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Does pain damage spinal cord neurons? Transsynaptic degeneration in rat following a surgical incision
N,'urosciencc L~ ::~'~.~. i ._' i 1993 j :, Si *: 1993 Elsevier Scientific Publishers ireland Ltd, All rights reserved 0304-394(119315 ()6 ~)
7g
NSL 09935
Does pain damage spinal cord neurons? Transsynaptic degeneration in rat following a surgical incision A n n K. N a c h e m s o n a, G a r y J. Bennett *'b aDepartment of Orthopaedics, University of G6teborg, Sahlgren Hospital GOteborg, Sweden bNeurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bldg 49, Room 1A11, 9000 Rockville Pike, Bethesda, MD 20892, USA (Received 7 June 1993; Revised version received 9 July 1993: Accepted 4 August 1993)
Key words." Excitotoxicity; Hyperalgesia; NMDA receptor; Painful peripheral neuropathy; Transsynaptic degeneration In prior studies, rats with a chronic constriction injury to the sciatic nerve have been found to have small- to medium-sized; pyknotic, and hyperchromatic neurons ('dark neurons'; DNs) in spinal dorsal horn laminae I-III. It has been proposed that DNs are produced by an excitotoxic insult involving N-methyl-D-aspartate receptor activation subsequent to ectopic nociceptor discharge, and that at least some DNs are inhibitory interneurons whose functional impairment or death contributes to a central state of hyperexcitability that underlies neuropathic hyperalgesia and allodynia. We show here that laminae I-III DNs are also present 2 days after a surgical procedure that does not include major nerve damage. We propose that this is also the result of a nociceptor-driven excitotoxic insult and that the functional impairment of the affected neurons may contribute to postoperative pain and tenderness.
'Dark neurons' (DNs) are found in laminae I-III of the spinal dorsal horn following certain peripheral nerve injuries. In particular, DNs have been detected in rats sacrificed 8 days after a chronic constriction injury (CCI) [2] to the sciatic nerve [6, 7]. It has been proposed that DNs are the result of an excitotoxic insult due to high levels of ectopic discharge in injured primary afferent neurons, and that the mechanism involves glutaminergic transmission at N-methyl-D-aspartate (NMDA) receptors. Moreover, it has been hypothesized that at least some DNs are inhibitory interneurons whose functional impairment or death creates a state of central hyperexcitability that contributes to the neuropathic pains that accompany the CCI [4, 6, 7]. Abnormal pain sensations are first noted in CCI animals on the second postoperative day [2]. If the hypothesis is correct, then DNs should be detected 48 h after the nerve injury. We began an examination of this question by analyzing a control group of animals that had received a bilateral sham-operation 2 days before sacrifice. To our surprise, we saw a clearly increased incidence of DNs in the lumbar dorsal horn. We now report data that suggest that a subpopulation of spinal cord neurons in laminae I-III are damaged
*Corresponding author. Fax: (1) (301) 402-0667.
by the nociceptor input that accompanies and/or follows ordinary surgery. Four groups (each n = 5) of adult (ca. 300 g) male Sprague Dawley rats were prepared using sodium pentobarbital anesthesia (50 mg/kg, i.p.). Rats in group l received a bilateral surgical exposure of the sciatic nerve. The nerve was approached from the lateral surface of the thigh through a skin incision that was 5 6 cm long and a blunt dissection (about 3 cm long) through the belly of the biceps femoris muscle. The sciatic nerve was gently mobilized from surrounding tissue (as would be done to create the CCI [2]). The wound was then closed in layers. Rats in group 2 were treated in the same way except that the nerve was not mobilized (i.e. a control for inadvertent nerve damage). Rats in group 3 received only a unilateral surgery (with nerve mobilization). Rats in group 4 were anesthetized but not operated on. Using procedures described previously [6, 7], all animals were sacrificed 48 h later and tissue blocks from the lumbar (L4-L5 junction) and cervical (C6) cord were embedded in plastic, thin sectioned (1 pm), and stained with Toluidine blue. DNs were identified (100x objective) according to our standard criteria [6, 7]. The incidence of DNs in each rat was taken to be the mean of the counts in each of 3-5 sections per block (sections separated by at least 75 g m in order to avoid double-counting). The locations of DNs were
79
plotted onto camera lucida drawings of each section. The group incidence data are given as the mean + S.D. of the number of DNs per lumbar hemisection (both sides averaged together for the groups with bilateral surgery or anesthesia alone; each side separately for the unilateral surgery group). The means for the cervical incidences are from counts made in one hemisection only (left or right side at random, except in the group with unilateral surgery where the ipsilateral side was examined). The statistical analysis used ANOVA followed by pair-wise comparisons with the Duncan multiple range test. Significance was accepted at P < 0.05. Counting accuracy was checked by comparing our counts with those of a blinded observer who examined 10 randomly selected sections (approximately 100 DNs). The two counts agreed to within 10%; most of the discrepancies involved one of the observers not counting especially small cells with scant cytoplasm. The light-microscopic appearance of the DNs seen 2 days after surgery was identical to what is seen in CCI rats 8 days after nerve injury (see Figs. 1-3 in ref. 7). The average incidence of DNs in each of the groups is shown in Fig. 1. The results from the cervical sections were pooled because there were no statistically significant between-group differences at this level (groups 1~,, respectively, DNs/hemisection: 2.8 + 1.8; 3.6 + 1.5; 2.5 + 1.0; 3.0 + 1.4). The animals receiving anesthesia-only (group 4) had a low incidence of lumbar DNs (2.8 + 1.0) that
Bilat. with Bilat. w/o
q¢'k
111111111[111111111111111111111 Ipsl.
Unilat, with
J Contra,
Anesth. only Cervical (all)
0
2
4
6
8
10
12
DNs per Hemisection (mean + S.D.) Fig. I. The incidence (mean + S.D.) of DNs in the lumbar dorsal horn of'the four experimental groups and the incidence in the cervical dorsal h~)rn of all groups combined (bottom row). Bilat. with: bilateral surgery with nerve manipulation (group 1). Bilat. w/o: bilateral surgery without nerve manipulation (group 2). Unilat. with (ipsi- and contralateral counts shown separately): unilateral surgery with nerve manipulation (group 3). Anesth. only: anesthesia but no surgery on either side (group 4). "*P < 0.05: significantly greater than either cervical control (withingroup and between-groups comparisons) and significantly greater than the anesthesia-only lumbar control group. "P < 0.05: as above, but also significantly smaller than the lumbar counts from either group with bilateral surgery.
was not significantly different from their own cervical counts or from the cervical counts of all the groups combined. Animals with bilateral surgery with nerve manipulation (group 1) had about a 3-fold increase (relative to the anesthesia-only group's lumbar counts and to the cervical controls) in the average incidence of lumbar DNs (8.1 + 2.3). This is significantly greater than the incidence in their own cervical sections (within-group comparison), to the combined cervical data (between-groups comparison), and relative to the anesthesia-only lumbar controls. The side-to-side differences in the lumbar counts of group 1 were not significantly different. Bilateral surgery without nerve manipulation (group 2) also produced an increased incidence of lumbar DNs (6.6 + 2.5). The incidence is significantly different relative to both cervical comparisons (within-group and between-groups) and relative to the anesthesia-only lumbar controls. The side-to-side differences in the lumbar counts of group 2 were not significantly different. The increase seen in group 2 was smaller than that of group 1, but the difference was not significant. Unilateral surgery with nerve manipulation (group 3) produced an increased incidence of DNs on both sides of the lumbar spinal cord. The ipsilateral incidence (5.3 + 2.0) was larger than the contralateral incidence (4.3 + 1,2), but this was not statistically significant. Each side was increased significantly relative to either cervical comparison (within-group and between-groups) and relative to the anesthesia-only lumbar control. The increases on each side of the unilateral surgery group were significantly less than the increases seen in either of the bilateral surgery groups. In each of the groups receiving surgery, nearly all of the DNs were found in laminae I-1II. Some DNs were in the lateral-most third of these laminae. This region is innervated by the posterior cutaneous nerve of the lhigh [8]; the cutaneous incision was at least partly within the territory of this nerve. In contrast, the lateral-most third of these laminae is conspicuously lacking DNs in CCI rats sacrificed 8 days post-surgery. In the CCI rats, DNs are confined to the damaged sciatic nerve's territory in the medial two-thirds of the laminae [6, 7]. Surgery must activate nociceptors and it is very likely that nociceptors are sensitized during the early postoperative period. Our hypothesis on the cause of DNs and the results given here suggest that surgically-evoked nociceptot input is sufficient to produce at least a temporary excitotoxic insult in a subpopulation of laminae I Ill neurons. All the groups receiving surgery had significantly more lumbar DNs than the group receiving anesthesia alone. There was no cervical versus lumbar difference in the anesthesia-only group, which shows that
80 there is no differential segmental tendency to preparation artefact (e.g. unequal fixation). Significant differences between the surgical groups's lumbar counts and their own cervical counts (the within-group control) rule out general systemic effects, such as stress. Unilateral surgery (with nerve manipulation) produced an increased incidence of lumbar D N s on both sides of the cord. If the presence of D N s is due to nociceptor input, then the number of D N s should be distinctly larger if the amount of nociceptor input is increased by having surgery on both sides. Specifically, one would predict that each side of a case with bilateral surgery would have an incidence of D N s equal to the sum of the ipsi- and contra-lateral effects of an unilateral surgery. In the animals with unilateral surgery (plus nerve manipulation), we found an average of 5.3 D N s per section ipsilaterally and 4.3 D N s per section contralaterally. The prediction would therefore be: 5.3 + 4.3 = 9.6 D N s per side in a case with bilateral surgery. We found an average of 8.1 + 2.3 D N s per side in the group with bilateral surgery (plus nerve manipulation; group 1), a result that is reasonably close to the prediction. In the CCI case, we have speculated that at least some D N s are inhibitory interneurons whose functional impairment or death contributes to the production of a central hyperexcitable state. The appearance of D N s following surgery suggests that such disinhibition might also contribute to postoperative pain and tenderness, Hyperalgesic responses to heat and cold have recently been shown to be present 2 days after a bilateral sham operation like that used here [5]. The increased incidence of D N s contralateral to a surgery on one side is consistent with previous results in the CCI model [6, 7]. We know of no data for hyperalgesia contralateral to a surgical incision, although it has been detected contralateral to the CCI [1]. It m a y be that the presence of D N s is not itself sufficient for the production of hyperalgesia; the appearance of hyperalgesia may require some additional factor, e.g. spontaneous discharge from damaged or sensitized nociceptors. This would be consistent with a recent clinical investigation that showed that both peripheral and central mechanisms were necessary for the appearance of neuropathic pain sensations [4]. Previous studies [6, 7] found a 'basal' (control) incidence of D N s in the cervical and lumbar dorsal horns of 0.5-1.0 cells per hemisection when animals of similar age were sacrificed 8 days post-surgery. The basal incidence in the present study, i.e. the incidence in the cervical dorsal horn of every group and in the lumbar dorsal horn of the rats receiving anesthesia alone, was about 3.0 cells per hemisection. We do not know the cause of the larger basal incidence in the present study; it may be an effect of
barbiturate anesthesia. D N s may recover, remain in an atrophic state, or die. In previous studies with a survival interval of 8 days, rats receiving a bilateral sham operation (with nerve manipulation) did not have an elevated incidence of lumbar D N s [7]. Thus, DNs produced by a surgical incision disappear between 2 and 8 days after surgery, and we may discount the possibility that they remain in an atrophic state. They may recover (completely or partially) or they may die. Their death would be difficult to detect because they are generally small and would leave behind little debris. It now seems probable that there are two mechanisms producing D N s in the CCI animals. First, there is the nociceptor input arising from the surgery. By itself, this mechanism produces D N s that are detectable for only a few days and it resolves when the tissue injury has healed. However, if the injury includes significant nerve damage, a second mechanism appears ongoing ectopic discharge in damaged primary afferent neurons [4]. It is presumably this second mechanism, ectopic discharge, acting alone or with its effect exaggerated by the impact of the surgery, that results in the appearance of D N s 8 days post-surgery in CCI rats. We thank R. Caudle, R. Dubner, and M.A. Ruda for reviewing the manuscript, Mrs. E. Franklin for techincal assistance, and J.M.A. Laird for helpful discussions. 1 Attal, N., Jazat, F., Kayser, V. and Guilbaud, G., Further evidence for 'pain-related' behaviours in a model of unilateral peripheral mononeuropathy, Pain, 41 (1990) 235-251. 2 Bennett, G.J. and Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87 108. 3 Gracely, R.H., Lynch, S.A. and Bennett, G.J., Painful neuropathy: altered central processing maintained dynamically by peripheral input, Pain, 51 (1992) 175 194. 4 Kajander, K.C. and Bennett, G.J., The onset of a painful peripheral neuropathy in rat: a partial and differential deafferentation and spontaneous discharge in Aft and A~ primary afferent neurons, J. Neurophysiol., 68 (1992) 734~74& 5 Ro, L.-S. and Jacobs, J.M., The role of the saphenous nerve in experimental sciatic nerve mononeuropathy produced by loose ligatures: a behavioural study, Pain, 52 (1993) 359 369. 6 Sugimoto, T., Bennett, G.J. and Kajander, K.C., Strychnine-enhanced transsynaptic degeneration of dorsal horn neurons in rats with an experimentalpainful peripheral neuropathy, Neurosci. Lett., 98 (1989) 139 143. 7 Sugimoto, T., Bennett, G.J. and Kajander, K,C., Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine, Pain, 42 (1990) 205-213. 8 Swett, J.E. and Woolf, C.J., The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 (1985) 66-77.
7g
NSL 09935
Does pain damage spinal cord neurons? Transsynaptic degeneration in rat following a surgical incision A n n K. N a c h e m s o n a, G a r y J. Bennett *'b aDepartment of Orthopaedics, University of G6teborg, Sahlgren Hospital GOteborg, Sweden bNeurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bldg 49, Room 1A11, 9000 Rockville Pike, Bethesda, MD 20892, USA (Received 7 June 1993; Revised version received 9 July 1993: Accepted 4 August 1993)
Key words." Excitotoxicity; Hyperalgesia; NMDA receptor; Painful peripheral neuropathy; Transsynaptic degeneration In prior studies, rats with a chronic constriction injury to the sciatic nerve have been found to have small- to medium-sized; pyknotic, and hyperchromatic neurons ('dark neurons'; DNs) in spinal dorsal horn laminae I-III. It has been proposed that DNs are produced by an excitotoxic insult involving N-methyl-D-aspartate receptor activation subsequent to ectopic nociceptor discharge, and that at least some DNs are inhibitory interneurons whose functional impairment or death contributes to a central state of hyperexcitability that underlies neuropathic hyperalgesia and allodynia. We show here that laminae I-III DNs are also present 2 days after a surgical procedure that does not include major nerve damage. We propose that this is also the result of a nociceptor-driven excitotoxic insult and that the functional impairment of the affected neurons may contribute to postoperative pain and tenderness.
'Dark neurons' (DNs) are found in laminae I-III of the spinal dorsal horn following certain peripheral nerve injuries. In particular, DNs have been detected in rats sacrificed 8 days after a chronic constriction injury (CCI) [2] to the sciatic nerve [6, 7]. It has been proposed that DNs are the result of an excitotoxic insult due to high levels of ectopic discharge in injured primary afferent neurons, and that the mechanism involves glutaminergic transmission at N-methyl-D-aspartate (NMDA) receptors. Moreover, it has been hypothesized that at least some DNs are inhibitory interneurons whose functional impairment or death creates a state of central hyperexcitability that contributes to the neuropathic pains that accompany the CCI [4, 6, 7]. Abnormal pain sensations are first noted in CCI animals on the second postoperative day [2]. If the hypothesis is correct, then DNs should be detected 48 h after the nerve injury. We began an examination of this question by analyzing a control group of animals that had received a bilateral sham-operation 2 days before sacrifice. To our surprise, we saw a clearly increased incidence of DNs in the lumbar dorsal horn. We now report data that suggest that a subpopulation of spinal cord neurons in laminae I-III are damaged
*Corresponding author. Fax: (1) (301) 402-0667.
by the nociceptor input that accompanies and/or follows ordinary surgery. Four groups (each n = 5) of adult (ca. 300 g) male Sprague Dawley rats were prepared using sodium pentobarbital anesthesia (50 mg/kg, i.p.). Rats in group l received a bilateral surgical exposure of the sciatic nerve. The nerve was approached from the lateral surface of the thigh through a skin incision that was 5 6 cm long and a blunt dissection (about 3 cm long) through the belly of the biceps femoris muscle. The sciatic nerve was gently mobilized from surrounding tissue (as would be done to create the CCI [2]). The wound was then closed in layers. Rats in group 2 were treated in the same way except that the nerve was not mobilized (i.e. a control for inadvertent nerve damage). Rats in group 3 received only a unilateral surgery (with nerve mobilization). Rats in group 4 were anesthetized but not operated on. Using procedures described previously [6, 7], all animals were sacrificed 48 h later and tissue blocks from the lumbar (L4-L5 junction) and cervical (C6) cord were embedded in plastic, thin sectioned (1 pm), and stained with Toluidine blue. DNs were identified (100x objective) according to our standard criteria [6, 7]. The incidence of DNs in each rat was taken to be the mean of the counts in each of 3-5 sections per block (sections separated by at least 75 g m in order to avoid double-counting). The locations of DNs were
79
plotted onto camera lucida drawings of each section. The group incidence data are given as the mean + S.D. of the number of DNs per lumbar hemisection (both sides averaged together for the groups with bilateral surgery or anesthesia alone; each side separately for the unilateral surgery group). The means for the cervical incidences are from counts made in one hemisection only (left or right side at random, except in the group with unilateral surgery where the ipsilateral side was examined). The statistical analysis used ANOVA followed by pair-wise comparisons with the Duncan multiple range test. Significance was accepted at P < 0.05. Counting accuracy was checked by comparing our counts with those of a blinded observer who examined 10 randomly selected sections (approximately 100 DNs). The two counts agreed to within 10%; most of the discrepancies involved one of the observers not counting especially small cells with scant cytoplasm. The light-microscopic appearance of the DNs seen 2 days after surgery was identical to what is seen in CCI rats 8 days after nerve injury (see Figs. 1-3 in ref. 7). The average incidence of DNs in each of the groups is shown in Fig. 1. The results from the cervical sections were pooled because there were no statistically significant between-group differences at this level (groups 1~,, respectively, DNs/hemisection: 2.8 + 1.8; 3.6 + 1.5; 2.5 + 1.0; 3.0 + 1.4). The animals receiving anesthesia-only (group 4) had a low incidence of lumbar DNs (2.8 + 1.0) that
Bilat. with Bilat. w/o
q¢'k
111111111[111111111111111111111 Ipsl.
Unilat, with
J Contra,
Anesth. only Cervical (all)
0
2
4
6
8
10
12
DNs per Hemisection (mean + S.D.) Fig. I. The incidence (mean + S.D.) of DNs in the lumbar dorsal horn of'the four experimental groups and the incidence in the cervical dorsal h~)rn of all groups combined (bottom row). Bilat. with: bilateral surgery with nerve manipulation (group 1). Bilat. w/o: bilateral surgery without nerve manipulation (group 2). Unilat. with (ipsi- and contralateral counts shown separately): unilateral surgery with nerve manipulation (group 3). Anesth. only: anesthesia but no surgery on either side (group 4). "*P < 0.05: significantly greater than either cervical control (withingroup and between-groups comparisons) and significantly greater than the anesthesia-only lumbar control group. "P < 0.05: as above, but also significantly smaller than the lumbar counts from either group with bilateral surgery.
was not significantly different from their own cervical counts or from the cervical counts of all the groups combined. Animals with bilateral surgery with nerve manipulation (group 1) had about a 3-fold increase (relative to the anesthesia-only group's lumbar counts and to the cervical controls) in the average incidence of lumbar DNs (8.1 + 2.3). This is significantly greater than the incidence in their own cervical sections (within-group comparison), to the combined cervical data (between-groups comparison), and relative to the anesthesia-only lumbar controls. The side-to-side differences in the lumbar counts of group 1 were not significantly different. Bilateral surgery without nerve manipulation (group 2) also produced an increased incidence of lumbar DNs (6.6 + 2.5). The incidence is significantly different relative to both cervical comparisons (within-group and between-groups) and relative to the anesthesia-only lumbar controls. The side-to-side differences in the lumbar counts of group 2 were not significantly different. The increase seen in group 2 was smaller than that of group 1, but the difference was not significant. Unilateral surgery with nerve manipulation (group 3) produced an increased incidence of DNs on both sides of the lumbar spinal cord. The ipsilateral incidence (5.3 + 2.0) was larger than the contralateral incidence (4.3 + 1,2), but this was not statistically significant. Each side was increased significantly relative to either cervical comparison (within-group and between-groups) and relative to the anesthesia-only lumbar control. The increases on each side of the unilateral surgery group were significantly less than the increases seen in either of the bilateral surgery groups. In each of the groups receiving surgery, nearly all of the DNs were found in laminae I-1II. Some DNs were in the lateral-most third of these laminae. This region is innervated by the posterior cutaneous nerve of the lhigh [8]; the cutaneous incision was at least partly within the territory of this nerve. In contrast, the lateral-most third of these laminae is conspicuously lacking DNs in CCI rats sacrificed 8 days post-surgery. In the CCI rats, DNs are confined to the damaged sciatic nerve's territory in the medial two-thirds of the laminae [6, 7]. Surgery must activate nociceptors and it is very likely that nociceptors are sensitized during the early postoperative period. Our hypothesis on the cause of DNs and the results given here suggest that surgically-evoked nociceptot input is sufficient to produce at least a temporary excitotoxic insult in a subpopulation of laminae I Ill neurons. All the groups receiving surgery had significantly more lumbar DNs than the group receiving anesthesia alone. There was no cervical versus lumbar difference in the anesthesia-only group, which shows that
80 there is no differential segmental tendency to preparation artefact (e.g. unequal fixation). Significant differences between the surgical groups's lumbar counts and their own cervical counts (the within-group control) rule out general systemic effects, such as stress. Unilateral surgery (with nerve manipulation) produced an increased incidence of lumbar D N s on both sides of the cord. If the presence of D N s is due to nociceptor input, then the number of D N s should be distinctly larger if the amount of nociceptor input is increased by having surgery on both sides. Specifically, one would predict that each side of a case with bilateral surgery would have an incidence of D N s equal to the sum of the ipsi- and contra-lateral effects of an unilateral surgery. In the animals with unilateral surgery (plus nerve manipulation), we found an average of 5.3 D N s per section ipsilaterally and 4.3 D N s per section contralaterally. The prediction would therefore be: 5.3 + 4.3 = 9.6 D N s per side in a case with bilateral surgery. We found an average of 8.1 + 2.3 D N s per side in the group with bilateral surgery (plus nerve manipulation; group 1), a result that is reasonably close to the prediction. In the CCI case, we have speculated that at least some D N s are inhibitory interneurons whose functional impairment or death contributes to the production of a central hyperexcitable state. The appearance of D N s following surgery suggests that such disinhibition might also contribute to postoperative pain and tenderness, Hyperalgesic responses to heat and cold have recently been shown to be present 2 days after a bilateral sham operation like that used here [5]. The increased incidence of D N s contralateral to a surgery on one side is consistent with previous results in the CCI model [6, 7]. We know of no data for hyperalgesia contralateral to a surgical incision, although it has been detected contralateral to the CCI [1]. It m a y be that the presence of D N s is not itself sufficient for the production of hyperalgesia; the appearance of hyperalgesia may require some additional factor, e.g. spontaneous discharge from damaged or sensitized nociceptors. This would be consistent with a recent clinical investigation that showed that both peripheral and central mechanisms were necessary for the appearance of neuropathic pain sensations [4]. Previous studies [6, 7] found a 'basal' (control) incidence of D N s in the cervical and lumbar dorsal horns of 0.5-1.0 cells per hemisection when animals of similar age were sacrificed 8 days post-surgery. The basal incidence in the present study, i.e. the incidence in the cervical dorsal horn of every group and in the lumbar dorsal horn of the rats receiving anesthesia alone, was about 3.0 cells per hemisection. We do not know the cause of the larger basal incidence in the present study; it may be an effect of
barbiturate anesthesia. D N s may recover, remain in an atrophic state, or die. In previous studies with a survival interval of 8 days, rats receiving a bilateral sham operation (with nerve manipulation) did not have an elevated incidence of lumbar D N s [7]. Thus, DNs produced by a surgical incision disappear between 2 and 8 days after surgery, and we may discount the possibility that they remain in an atrophic state. They may recover (completely or partially) or they may die. Their death would be difficult to detect because they are generally small and would leave behind little debris. It now seems probable that there are two mechanisms producing D N s in the CCI animals. First, there is the nociceptor input arising from the surgery. By itself, this mechanism produces D N s that are detectable for only a few days and it resolves when the tissue injury has healed. However, if the injury includes significant nerve damage, a second mechanism appears ongoing ectopic discharge in damaged primary afferent neurons [4]. It is presumably this second mechanism, ectopic discharge, acting alone or with its effect exaggerated by the impact of the surgery, that results in the appearance of D N s 8 days post-surgery in CCI rats. We thank R. Caudle, R. Dubner, and M.A. Ruda for reviewing the manuscript, Mrs. E. Franklin for techincal assistance, and J.M.A. Laird for helpful discussions. 1 Attal, N., Jazat, F., Kayser, V. and Guilbaud, G., Further evidence for 'pain-related' behaviours in a model of unilateral peripheral mononeuropathy, Pain, 41 (1990) 235-251. 2 Bennett, G.J. and Xie, Y.-K., A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man, Pain, 33 (1988) 87 108. 3 Gracely, R.H., Lynch, S.A. and Bennett, G.J., Painful neuropathy: altered central processing maintained dynamically by peripheral input, Pain, 51 (1992) 175 194. 4 Kajander, K.C. and Bennett, G.J., The onset of a painful peripheral neuropathy in rat: a partial and differential deafferentation and spontaneous discharge in Aft and A~ primary afferent neurons, J. Neurophysiol., 68 (1992) 734~74& 5 Ro, L.-S. and Jacobs, J.M., The role of the saphenous nerve in experimental sciatic nerve mononeuropathy produced by loose ligatures: a behavioural study, Pain, 52 (1993) 359 369. 6 Sugimoto, T., Bennett, G.J. and Kajander, K.C., Strychnine-enhanced transsynaptic degeneration of dorsal horn neurons in rats with an experimentalpainful peripheral neuropathy, Neurosci. Lett., 98 (1989) 139 143. 7 Sugimoto, T., Bennett, G.J. and Kajander, K,C., Transsynaptic degeneration in the superficial dorsal horn after sciatic nerve injury: effects of a chronic constriction injury, transection, and strychnine, Pain, 42 (1990) 205-213. 8 Swett, J.E. and Woolf, C.J., The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord, J. Comp. Neurol., 231 (1985) 66-77.