Authier N., Gillet J.P., Fialip J., et al. Description of a short-term Taxol-induced nociceptive neuropathy in rats. Brain Research. 2000;887:239–249.

Bennett G.J., Xie Y.K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 1988;33:87–107.

Calcutt N.A. Experimental models of painful diabetic neuropathy. Journal of the Neurological Sciences. 2004;220:137–139.

Campbell J.N., Meyer R.A. Mechanisms of neuropathic pain. Neuron. 2006;52:77–92.

Chaplan S.R., Bach F.W., Pogrel J.W., et al. Quantitative assessment of tactile allodynia in the rat paw. Journal of Neuroscience Methods. 1994;53:55–63.

Courteix C., Bardin M., Massol J., et al. Daily insulin treatment relieves long-term hyperalgesia in streptozocin diabetic rats. Neuroreport. 1996;7:1922–1924.

Dalziel R.G., Bingham S., Sutton D., et al. Allodynia in rats infected with varicella zoster virus—a small animal model for post-herpetic neuralgia. Brain Research. Brain Research Reviews. 2004;46:234–242.

Decosterd I., Woolf C.J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain. 2000;87:149–158.

Devor M. Sensory basis of autotomy in rats. Pain. 1991;45:109–110.

Field M.J., Bramwell S., Hughes J., et al. Detection of static and dynamic components of mechanical allodynia in rat models of neuropathic pain: are they signalled by distinct primary sensory neurones? Pain. 1999;83:303–311.

Fleetwood-Walker S.M., Quinn J.P., Wallace C., et al. Behavioural changes in the rat following infection with varicella-zoster virus. Journal of General Virology. 1999;80:2433–2436.

Gracely R.H., Lynch S.A., Bennett G.J. Painful neuropathy: altered central processing maintained dynamically by peripheral input. Pain. 1992;51:175–194.

Kim S.H., Chung J.M. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain. 1992;50:355–363.

King T., Vera-Portocarrero L., Gutierrez T., et al. Unmasking the tonic-aversive state in neuropathic pain. Nature Neuroscience. 2009;12:1364–1366.

LaBuda C.J., Fuchs P.N. A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats. Experimental Neurology. 2000;163:490–494.

Martin T.J., Kim S.A., Eisenach J.C. Clonidine maintains intrathecal self-administration in rats following spinal nerve ligation. Pain. 2006;125:257–263.

Qu C., King T., Okun A., et al. Lesion of the rostral anterior cingulate cortex eliminates the aversiveness of spontaneous neuropathic pain following partial or complete axotomy. Pain. 2011;152:1641–1648.

Rice A.S., Cimino-Brown D., Eisenach J.C., et al. Animal models and the prediction of efficacy in clinical trials of analgesic drugs: a critical appraisal and call for uniform reporting standards. Pain. 2008;139:243–247.

Rowbotham M.C., Fields H.L. The relationship of pain, allodynia and thermal sensation in post-herpetic neuralgia. Brain. 1996;119:347–354.

Siegel S.M., Lee J.W., Oaklander A.L. Needlestick distal nerve injury in rats models symptoms of complex regional pain syndrome. Anesthesia and Analgesia. 2007;105:1820–1829.

Seminowicz D.A., Laferriere A.L., Millecamps M., et al. MRI structural brain changes associated with sensory and emotional function in a rat model of long-term neuropathic pain. NeuroImage. 2009;47:1007–1014.