[1] 15w The resolution of neuropathic hyperalgesia following motor and sensory functional recovery in sciatic axonotmetic mononeuropathies

[1] 381w Nerve lesions producing extensive axonal loss can induce painful hyperalgesic states in man. The affect of axonal regeneration and end-organ reinnervation on hyperalgesia and pain is controversial. This study used two axonotmetic models, the sciatic crush injury (CI) and the sciatic chronic constrictive injury (CCI), to investigate the affects of nerve regeneration and reinnervation on hyperalgesia and presumed painful behavior in rats. The sciatic CI resulted in a transient loss of both sciatic motor function and the withdrawal response to pinch and heat in the sciatic distribution. Extensive recovery of motor function, pinch and heat response occurred over days 23-38 post-crush injury. This temporally corresponded with a plateau in the hindpaw autotomy score and a resolution of the saphenous-mediated pressure and heat hyperalgesia (adjacent neuropathic hyperalgesia; ANH) which developed over the medial dorsum of the hindpaw following the sciatic CI. In contrast, with sciatic transection and distal stump excision, no motor recovery occurs, large areas of the hindpaw remain unresponsive to heat and pinch, and the saphenous mediated ANH fails to resolve over a period of 3 months. When sciatic CI was compared to contralateral sciatic transection within the same rat, the bilateral saphenous-mediated pressure and heat thresholds were initially identical, but by 23-27 days post-crush, the crush side thresholds became hypoalgesic relative to the section side. This demonstrates an attenuation of the crush-induced ANH which temporally corresponds to the recovery of motor and sensory function.When the sciatic nerve was proximally crushed and distally transected (3 cm below the crush site), the saphenous-mediated pressure and heat threshold changes were identical (over 6 weeks of serial testing) to those produced by a contralateral sciatic transection within the same rat. This indicates that the microenvironments surrounding the regenerating axon tips did not differentially affect the development of ANH following sciatic CI or transection.The sciatic CC1 resulted in a transient loss of hindpaw motor function without the loss of pinch or heat withdrawal responses in the sciatic distribution. Motor function recovery occurred primarily over days 23-59 post-ligature. During this prolonged period of motor function recovery there was a resolution of the sciatic-mediated plantar surface heat hyperalgesia and the saphenous-mediated heat ANH. The above data support the hypothesis that the successful regeneration of distal axons after axonotmetic lesions can initiate the resolution of neuropathic hyperalgesia.

[1] 34w A high sciatic CI resulted in an initial complete loss of distal hindpaw motor function. Functional motor recovery occurred primarily over postoperative days 23-38, with no animals demonstrating deficits after 62 days (Fig. 1A).

[2] 216w The temporal recovery of hindpaw surface area responsive to pinch was examined for both sciatic CI and sciatic section injuries. This allowed differentiation between recovery of withdrawal responses due to collateral sprouting from the adjacent intact saphenous nerve, and the return due to sciatic regeneration (Devor et al. 1979). The area responding to pinch gradually increased following both sciatic crush or section. Between days 23-38 postoperatively the CI hindpaw's pinch response area greatly expanded, relative to the section hindpaw (Fig. 1B). All the CI rats responded at all test sites after 38 days, while sciatic-sectioned animals continued to demonstrate extensive loss of hindpaw pinch response at 62 days postoperatively. Fig. IC demonstrates the loss of heat response over the lateral plantar surface of the paw following sciatic CI. Heat response returned gradually over days 21-42 postoperatively, but the withdrawal threshold remained elevated relative to control thresholds for the duration of the study. Sciatic transection with distal stump resection resulted in only a transient plantar analgesia, and within 1 week some rats began withdrawing to heat over the medial plantar surface. The lateral plantar surface never became responsive to noxious heating over 1 I weeks following sciatic transection, so the lateral plantar surface of the paw was used to measure recovery of heat nociception after sciatic CI.

[3] 58w Autotomy behavior gradually increased after both sciatic CI and sciatic transection (Fig. 2). The autotomy behavior plateaued after day 23 for the CI rats, and after day 38 in the transected rats. The sectioned rats appeared to have higher autotomy scores, but when tested by repeated-measures ANOVA there were no significant differences between the CI and section rats.

[4] 134w Pressure thresholds over the lateral dorsum transiently increased following sciatic Cl (Fig. 3A,C). The LDP was increased only at 7 days post-crush, but the LDP/vF was usually unresponsive at 7 days postinjury, and significantly elevated on days 7-18. The LDP/vF gradually returned to baseline over days 14-33. The MDP and MDP/vF thresholds dropped following sciatic CI, and were significantly hyperalgesic over days 13-23, gradually returning to normal over days 23-32 for the MDP and days 23-44 for the MDP/vF (Fig. 3B.D). At 7 days post-crush there was no plantar response to noxious heat. The PSH thresholds remained hypoalgesic over the entire test period, although thresholds did gradually decline over days 18-32 (Fig. 4A). Fig. 4B illustrates the gradual decline of MDH thresholds following sciatic CI, with hyperalgesia at days 14-23, resolving by day 32.

[5] 53w To test for the possible development of a "mirror" hyperalgesia developing in the contralatera1 sham surgery paw, the contralateral paw withdrawal thresholds (LDP, LDP/vF, MDP, MDP/vF, PSH, MDH) were compared on days 7-23 to the pre-operative baseline thresholds. Using a l-factor repeated-measures ANOVA, no drop in thresholds were observed on any test date.

[6] 82w Following both the unilateral sciatic CI and the contralateral sciatic section, the saphenous-mediated MDP and MDH withdrawal thresholds gradually declined over the ensuing 3 weeks. When thresholds were compared for CI versus section, the threshold differences were not significant over postoperative days 9-23 (Fig. 5A,B). After the initial decline, the CI thresholds rose over post-crush days 23-30, and then plateaued over days 30-51. Between days 23 and 51 the CI thresholds were usually significantly elevated relative to the section thresholds (Fig. 5).

[7] 70w No significant differences in the MDP and MDH thresholds were observed between unilateral sciatic CI with distal section and contralateral sciatic section over postoperative days l-42 (Fig. 6A,B). Autotomy behavior plateaued at day 23 for crush and day 38 for section. The section rats had higher final autotomy scores, but the difference between crush and section was not significant. The cross-hatched bar represents the time of maximal sciatic-mediated functional recovery.

[8] 55w days 23-59, and then more gradually over days 59-80, and plateauing after that. Several rats failed to regain full digit abduction over the 14 week duration of this experiment. There was no loss of pinch response over the 10 standard test sites and no elevation of plantar surface heat withdrawal thresholds following loose sciatic ligature.

[9] 52w For most test days, the unilateral ligature rats had a slightly lower LDP threshold on the ligature side compared with the control side, but this was not significant (Fig. 8A). A similar hyperalgesic tendency was seen in the MDP thresholds following loose ligature (Fig. 8B1, but again the difference was not significant.

[10] 30w Following loose sciatic ligature there was an initial no significant difference between the ligature and control side thresholds by day 63 for the PSH and day 50 for the MDH.

[11] 73w To test for the possible development of a "mirror" hyperalgesia developing in the contralateral sham surgery paw, the contralateral paw withdrawal thresholds (LDP, MDP, PSH, MDH) were compared on days 14-59 to the pre-operative baseline thresholds. Using a l-factor repeated-measures ANOVA, no drop in thresholds were observed on any test date, replicating our findings of no contralateral mechanical or heat hyperalgesia developing after unilateral CC1 with chromic gut ligatures Wngery et al. 1994).

[12] 49w drop in heat withdrawal thresholds over the plantar surface and medial dorsum of the hindpaw (Fig. 8C,D). Over postoperative days 14-59 for the PSH and days 28-43 for the MDH, the ligature side thresholds were significantly lower than the contralateral controls. This thermal hyperalgesia gradually resolved and there was

[1] 29w The first experiment demonstrated that a high sciatic crush injury in 300-350 g rats resulted in a loss of primarily between 23 and 38 days after CI (Fig. 1A).

[2] 62w Prior studies (Berenberg et al. 1977;Medinaceli et al. 1982;Kauppila et al. 1988;Kerns et al. 1991;Swett et al. 1991;Kerns and Lucchinetti 1992) have reported functional motor recovery occurring between days 14 and 28 post-crush, but it is difficult to compare our data with the results of these investigators due to discrepant levels of sciatic crush, animal size, and methods for measuring motor function.

[3] 19w The possibility that a hyperalgesic paw would affect evaluation of motor function recovery was considered unlikely for several reasons.

[4] 80w Prior investigators have sync CRUSH vs cormo~ 0 5 10 15 20 25 30 35 40 45 8. .51 l MEouLDoRsuY 2.51 0 5 lo I5 20 25 30 35 40 45 DAYS AFTER CRUSH A. '001 SclATlC CRUSH vs. sclAnc SECTION 0 MEDIAL DORSUW 401 0 10 20 30 40 50 60 6. 3 2.5 1 2 l 0 10 20 30 40 50 60 DAYS AFTER NERVE LESION Fig. 5. Saphenous-mediated medial dorsum withdrawal thresholds to pressure (analgesy-meter)

[5] 186w and heat following unilateral sciatic crush and contralateral sciatic transection. Positive threshold differences (above the line) indicate the withdrawal threshold on the side of the sciatic crush injury was higher then the threshold on the contralat-era1 section side. The cross-hatched bar represents the time of maximal sciatic-mediated functional recovery after crush. A: No initial difference in pressure thresholds up to day 27, then the crush thresholds elevated relative to the section side. B: Again. no initial difference in heat thresholds up to day 21. then there was a relative increase in the crush thresholds relative to the section side. * P < 0.05: ** P < 0.01; *** P < 0.001. demonstrated a temporal correlation between behavioral and electrophysiologic measures of motor function recovery after sciatic CI (Kerns et al. 1991;Swett et al. 1991). We observed complete motor loss immediately after sciatic CI, weeks before the onset of hyperalgesia. Although nociceptive threshold reductions were variable after sciatic CI, with some animals failing to demonstrate consistent hyperalgesia, all animals had complete motor function loss over the first 3 weeks, and extensive recovery over days 23-38 (Fig. 1A).

[6] 73w Over days 23-38 following the sciatic CI, the area responsive to pinch greatly expanded relative to the sciatic section injury (Fig. 1B). This would indicate the return of mechanical nociception mediated by the re-generated sciatic nerve, rather than collateral reinnervation from saphenous sprouts. Devor et al. (1979) found a similar latency for the recovery of pinch response following a mid-thigh sciatic crush injury and an ipsilateral saphenous nerve ligation in 250-350 g rats.

[7] 71w The sciatic-mediated lateral plantar heat withdrawal response primarily recovered over days 21-42 postcrush (Fig. 1C). Other investigators (Bijlsma et al. 1981;Kauppila et al. 1988;Kerns et al. 1991) have reported the reappearance of heat nociception in the hindpaw between 10 and 21 days following sciatic crush injury, but these studies did not did not limit nociceptive testing to the lateral plantar surface and may have also measured saphenous-mediated medial plantar heat nociception.

[8] 79w Both sciatic CI and section led to autotomy behavior in some rats within 7 days of injury (Fig. 2). Autotomy RECOVERY Of HOTOR FUNCTION 0 20 DAYS A:fER LI&"RE 80 loo Fig. 7. The recovery of sciatic-mediated motor function following sciatic chronic constrictive injury. No motor function was observed for 23 days following this injury, then a rapid recovery of function was observed from days 23 to 59, plateauing after day 80 with some animals demonstrating mild residual deficits.

[9] 282w increased for 23 days after CI, and for 38 days after section. The maximum autotomy score was higher in the section rats then the CI rats, but this failed to reach significance, probably due to the small numbers of animals used in this study. A prior study using larger numbers of rats also demonstrated diverging autotomy scores for sciatic CI and transection, appearing between days 21 and 28 after nerve lesion (Wall et al. 1979). These authors also reported a significantly lower degree of autotomy following sciatic CI compared with transection. Fig. 2 illustrates that functional recovery (cross-hatched bar) and the divergence of autotomy scores primarily occurred over days 23-38. This correspondence may be the result of nociceptive function returning in the lateral 3 digits, which would probably limit self-mutilation in those digits. The temporal correspondence of functional recovery with the resolution of autotomy behavior may also indicate an attenuating affect of nerve regeneration on presumptive pain behavior. Sciatic crush resulted in a marked decline in MDP, MDP/vF and MDH withdrawal thresholds over the first 14-18 days following surgery (Figs. 3B,D and 4B). There was significant medial dorsum pressure and heat hyperalgesia over days 14-23. Since sciatic CI resulted in a complete loss of sciatic-mediated mechanical and heat nociceptive sensory function during this period (Fig. lB,C), and transection of both the sciatic and saphenous nerves results in a complete hindpaw analgesia (Kingery et al. 19941, the medial dorsum hyperalgesia was saphenous-mediated and represents a new axonotmetic model of ANH. The gradual reversal of the MDP, MDP/vF, and MDH hyperalgesias on the crush side temporally corresponded with the recovery of motor and nociceptive function in the paw (Figs. 3B,D and 4B, cross-hatched bar).

[10] 112w Experiment 2 found that for days l-23, the drop in withdrawal thresholds in the CI paw were identical to those observed following sciatic section (Fig. 5A,B). This decline was arrested or reversed over days 23-36, with a resultant persistent hypoalgesia on the crush side relative to the section side. Following sciatic transection with distal segment excision a saphenous-mediated ANH develops which lasts at least 12 weeks (Kingery and Vallin 1989;Vallin and Kingery 1991). The first experiment demonstrated the early resolution of CI-induced ANH over days 23-38 postinjury (Figs. 3B,D and 4B). In Experiment 2 the development of a relative hypoalgesia on the CI side over days 23-51 corresponds with the resolving ANH.

[11] 93w Extensive axonal degeneration occurs distal to both sciatic transection or crush, but the CI does not disrupt the tubular, basal lamina framework which guides the regenerating axons across the injury site and allows extensive end-organ reinnervation (Hafteck and Thomas 1968;Swett et al. 1991), while the sciatic transection with a 1 cm distal stump resection prevents successful end-organ reinnervation. The different durations of CI and transection-induced ANH, with resolution of the crush ANH temporally corresponding with functional recovery, probably reflects the differences in the abilities of the crushed and transected axons to successfully regenerate.

[12] 186w Sciatic nerve transection and sciatic crush injury have different early effects on the neurophysiology of the primary afferents and their central terminals (Barbut et al. 1981;Devor and Wall 1981;Wall and Devor 1981;Lisney 1982;Wall 1982;McGregor et al. 1984;Nielsch et al. 1987;Kingery et al. 1988;Nielsch and Keen 1989). Crush injury has no or minimal effect on some of the histochemical and electrophysiological parameters which are profoundly altered by section. Because of these early differences between transection and crush, it has been proposed that differences in the immediate environment of the regenerating axon tips may modulate proximal neurophysiologic changes, per- haps by retrograde transport of trophic factors available in the distal nerve segment (Nielsch et al, 1987;Bhisitkul et al. 1990). To test this hypothesis a rat model has been used in which the sciatic nerve is proximally crushed and distally sectioned. Using this model, it has been reported that the axotorny induced loss of dorsal horn substance P is deIayed for 20 days postoperatively (Gorio et al. 19861, and the expected dorsal root GABA sensitivity loss is significantly attenuated between days 12 and 21 postinjury (Bhisitkul et al. 1990).

[13] 97w As Fig. 6 illustrates, no significant differences in the MDP and MDH thresholds were observed between the proximal sciatic crush with distal section, and the contralateral sciatic transection. This was true even between days 1 and 17 postoperatively, before the axon sprouts could have could have advanced 3 cm distal to the crush site and reached the transection point (Gorio et al. 1986;Krarup et al. 1988). This experiment demonstrates that following sciatic CI, the endoneurial environment of the regenerating axon sprouts does not attenuate or delay the onset of the ANH that develops after extensive axonal degeneration.

[14] 259w In Experiment 4, a transient complete loss of distal hindpaw motor function was observed following sciatic CCI, without any loss of nociceptive function (Figs. 7A,C and 8). This pattern would be expected on the basis of the histopathology of this lesion, which results in extensive degeneration of large-diameter fibers distal to the ligature, with a less extensive loss of smaller fibers (Gautron et al. 1990;Basbaum et al. 1991;Carlton et al. 1991;Munger et al. 1992). CC1 can also induce degeneration in proximal myelinated axons, proximal to the injury site (Guilbaud et al. 1993). Following CC1 with chromic suture, the maximum distal myelinated axon loss occurs within 14 days, and by 28 days post-ligature both myelinated and unmyelinated axon counts are normal at a point 1 cm dista1 to the ligature, although the size histogram of the myelinated fibers is down shifted for up to 15 weeks (Coggeshall et al. 1993;Guilbaud et al. 1993). Experiment 4 demonstrated a rapid recovery of motor function occurring over days 23-59, with a few animals demonstrating persistent weakness in digit abduction at 94 days postinjury (Fig. 7). The recovery of motor function (cross-hatched bars) temporally corresponded with the gradual resolution of PSH (Fig. 8C) and MDH (Fig. 8D) hyperalgesia over days 43-63 post-ligature. After sciatic CC1 the PSH threshold is mediated by the sciatic nerve, while the MDH threshold is mediated by the saphenous nerve (Kingery et al. 1994). Experiment 4 illustrates that both the sciatic and saphenous-mediated heat hyperalgesia gradually resolve following sciatic loose ligation, temporally co~esponding to the recovery of sciatic-mediated motor function.

[15] 303w The MDP and LOP thresholds (measured by analgesy-meter) tended to be lower on the CC1 side than the control thresholds, but this was not significant (Fig, SA,B). In a prior study we demonstrated a CCIinduced LDP and MDP hyperalgesia using von Frey fibers, which are probably a more sensitive method of measuring mechanical hyperalgesia than the analgesymeter (Kingery et al. 1994). This agrees with Attal et al. (19901, who found LDP hyperalgesia in CC1 rats using a vocalization threshold technique. Kupers et al. (1992) found that a sciatic CC1 induced an initial motor loss, with functional recovery occurring over days 14-42. These researchers reported resolution of PSH hyperalgesia by day 42, and a resolution of nociceptive scratching behavior by day 56. They also looked at the effects of reabsorbable (chromic) and non-reabsorbable (polypropylene) sutures. No differences in nociceptive behavior were observed between suture groups, but the motor function recovery in the non-reabsorbabIe suture rats plateaued after 42 days at an impaired level, while the reabsorbable group completely recovered after 42 days. These results are similar to ours, except the resolution of the motor deficits and PSH hyperalgesia occurred later in our study (Figs. 7 and 8). This may reflect the different methods used in measuring motor function, different ligature materials, or possible variability in the tightness of the ligatures. The temporal resolution of the PSH hyperalgesia in our study is similar to that reported by Bennett and Xie (1988) using a chromic CCL A recent study using silk ligatures applied to the sciatic nerve did not find PSH hyperalgesia (Maves et at. 1993). This discrepancy with our results may be related to the tightness with which the ligatures are tied, since Maves et al. did not find any axonal loss distal to the ligature site, indicating that this was not an axonotmetic lesion.

[16] 88w Both sciatic CI and sciatic CC1 result in extensive axon loss distal to the injury, which triggers the development of the sciatic and saphenous-mediated hyperalgesias. Both these lesions also induce chronic elcctrophysiological and histochemical changes in the central terminals of the Iesioned afferents (McGregor et al. 1984;Bennett et al. 1989;Cameron et al. 1991: Aanonsen et al. 1992). Such changes may lead to increased excitation of second-order neurons activated by the injured primary afferents, and the adjacent intact afferents, resulting in hyperalgesia (Williams et al. 1991;Mao et al. 1992).

[17] 149w Axon loss with subsequent regeneration and reconnection with target end-organs can lead to biphasic proximal changes in the primary afferents (Frizell and Sjostrand 1974;Davis et al, 1978;Barbut et al. 1981;Devor and Wall 1981;Horch and Lisney 1981;Bisby and Keen 1985;Nielsch et al. 1987;Nielsch and Keen 1989;Knyiahar-Csillik et al. 1990;Woolf et al. 1990). These electrophysiologic and histochemical changes are reversed or attenuated following end-organ reinnervation, but persist if reinnervation fails to occur. Reinnervation of the target-organ can influence central connectivity and be target-organ specific (Lewin and McMahon 1991). It has been proposed that a retrograde signal, possibly the interrupted axonal transport of a neurotrophic factor or factors from the peripheral target-organ to the cell body, triggers the central neurophysiologic changes described above. Restoration of axonal transport between the periphery and the cell body may reverse or attenuate these changes, and may reverse the neuropathic hyperalgesia seen following sciatic CI and CCI.

[18] 63w Surgical treatment outcomes for neuropathic pain provide mixed results. Neurectomy or rhizotomy are of limited benefit in neuropathic pain (Davies et al. 1991). Amputation or revision of a painful limb or stump is also usually unsuccessful in resolving pain complaints (Sherman et al. 1984;Campbell et al. 1987;Szeinberg-Arazi et al. 1993). Neuroma resection is rarely beneficial (Sherman et al. 1984;Wynn-Parry and Withrington 1984;Loeser 1990).

[19] 233w Surgical release of constrictive mononeuropathies, such as carpal tunnel syndrome (Duncan et al. 19871, or discectomy for nerve root compression injuries from lumbar disc herniation (Abramovitz and Neff 1991), can result in an initial rapid reduction of dysesthesia and pain which precedes regeneration. Later changes in neuropathic pain have also, been described. A bimodal reduction in pain following carpal tunnel release has been reported, with some' patients having immediate pain resolution, and in other patients the pain resolved between 6 and 26.weeks post-release, corresponding temporally with the recovery of motor function (Agee et al. 1992). Successful regeneration following transection can be accompanied by resolution, or a reduction in the area, of the pain and hyperalgesia. This has been reported following peripheral nerve transection and regeneration in man (Rivers and Head 1908; Denny-Brown 19681, and after nerve resection, graft insertion, and regeneration (Noordenbos and Wall 1981). There is a correlation between successful nerve regeneration and resolution of neuropathic pain and allodynia following complete peripheral nerve transections or brachial plexus avulsions. Following nerve repair by reanastomosis or grafting, patients who had extensive motor and sensory recovery were more likely to note decreased pain and allodynia than patients with minimal or absent recovery (Bruxelle et al. 1988;Ochs et al. 1989). Hyperalgesia to heat and cold is initially observed following median or ulnar nerve transection and reanastomosis, but not after regeneration is completed (Braune and Schady 1993).

[20] 105w In conclusion, our study demonstrates that following axonotmetic nerve injuries, functional recovery is associated with resolution of the hyperalgesias mediated by both the injured nerve, and the intact adjacent nerve. When nerve regeneration is prevented, following transection and stump excision, the hyperalgesia does not resolve. If the reversal of hyperalgesia following sciatic CI or CC1 correlates with the reversal of neuropathic pain, then these axonotmetic neuropathy models provide a rational for surgical interventions in human neuropathic pain states. Effective treatments for neuropathic pain may evolve from investigations of surgical or pharmacologic interventions that can mimic the effects that successful regeneration and reinnervation have on hyperalgesia.

[1] 42w were reviewed and approved by our institute's Animal Subjects Committee and followed the guidelines of the IASP (Zimmermann et al. 1983). The animals were carefully examined for signs of pain and distress and it appeared their symptoms were neither severe nor debilitating.

[2] 11w Adult (300-350 g) male Sprague-Dawley rats were used in all experiments.

[3] 30w The animals were housed postoperatively in groups of two in clear plastic cages with solid floors covered with 3-6 cm of soft bedding and were fed and watered ad libitum.

[4] 170w The rats were deeply anesthetized with sodium pentobarbital (50 mg/kg i.p.1 and under clean conditions the sciatic nerves were exposed in the upper thigh. The following sciatic nerve injuries were made just distal to the greater trochanter in the various experiments of this study. (1) A transection of the nerve, with a 1 cm distal stump excision. (2) The CI surgery, using Halsted-mosquito forceps with rounded ground smooth, 2 mm wide jaws to crush the nerve. The hemostats were clamped onto the nerve until the first clip engaged, and the nerve was compressed for 1 min at each of two sites, spaced 5 mm apart. (3) The CI as described above, plus transection of the nerve 3 cm distal to the crush site, with a 1 cm distal stump excision. (4) The CC1 surgery, with 4 ligatures of 4.0 silk tied loosely around the nerve as described by Bennett and Xie (1988). ( 5) Sham surgery, with exposure of the nerve and freeing it of tissue adhesions, without other intervention.

[5] 70w Following all of the above procedures the incisions were closed with wound clips, which were removed 10 days later. Most animals had bilateral surgery. The study and control sides were randomly alternated so that in one-half of the animals the intervention side was on the right and in the other half on the left. Side-to-side comparisons within the same rats were made, with the contralateral side serving as the control.

[6] 29w The weight or temperature at which the rat withdrew its hindpaw from a stimulus was measured, and the mean of 3 consecutive tests was recorded as the withdrawal threshold.

[7] 25w For pressure thresholds an Ugo Basile analgesy-meter (Stoelting, Chicago, IL) was used, which applied a linearly increasing pressure (16 g/secl via a dome-tip stylus (diameter:

[8] 49w 1-2 mm, depending on the depth of tissue displacement) to the hindpaw. The test was stopped if there was no withdrawal response at 250 g. An additional method of measuring pressure withdrawal thresholds utilizing von Frey hairs (North Coast Medical, San Jose, CA1 was used in one CI experiment.

[9] 76w A calibrated set of von Frey fibers were used to apply graded pressure over a range from 4.1 to 130 g to the dorsum of the hindpaw while the rat was manually supported with the hindpaw resting on a flat surface. Three consecutive tests were performed with each filament, and then the next larger filament was tested. This continued until at least 2 withdrawal responses were observed, and that filament's pressure was used as the threshold.

[10] 122w Heat nociceptive thresholds were determined using a linearly increasing temperature (l"C/sec, starting at 40°C) applied with a Peltier device to the hindpaw, as previously described (Vallin and Kingery 1991). The interstimulus interval was approximately 60 set and the test was stopped if there was no withdrawal response by 52°C. This testing procedure is similar to the methods used to demonstrate neuropathic heat hyperalgesia in clinical studies (Lindblom and Verrillo 1979;Verdugo and Ochoa 1992). While we cannot discard the possibility that nociceptors could have been sensitized by this method, this seems unlikely taking into account the limited nature (1 set, < 50°C) of the maximal heat stimuli used in the typical animal (Fitzgerald and Lynn 1977;Campbell et al. 1979;Lynn 1980;LaMotte et al. 19821.

[11] 23w Autotomy behavior was measured weekly, using the method described by Wall et al. (19791, and no animal had a score higher than 7.

[12] 95w A modified 4-point scale was used to measure sciatic motor function (Berenberg et al. 1977;Kalichman and Myers 1987). One point was given for each of the following motor functions: plantar flexion against resistance or active grasp, dorsiflexion against gravity, abduction of digit 5, and abduction of digit 1. The motor function was evaluated by repeatedly partially raising and lowering the animal over a flat surface, to observe ankle dorsiflexion and toe spreading. Plantar flexion and grasp were tested by pushing the plantar paw surface with the examiner's finger tip while the rat was partially suspended.

[13] 41w The area of cutaneous mechanical nociception was measured by testing for the presence or absence of a withdrawal response to forceful pinch with forceps over 10 standard test sites over the ventrum and digits of the hindpaw (Kingery and Vallin 1989).

[14] 107w The testing procedure always followed the same sequence, beginning with the right paw, first measuring the various pressure thresholds, and then the heat thresholds of the hindpaw. The same procedure was then repeated on the left paw. Following the sensory testing the functional testing was performed. The investigators making the measurements were blinded regarding which side the intervention was on, but in the studies utilizing unilateral surgical lesions and contralateral sham surgery there were obvious side-to-side differences in motor loss and autotomy behavior. The pressure, temperature, autotomy, and functional measurements were made on a weekly or biweekly basis postoperatively. All animals were killed after the final measurements.

[15] 114w The following hindpaw threshold sites were measured in some or all experiments: (1) medial dorsum pressure thresholds measured with the analgesy-meter (MDP) over the 1st and 2nd tarsometatarsal joints; (2) medial dorsum pressure thresholds measured with the von Frey fibers (MDP/vF) over the interossei between the 1st and 2nd metatarsals; (3) medial dorsum heat thresholds (MDHI over the medial dorsum of the hindpaw; (4) lateral dorsum pressure thresholds measured with the analgesy-meter (LDP) over the 4th and 5th tarsometatarsal joints; (5) lateral dorsum pressure thresholds measured with the von Frey fibers (LDP/vF) over the interossei between the 4th and 5th metatarsals; (6) plantar surface heat thresholds (PSH) over the plantar surface of the hindpaw.

[16] 66w Experimenf 1. A sciatic CI (n = 8) was compared to contralateral sham surgery within the same rat. Pressure (analgesy-meter and von Frey fiber) and heat thresholds, autotomy behavior, pinch response area, and motor function were evaluated pre-operatively, then weekly for 8 weeks postoperatively. The functional recovery and autotomy data was compared to data derived from a group of unilateral sciatic transection rats (n = 12).

[17] 29w Experiment 2. A sciatic CI was compared to a contralateral sciatic transection within the same rat (n = 12). MDP and MDH thresholds were measured biweekly for 7 weeks.

[18] 32w Experiment 3. A sciatic Cl with a 3 cm distal section was compared to a contralateral sciatic transection tn = 20). The MDP and MDH thresholds were measured biweekly for 6 weeks.

[19] 51w Experiment 4. A sciatic CC1 was compared to a contralateral sham operation (n = 15). Silk 4-O suture was used, instead of chromic gut, to delay regeneration and return of function (Kupers et al. 1992). The MDP, MDH, LDP, and PSH thresholds were measured pre-operatively and then weekly for 13 weeks.

[20] 195w For all experiments a repeated-measures analysis of variance (ANOVA) was performed on the absolute threshold values for each test date, comparing surgical groups, where the repeated measure was time. Then, the Student's t test was used to determine the source of differences among groups. A Wilcoxon signed-ranks test (2-tailed) was used to compare the von Frey fiber thresholds in Experiment 1. All data are presented as the mean f standard error of the mean (S.E.M.), and differences are considered significant for P values less than 0.05. Withdrawal responses were tested to forceps pinch over the plantar paw surface and all digits. Comparing sciatic crush with sciatic section allowed differentiation between sciatic reinnervation and collateral sprouting from the sapbenous nerve. Between days 23 and 38 there was a sharp divergence in the pinch responsive areas for the crush and section animals. From days 32 to 62 the number of pinch-responsive sites was greater for crush than section, with all sites responding after day 38 in the crush animals. C: No heat withdrawal response was observed over the lateral plantar surface for 21 days following sciatic crush. Heat response gradually returned over days 21-42, and then plateaued.

[1] 151w Surgical treatment of nerve injury-induced pain is controversial, with little consensus among clinical specialists experienced in the treatment of chronic pain (Davies et al. 1991). One of these surgical pain treatments is the resection and repair of damaged nerves, either by direct reanastamosis or by bridging the resected ends with a nerve graft. These interventions can result in the regeneration of axons across the injury site and through the distal endoneurium, with some reinnervation of target end-organs. It has been proposed that successful distal reinnervation can initiate neurophysiologic changes in the central terminals of the primary afferents, with a reduction of central hypersensitivity and a subsequent reduction or resolution of pain and hyperalgesia (Fitzgerald et al. 1985). While some authors have recommended reanastamosis or grafting for treating neuropathic pain and hyperalgesia (Campbell et al. 1988;Portenoy 19911, others have had minimal success with such procedures (Noordenbos and Wall 1981;Wynn-Parry and Withrington 1984).

[2] 163w Axonotmesis is the interruption of the nerve's axons with degeneration of the distal segment, without severance of the surrounding connective tissues. When the axons regenerate they are guided through the intact endoneurium and usually reinnervate their respective end-organs, although some axons fail to regenerate, or reinnervate inappropriate targets (Swett et al. 1991). Such lesions may result from traction, crushing, or prolonged pressure. In this study two axonotmetic mononeuropathy models were used to evaluate the temporal correspondence between functional recovery and nociceptive threshold changes. Extensive distal axonal loss is produced in the rat's sciatic nerve with a forceps induced crush injury (CI) or with loose ligature ligation (chronic constrictive injury; CCI). Both injuries resulted in a transient functional loss (sciatic motor and sensory loss after CI, motor loss only after CC11 and a saphenous-mediated adjacent neuropathic hyperalgesia (ANH). The CC1 mononeuropathy also resulted in a transient sciatic-mediated heat hyperalgesia. There was a temporal correspondence between the recovery of function and the resolution of hyperalgesia.

[3] 76w Sciatic transection, with distal segment excision to prevent regeneration, prevents any sciatic-mediated motor or sensory recovery, and results in a long-term ANH mediated by the saphenous nerve (Kingery and Vallin 1989;Vallin and Kingery 1991). The resolution of the hyperalgesia in the CI and CC1 axonopathies, which accompanied functional recovery, and the persistent hyperalgesia observed when reinnervation is prevented by sciatic section and excision, supports the hypothesis that distal axon regeneration can attenuate or resolve neuropathic hyperalgesia.