Pnina Raber

Corticosteroids suppress ectopic neural discharge originating in experimental neuromas

&NA; Some injured sensory fibers ending in an experimental neuroma in the rat sciatic nerve discharge spontaneously. Furthermore, many become sensitive to a range of physical and chemical stimuli. The resulting afferent barrage is thought to contribute to paresthesias and pain associated with peripheral nerve injury. We report that the development of such ectopic neuroma discharge is largely prevented when the freshly cut nerve end is treated with any of 3 commercially available corticosteroid preparations including two in depot form, triamcinolone hexacetonide (Lederspan) and triamcinolone diacetate (Ledercort), and one in soluble form, dexamethasone (Dexacort). These corticosteroids also produce a rapid and prolonged suppression of ongoing discharge in chronic neuromas that have already become active. The kinetics of corticosteroid suppression of neuroma discharge suggest a direct membrane action rather than an anti‐inflammatory action.

Proliferation of Primary Sensory Neurons in Adult Rat Dorsal Root Ganglion and the Kinetics of Retrograde Cell Loss after Sciatic Nerve Section

This study was aimed at measuring the kinetics of retrograde death among primary sensory neurons axotomized by transection of the ipsilateral sciatic nerve in adult rats. Using electrophysiological and retrograde transport methods, we first determined that most sciatic afferents enter the spinal cord along the L4 and L5 dorsal roots (DRs), and that about 54% of the cells in the L4 and L5 dorsal root ganglia (DRGs) project an axon into the sciatic nerve. Knowing this value, we could then calculate the rate of loss of axotomized neurons from the overall rate of neuron loss in the DRGs at different times after the lesion. Following unilateral sciatic neurectomy, we found a steady falloff in the ratio of DRG neurons on the operated versus the intact control sides in cresyl-violet-stained serial paraffin sections. We were surprised to note, however, that on the control side there was a steady increase in the cell count with age. Counts done on a series of unoperated rats of various ages confirmed this natural increase. Overall, new neurons accrete at an average rate of 18.1 cells per day to the combined L4 and L5 DRGs, nearly doubling their numbers during the adult life of the animal. The new cells add mostly to the small-diameter neuronal compartment. Evidence from neonatally operated rats indicates that the decline in the ratio of neurons in operated versus control DRGs following sciatic nerve section in the adult results more from a halt in the accretion of new neurons to the sciatic compartment than from frank cell death. From our data, we calculate that the loss of axotomized neurons occurs at a rate of only about 8% per 100 postoperative days.

Heritability of symptoms in an experimental model of neuropathic pain

&NA; Male and female rats underwent transection and ligation of the sciatic and saphenous nerves, and the development of autotomy was monitored. The deafferented animals were then interbred, always selecting males and females that expressed relatively high and, alternatively, relatively low levels of autotomy. Offspring were similarly operated and interbred. By the sixth generation of selective breeding, lines were achieved in which autotomy was consistently high (HA) or consistently low (LA). There was no indication of sex linkage. Thermal and mechanical nocifensive responsiveness co‐selected with propensity to express autotomy following nerve injury: response thresholds were lower in HA than in LA rats. F1 hybrids formed by crossing homozygous HA and LA animals showed low levels of autotomy, similar to LA stock. This indicates recessive inheritance of the autotomy trait. Backcrossing F1 hybrids onto the LA line yielded a low autotomy phenotype in almost all cases; backcrossing F1 hybrids onto HA stock yielded about 50% high autotomy and 50% low autotomy. These ratios are consistent with simple mendelian inheritance of a single gene. Taken together, the data suggest that autotomy is inherited as a single‐gene autosomal recessive trait.

Key role of the dorsal root ganglion in neuropathic tactile hypersensibility

Cutting spinal nerves just distal to the dorsal root ganglion (DRG) triggers, with rapid onset, massive spontaneous ectopic discharge in axotomized afferent A‐neurons, and at the same time induces tactile allodynia in the partially denervated hindlimb. We show that secondary transection of the dorsal root (rhizotomy) of the axotomized DRG, or suppression of the ectopia with topically applied local anesthetics, eliminates or attenuates the allodynia. Dorsal rhizotomy alone does not trigger allodynia. These observations support the hypothesis that ectopic firing in DRG A‐neurons induces central sensitization which leads to tactile allodynia. The question of how activity in afferent A‐neurons, which are not normally nociceptive, might induce allodynia is discussed in light of the current literature.

Correlation of intact sensibility and neuropathic pain-related behaviors in eight inbred and outbred rat strains and selection lines

&NA; In some rat strains, total hindpaw denervation triggers autotomy, a behavior of self mutilation presumably related to neuropathic pain. Partial sciatic ligation (PSL) in rats produces tactile allodynia and heat hyperalgesia but not autotomy. Our aims in this study were to examine: (1) whether sensibility of intact rats to noxious and non‐noxious stimuli is strain‐dependent; (2) whether sensibility of intact rats could predict levels of autotomy, or of allodynia and hyperalgesia in the PSL model; and (3) whether autotomy levels are correlated with levels of allodynia or hyperalgesia. Here we report that in two inbred rat strains (Lewis and Fisher 344), two outbred rat strains (Sabra and Sprague–Dawley) and four selection lines of rats (Genetically Epilepsy‐Prone Rats, High Autotomy, Low Autotomy and Flinders Sensitive Line), tactile sensitivity and response duration to noxious heat of intact animals were strain‐dependent. Levels of autotomy following hindpaw denervation and of allodynia and hyperalgesia in the PSL model were also strain‐dependent. Thus, these traits are determined in part by genetic factors. Sensory sensibility of intact rats was not correlated with levels of autotomy following total denervation, or allodynia and hyperalgesia following partial denervation. We suggest that preoperative sensibility of intact rats is not a predictor of levels of neuropathic disorders following nerve injury. Likewise, no correlation was found between autotomy, allodynia and hyperalgesia, suggesting that neuropathic pain behaviors triggered by nerve injury of different etiologies are mediated by differing mechanisms.

Social variables affect phenotype in the neuroma model of neuropathic pain

&NA; When the degree of genetic determination of a trait (i.e. its heritability) is high, one tends to presume that environmental factors will not modify its expression by much. Contrary to this expectation, we show here in rats that a psychosocial‐behavioral variable, the identity of cagemates, can largely over‐ride genetic predisposition to pain behavior. We used selection‐line rats that consistently show high (HA) or low (LA) pain phenotype (autotomy) in the neuroma model of neuropathic pain. Normally, HA animals show autotomy after nerve injury while LA animals do not. However, when caged together with HA rats, LA rats showed high levels of autotomy. This occurred even when the individual HA cagemates were familiar preoperatively, and it did not depend on the actual performance of autotomy by the HA rats. Indeed, cage bedding soiled by HA rats was sufficient to induce a modest level of autotomy in LA animals. Chemical cues associated with HA rats, perhaps in combination with behavioral characteristics, are apparently able to induce pain phenotype despite the powerful protection otherwise rendered by the LA genotype. Social factors must be considered in behavior‐related research on rodents that have undergone genetic modification. More generally, the overwhelming influence that psychosocial‐behavioral variables have on pain perception, and on pain behavior, in humans may have evolutionary roots deeper than has previously been appreciated.

Hyperexcitability in sensory neurons of rats selected for high versus low neuropathic pain phenotype.

Selection line rats congenitally high or low for autotomy in the neuroma model of neuropathic pain (HA and LA rats) were found to be correspondingly high and low in a second type of neuropathic pain, the Chung model, which employs an alternative phenotypic endpoint, tactile allodynia. It has been proposed that both phenotypes reflect ectopic hyperexcitability in axotomized primary sensory neurons. To test this hypothesis we made in vitro recordings from sensory neurons in the L4 and 5 dorsal root ganglia. Baseline excitability was similar in HA and LA rats, and axotomy caused an increase in both lines. However, in the one neuronal subclass previously linked to neuropathic pain in these models the increase was significantly greater in HA than LA rats, and only at the time when pain scores in the two lines were diverging. Heritable differences in electrical response to axotomy in a specific afferent cell type appear to be a fundamental determinant of neuropathic pain.

Autotomy after nerve injury and its relation to spontaneous discharge originating in nerve-end neuromas.

Following transection and ligation of the sciatic and saphenous nerves, rodents frequently scratch and bite their anesthetic foot (autotomy). Many authors have suggested that autotomy is related to uncomfortable paresthesias induced by abnormal afferent discharge known to be generated in myelinated afferents in nerve-end neuromas. We report that preventing the development of ectopic neuroma discharge in rats by treating the severed nerves with colchicine or vinblastine does not prevent, or even detectably reduce, autotomy directed toward the denervated hindlimb. We conclude that abnormal discharge in myelinated afferents is not necessary for the expression of autotomy following nerve injury in rats.

pain1: A neuropathic pain QTL on mouse chromosome 15 in a C3H×C58 backcross

&NA; We have produced a backcross (BC) population of 267 mice from the parental strains C3H/HeN and C58/J. The mice were phenotyped for neuropathic pain using the neuroma model. Subsequently all BC mice were genotyped in a region of chromosome 15 that has been previously suggested to contain a quantitative trait locus (QTL) for this trait. We have confirmed the linkage of the QTL, named pain1, to the central region of chromosome 15. Our finding provides the necessary robustness to justify efforts towards identification of the underlying gene.

Sex‐specific variability and a ‘cage effect’ independently mask a neuropathic pain quantitative trait locus detected in a whole genome scan

Sex and environment may dramatically affect genetic studies, and thus should be carefully considered. Beginning with two inbred mouse strains with contrasting phenotype in the neuroma model of neuropathic pain (autotomy), we established a backcross population on which we conducted a genome‐wide scan. The backcross population was partially maintained in small social groups and partially in isolation. The genome scan detected one previously reported quantitative trait locus (QTL) on chromosome 15 (pain1), but no additional QTLs were found. Interestingly, group caging introduced phenotypic noise large enough to completely mask the genetic effect of the chromosome 15 QTL. The reason appears to be that group‐caging animals from the low‐autotomy strain together with animals from the high‐autotomy strain dramatically increases autotomy in the otherwise low‐autotomy mice (males or females). The converse, suppression of pain behaviour in the high‐autotomy strain when caged with the low‐autotomy strain was also observed, but only in females. Even in isolated mice, the genetic effect of the chromosome 15 QTL was significant only in females. To determine why, we evaluated autotomy levels of females in 12 different inbred stains of mice and compared them to previously reported levels for males. Strikingly larger environmental variation was observed in males than in females for this pain phenotype. The high baseline variance in males can explain the difficulty in detecting the genetic effect, which was readily seen in females. Our study emphasizes the importance of sex and environment in the genetic analysis of pain.