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Neuroma formation and numbers of axons in a rat model of experimental peripheral neuropathy
88
Neuroscience Letters, 131 (199l) 88-92 1991 Elsevier Scientific Publishers Ireland Ltd. ADONIS 030439409100549M
NSL 08067
Neuroma formation and numbers of axons in a rat model of experimental peripheral neuropathy Susan M. Carlton, Patrick M. D o u g h e r t y , C a r o l y n M. Pover a n d R i c h a r d E. Coggeshall Marine Biomedical Institute, Galveston, TX 77550 (U.S.A.) (Received 24 April 1991; Revised version received 20 June 1991; Accepted 21 June 1991) Key words: Peripheral nerve; Hyperalgesia; Partial nerve constriction; Rat Two weeks following chronic partial constriction of rat sciatic nerve, the perineurium was disrupted and a neuroma had formed at the constriction site in all nerves (n = 5). Axon counts demonstrated an 84-99% and a 62-84% decrease in myelinated and unmyelinated axons respectively, distal to the lesion. Distally, the majority of surviving myelinated axons had diameters of less than 5.0/am. There was considerable disparity in fiber loss from animal to animal, but similar behavioral changes were demonstrated by all animals. These results are discussed with reference to previously published data and possible mechanisms underlying the behavioral manifestations of this neuropathy model.
Chronic partial constriction of the sciatic nerve produces a neuropathy characterized by hyperalgesia and allodynia in the affected limb [3]. Important questions to be investigated in this pain model are the degree of fiber damage in the sciatic nerve distal to the injury and the nature of the lesion at the site of the constriction. With these issues in mind, the goals of the present study are to document: (1) changes in the unmyelinated and myelinated fiber populations distal to the lesion, particularly the numbers and diameters of surviving fibers, (2) neuroma formation at the site of constriction and (3) correlations between fiber loss and behavioral changes. Five rats (male, Sprague-Dawley, 250-300 g) were tested for paw withdrawal latency (PWL) to radiant heat, anesthetized (Nembutal, 35 mg/kg i.p.), and then one sciatic nerve was constricted by 4 loose ligatures [3]. A sham operation was done contralaterally. The PWLs were determined again on days 7 and 14 postsurgery. After the final test, the animals were anesthetized and perfused with mixed aldehydes containing 0.1% picric acid. Segments of injured sciatic nerve within 1 cm distal and proximal to the constricted region and the constricted region itself were prepared for light (LM) and electron microscopy (EM). Montages (× 900) of the proximal and distal nerves were prepared from thin sections from 4 animals. A grid consisting of 10 cm squares was placed Correspondence: S.M. Carlton, 200 University Blvd., Marine Biomedical Institute, Galveston, TX 77550, U.S.A.
randomly over each montage and the squares numbered sequentially. Using even or odd numbers (chosen at random), all axons in every alternate square were counted. The counts were then multiplied by two to give the final estimates. To verify our sampling method, every square was counted in one nerve and the numbers compared with estimates obtained by counting every 2nd, every 3rd square, etc. Counting every other square, our estimates were within 5% of the true number. Counting fewer squares gave unacceptable variability. Ultrathin sections through the constricted region were analyzed for general morphology. Diameter measurements of approximately 100 randomly selected myelinated axons were obtained for each proximal and distal segment. Presurgery, there was no significant difference between left and right PWLs. However, on days 7 and 14 postsurgery, all 5 animals showed significantly faster PWLs on the operated compared to the sham-operated side (paired t-test, P < 0.05). By 2 days postsurgery, each animal demonstrated the characteristic guarding behavior described previously [1, 3, 9]. At the LM level, each proximal nerve appeared normal (Fig. I A). Distal to the injury, each nerve had fewer myelinated fibers, much degenerating myelin, many activated Schwann cells and macrophages laden with membraneous debris (Fig. I B). The region of constriction revealed a loss of the perineurium around part or all of the nerve. At the EM level, most myelinated axons in the proxi-
89
Fig. I. Proximal (A) and distal (B) segments from an injured sciatic nerve. In the distal segment, note the decrease in the number of myelinated fibers and signs of degeneration in those that remain. Bars for A and B = 50/zm. Electron micrographs demonstrating relatively normal unmyelinated axons in Remak bundles (arrows, C) and proliferating Schwann cell processes (arrows, D) which are commonly observed in the distal segments. Regenerating myelinated axons (M), with thin myelin sheaths are shown in E (distal segment) and F (site of constriction). Axonal growth cones are also demonstrated in F (arrows). Bars for C-F = 0.5/zm.
90 mal nerves appeared normal, but an occasional degenerating axon could be seen. Many unmyelinated axons and attending Schwann cells were normal, but there was also Schwann cell process proliferation. The distal nerve segments showed much Waller±an degeneration but some myelinated and unmyelinated axons survived (Fig. 1C). Surviving unmyelinated axons had to be carefully differentiated from proliferating Schwann cell processes (Fig. I D) by the criteria of lighter cytoplasm and a typical relation with Schwann cells [11], Some myelinated axons had thin myelin sheaths (Fig. 1E), and were regarded as regenerating. The perineurium was disrupted at the site of constriction. Many axons, presumed growth cones and Schwann cells could be seen in the connective tissue around each nerve (Fig. 1F). Axon counts demonstrated a 62-84% decrease in unmyelinated fibers and an 84-99% decrease in myelinated fibers distally (Table I). These decreases were statistically significant (paired t-test, P < 0.01). A significant decrease in the proportion of large diameter fibers ( t> 5.0 /tm) was noted in the distal nerves (Mann-Whitney U-test, P<0.05, Fig. 2A). There was, however, great variability in the decrease in large diameter fibers (Fig. 2B and C). In the distal segment, the small myelinated fibers (< 5.0/tm), had a mean density of 390/mm 2, with a range of 25--869/mm 2, and the large fibers (> 5.0/~m) had a mean density of 174/mm 2, with a range of 0-594/ mm 2. The findings of the present study are: (1) a 84-99% decrease in myelinated axons and a 62-84% decrease in unmyelinated axons in the distal nerve segment as compared to the proximal, (2) the majority of myelinated axons in the distal nerve are Ats ( < 5.0/tm in diameter), (3) a neuroma forms at the site of the injury, (4) regardless of the difference in fiber loss, each animal demonstrates hyperalgesia and guarding behavior. Our data confirm Basbaum et al., [2], that unmyeli-
nated fibers are lost and Gautron et al., [7], that large diameter fibers are greatly reduced. However, our specific counts differ from their estimates. We find a much greater loss of unmyelinated axons, up to an 84% decrease compared to the 33% decrease reported by Basbaum et al., [2]. Gautron et al., [7] reported the density of A8 fibers in the distal segment to be more than 5000/ mm 2 and hypothesized that this fiber population was 'partly responsible for most of the pain-related behavior observed in these rats'. In the present study, we found the mean density of A8 fibers to be only 390/mm 2. We wish to emphasize that in 3 out of 4 animals, 97-99% of the total myelinated fiber populations were lost distal to the injury, indicating that myelinated fibers, Ats included, are probably not critical for the pathogenesis of pain in this model. Differences between our counts and those previously reported may be variability in surgical techniques or different methods of counting axons. Sections through the site of injury demonstrated neuroma characterized by a disrupted perineurium and numerous growth cones [12]. These findings confirm the previous suggestion that fibers 'evocative' of a neuroma are present [7]. Furthermore, since a portion of dorsal root ganglion cells maintain contact with the periphery, this is a 'neuroma-in-continuity' [12]. It is thus of interest that the time course of our behavioral hyperalgesia parallels the time course of a 'neuroma discharge'. In a typical neuroma, there is virtually no recordable activity until two days following injury, then fiber activity rises to high levels for about two weeks after which it falls back to a low sustained level [5, 8]. This is the same time course as the behavioral changes in this neuropathy model [7, 9, the present paper], in that the animals demonstrate guarding or hyperalgesia 2-3 days postsurgery and these behaviors persist for approximately 14 days, after which the animals trend toward normal PWLs for the affected limb. In our opinion, the close
TABLE I COUNTS OF MYELINATED AND UNMYEL1NATED AXONS PROXIMAL AND DISTAL TO CONSTRICTION OF THE SCIATIC NERVE M = myelinated,UM = unmyelinated. Proximal
Distal
%Decrease
Rat
M
UM
M
UM
M
UM
1 2 3 4 2±S.E.M.
7940 8240 8398 8312 8222± 100
20,310 21,122 14,772 11,456 16,915±2302
280 1288 24 219 452± 284
4874 8134 4312 1873 4798± 1289
97 84 99 97 94± 3
76 62 71 84 73 ± 5
91
HISTOGRAMS OF FIBER DIAMETERS
A
30
PROXIMALme DISTAL
25 20
l tl ,
15 10 5 0
>
LU CO m 0
40 30
7
2O
W ~
0
UJ
25 20 15 10 5 0
~B
~ I
I
HIlllfp,, O)
o)
O)
~)
O)
0')
I
I
I
I
l I,..
I I QO o)
DIAMETER
O)
0
(pm)
Fig. 2. Histograms demonstrating the distribution of myelinated axons by diameter in the distal segments. A: for the whole population (n = 5), note a significant decrease in large myelinated axons (/> 5.0 gm) and a relative increase in small myelinated axons ( < 5.0 #m). Also note the great variability between animals as demonstrated between rat 3 (B) and rat 4 (C).
correspondence of neuroma activity and behavior in our paradigm suggests that peripheral mechanisms involving the neuroma must be considered in the generation of the behavior. The other factors we wish to consider are the numbers and ratios of myelinated and unmyelinated axons distal to the lesion. First, the numbers of axons are quite variable' from animal to animal. Myelinated axons vary by almost 60 fold (24 in rat 3 as compared to 1288 in rat 2) and unmyelinated axons by approximately 7 fold.
Nevertheless there are some common morphological features observed in the distal segments. For example, all animals show a considerable loss in small myelinated and unmyelinated axons with a greater loss in large myelinated axons compared to the proximal segment. Increased pain (hyperalgesia) associated with loss of large myelinated axons is consistent with the gate theory of pain [10], and the marked reduction of both small myelinated and unmyelinated axons is consistent with the hypothesis of Dyck et al., [6] that pain occurs in humans when both small myelinated and unmyelinated axons are lost. Furthermore, the loss of small myelinated fibers (A&s) may lead to disinhibition of spinothalamic tract cells [4], one of the most important tracts transmitting nociceptive input to higher brain centers [13]. Such correlations do not imply causal relationships, but they do indicate that the axons that pass through the lesion into the distal nerve are probably of importance in the generation of the symptoms. Experiments separating the effects of neuroma activity from those of the axon activity in the distal nerve would be desirable. At present, we hypothesize that input from surviving axons in the distal segment results in increased activity in the neuroma which in turn sends signals centrally that are interpreted as nociceptive by the animal. We focus particularly on the unmyelinated axons because fewer of these are lost and also because hyperalgesia occurred in an animal with only 24 surviving myelinated axons distal to the injury. The authors thank Elizabeth Hayes and Gregory Hargett for technical assistance. This study was supported by NS 27910 (S.M.C.), NSl1255 (S.M.C., R.E.C.), NS10161 (R.E.C.) and NS08860 (P.M.D.). 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 Basbaum, A.I., Jazat, F. and Guilbaud, G., The spectrum of fiber loss in a model of neuropathic pain in the rat, Am. Pain Soc. Abstr., 8 (1990) 7. 3 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) 87-107. 4 Chung, J.M., Lee, K.H., Hori, Y., Endo, K. and Willis, W.D., Factors influencing peripheral nerve stimulation produced inhibition of primate spinothalamic tract cells, Pain, (1984) 277-293. 5 Devor, M., The pathophysiology and anatomy of damaged nerve. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, New York, 1984, pp. 49~a4. 6 Dyck, P.J., Lambert, E.H. and O'Brien, P.C., Pain in peripheral neuropathy related to rate and kind of fiber degeneration, Neurology, 26 (1976) 466-471. 7 Gautron, M., Jazat, F., Ratinahirana, H., Hauw, J.J. and Guilbaud, G., Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of
92 abnormal small myelinated fibres and pain-related behavior, Neurosci. Lett., 111 (1990)28-33. 8 Govrin-Lippmann, R. and Devor, M., Ongoing activity in severed nerves: source and variation with time, Brain Res., 159 (1978) 406410. 9 Kajander, K.C., Sahara, Y., ladarola, M.J. and Bennett, G.J., Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy, Peptides, 11 (1990) 719-728.
10 Melzack, R. and Wall, P.D., Pain mechanisms: a new theory, Science, 150 (1965) 971-979. 11 Payer, A.F., An ultrastructural study of Schwann cell response to axonal degeneration, J. Comp. Neurol., 183 (1979) 365-384. 12 Sunderland, S., Nerves and nerve injuries, 2nd edn., Churchill Livingstone, New York, 1988, pp. 188-193. 13 Willis, W.D., The Pain System, Karger, New York, 1985, pp. 145 206.
Neuroscience Letters, 131 (199l) 88-92 1991 Elsevier Scientific Publishers Ireland Ltd. ADONIS 030439409100549M
NSL 08067
Neuroma formation and numbers of axons in a rat model of experimental peripheral neuropathy Susan M. Carlton, Patrick M. D o u g h e r t y , C a r o l y n M. Pover a n d R i c h a r d E. Coggeshall Marine Biomedical Institute, Galveston, TX 77550 (U.S.A.) (Received 24 April 1991; Revised version received 20 June 1991; Accepted 21 June 1991) Key words: Peripheral nerve; Hyperalgesia; Partial nerve constriction; Rat Two weeks following chronic partial constriction of rat sciatic nerve, the perineurium was disrupted and a neuroma had formed at the constriction site in all nerves (n = 5). Axon counts demonstrated an 84-99% and a 62-84% decrease in myelinated and unmyelinated axons respectively, distal to the lesion. Distally, the majority of surviving myelinated axons had diameters of less than 5.0/am. There was considerable disparity in fiber loss from animal to animal, but similar behavioral changes were demonstrated by all animals. These results are discussed with reference to previously published data and possible mechanisms underlying the behavioral manifestations of this neuropathy model.
Chronic partial constriction of the sciatic nerve produces a neuropathy characterized by hyperalgesia and allodynia in the affected limb [3]. Important questions to be investigated in this pain model are the degree of fiber damage in the sciatic nerve distal to the injury and the nature of the lesion at the site of the constriction. With these issues in mind, the goals of the present study are to document: (1) changes in the unmyelinated and myelinated fiber populations distal to the lesion, particularly the numbers and diameters of surviving fibers, (2) neuroma formation at the site of constriction and (3) correlations between fiber loss and behavioral changes. Five rats (male, Sprague-Dawley, 250-300 g) were tested for paw withdrawal latency (PWL) to radiant heat, anesthetized (Nembutal, 35 mg/kg i.p.), and then one sciatic nerve was constricted by 4 loose ligatures [3]. A sham operation was done contralaterally. The PWLs were determined again on days 7 and 14 postsurgery. After the final test, the animals were anesthetized and perfused with mixed aldehydes containing 0.1% picric acid. Segments of injured sciatic nerve within 1 cm distal and proximal to the constricted region and the constricted region itself were prepared for light (LM) and electron microscopy (EM). Montages (× 900) of the proximal and distal nerves were prepared from thin sections from 4 animals. A grid consisting of 10 cm squares was placed Correspondence: S.M. Carlton, 200 University Blvd., Marine Biomedical Institute, Galveston, TX 77550, U.S.A.
randomly over each montage and the squares numbered sequentially. Using even or odd numbers (chosen at random), all axons in every alternate square were counted. The counts were then multiplied by two to give the final estimates. To verify our sampling method, every square was counted in one nerve and the numbers compared with estimates obtained by counting every 2nd, every 3rd square, etc. Counting every other square, our estimates were within 5% of the true number. Counting fewer squares gave unacceptable variability. Ultrathin sections through the constricted region were analyzed for general morphology. Diameter measurements of approximately 100 randomly selected myelinated axons were obtained for each proximal and distal segment. Presurgery, there was no significant difference between left and right PWLs. However, on days 7 and 14 postsurgery, all 5 animals showed significantly faster PWLs on the operated compared to the sham-operated side (paired t-test, P < 0.05). By 2 days postsurgery, each animal demonstrated the characteristic guarding behavior described previously [1, 3, 9]. At the LM level, each proximal nerve appeared normal (Fig. I A). Distal to the injury, each nerve had fewer myelinated fibers, much degenerating myelin, many activated Schwann cells and macrophages laden with membraneous debris (Fig. I B). The region of constriction revealed a loss of the perineurium around part or all of the nerve. At the EM level, most myelinated axons in the proxi-
89
Fig. I. Proximal (A) and distal (B) segments from an injured sciatic nerve. In the distal segment, note the decrease in the number of myelinated fibers and signs of degeneration in those that remain. Bars for A and B = 50/zm. Electron micrographs demonstrating relatively normal unmyelinated axons in Remak bundles (arrows, C) and proliferating Schwann cell processes (arrows, D) which are commonly observed in the distal segments. Regenerating myelinated axons (M), with thin myelin sheaths are shown in E (distal segment) and F (site of constriction). Axonal growth cones are also demonstrated in F (arrows). Bars for C-F = 0.5/zm.
90 mal nerves appeared normal, but an occasional degenerating axon could be seen. Many unmyelinated axons and attending Schwann cells were normal, but there was also Schwann cell process proliferation. The distal nerve segments showed much Waller±an degeneration but some myelinated and unmyelinated axons survived (Fig. 1C). Surviving unmyelinated axons had to be carefully differentiated from proliferating Schwann cell processes (Fig. I D) by the criteria of lighter cytoplasm and a typical relation with Schwann cells [11], Some myelinated axons had thin myelin sheaths (Fig. 1E), and were regarded as regenerating. The perineurium was disrupted at the site of constriction. Many axons, presumed growth cones and Schwann cells could be seen in the connective tissue around each nerve (Fig. 1F). Axon counts demonstrated a 62-84% decrease in unmyelinated fibers and an 84-99% decrease in myelinated fibers distally (Table I). These decreases were statistically significant (paired t-test, P < 0.01). A significant decrease in the proportion of large diameter fibers ( t> 5.0 /tm) was noted in the distal nerves (Mann-Whitney U-test, P<0.05, Fig. 2A). There was, however, great variability in the decrease in large diameter fibers (Fig. 2B and C). In the distal segment, the small myelinated fibers (< 5.0/tm), had a mean density of 390/mm 2, with a range of 25--869/mm 2, and the large fibers (> 5.0/~m) had a mean density of 174/mm 2, with a range of 0-594/ mm 2. The findings of the present study are: (1) a 84-99% decrease in myelinated axons and a 62-84% decrease in unmyelinated axons in the distal nerve segment as compared to the proximal, (2) the majority of myelinated axons in the distal nerve are Ats ( < 5.0/tm in diameter), (3) a neuroma forms at the site of the injury, (4) regardless of the difference in fiber loss, each animal demonstrates hyperalgesia and guarding behavior. Our data confirm Basbaum et al., [2], that unmyeli-
nated fibers are lost and Gautron et al., [7], that large diameter fibers are greatly reduced. However, our specific counts differ from their estimates. We find a much greater loss of unmyelinated axons, up to an 84% decrease compared to the 33% decrease reported by Basbaum et al., [2]. Gautron et al., [7] reported the density of A8 fibers in the distal segment to be more than 5000/ mm 2 and hypothesized that this fiber population was 'partly responsible for most of the pain-related behavior observed in these rats'. In the present study, we found the mean density of A8 fibers to be only 390/mm 2. We wish to emphasize that in 3 out of 4 animals, 97-99% of the total myelinated fiber populations were lost distal to the injury, indicating that myelinated fibers, Ats included, are probably not critical for the pathogenesis of pain in this model. Differences between our counts and those previously reported may be variability in surgical techniques or different methods of counting axons. Sections through the site of injury demonstrated neuroma characterized by a disrupted perineurium and numerous growth cones [12]. These findings confirm the previous suggestion that fibers 'evocative' of a neuroma are present [7]. Furthermore, since a portion of dorsal root ganglion cells maintain contact with the periphery, this is a 'neuroma-in-continuity' [12]. It is thus of interest that the time course of our behavioral hyperalgesia parallels the time course of a 'neuroma discharge'. In a typical neuroma, there is virtually no recordable activity until two days following injury, then fiber activity rises to high levels for about two weeks after which it falls back to a low sustained level [5, 8]. This is the same time course as the behavioral changes in this neuropathy model [7, 9, the present paper], in that the animals demonstrate guarding or hyperalgesia 2-3 days postsurgery and these behaviors persist for approximately 14 days, after which the animals trend toward normal PWLs for the affected limb. In our opinion, the close
TABLE I COUNTS OF MYELINATED AND UNMYEL1NATED AXONS PROXIMAL AND DISTAL TO CONSTRICTION OF THE SCIATIC NERVE M = myelinated,UM = unmyelinated. Proximal
Distal
%Decrease
Rat
M
UM
M
UM
M
UM
1 2 3 4 2±S.E.M.
7940 8240 8398 8312 8222± 100
20,310 21,122 14,772 11,456 16,915±2302
280 1288 24 219 452± 284
4874 8134 4312 1873 4798± 1289
97 84 99 97 94± 3
76 62 71 84 73 ± 5
91
HISTOGRAMS OF FIBER DIAMETERS
A
30
PROXIMALme DISTAL
25 20
l tl ,
15 10 5 0
>
LU CO m 0
40 30
7
2O
W ~
0
UJ
25 20 15 10 5 0
~B
~ I
I
HIlllfp,, O)
o)
O)
~)
O)
0')
I
I
I
I
l I,..
I I QO o)
DIAMETER
O)
0
(pm)
Fig. 2. Histograms demonstrating the distribution of myelinated axons by diameter in the distal segments. A: for the whole population (n = 5), note a significant decrease in large myelinated axons (/> 5.0 gm) and a relative increase in small myelinated axons ( < 5.0 #m). Also note the great variability between animals as demonstrated between rat 3 (B) and rat 4 (C).
correspondence of neuroma activity and behavior in our paradigm suggests that peripheral mechanisms involving the neuroma must be considered in the generation of the behavior. The other factors we wish to consider are the numbers and ratios of myelinated and unmyelinated axons distal to the lesion. First, the numbers of axons are quite variable' from animal to animal. Myelinated axons vary by almost 60 fold (24 in rat 3 as compared to 1288 in rat 2) and unmyelinated axons by approximately 7 fold.
Nevertheless there are some common morphological features observed in the distal segments. For example, all animals show a considerable loss in small myelinated and unmyelinated axons with a greater loss in large myelinated axons compared to the proximal segment. Increased pain (hyperalgesia) associated with loss of large myelinated axons is consistent with the gate theory of pain [10], and the marked reduction of both small myelinated and unmyelinated axons is consistent with the hypothesis of Dyck et al., [6] that pain occurs in humans when both small myelinated and unmyelinated axons are lost. Furthermore, the loss of small myelinated fibers (A&s) may lead to disinhibition of spinothalamic tract cells [4], one of the most important tracts transmitting nociceptive input to higher brain centers [13]. Such correlations do not imply causal relationships, but they do indicate that the axons that pass through the lesion into the distal nerve are probably of importance in the generation of the symptoms. Experiments separating the effects of neuroma activity from those of the axon activity in the distal nerve would be desirable. At present, we hypothesize that input from surviving axons in the distal segment results in increased activity in the neuroma which in turn sends signals centrally that are interpreted as nociceptive by the animal. We focus particularly on the unmyelinated axons because fewer of these are lost and also because hyperalgesia occurred in an animal with only 24 surviving myelinated axons distal to the injury. The authors thank Elizabeth Hayes and Gregory Hargett for technical assistance. This study was supported by NS 27910 (S.M.C.), NSl1255 (S.M.C., R.E.C.), NS10161 (R.E.C.) and NS08860 (P.M.D.). 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 Basbaum, A.I., Jazat, F. and Guilbaud, G., The spectrum of fiber loss in a model of neuropathic pain in the rat, Am. Pain Soc. Abstr., 8 (1990) 7. 3 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) 87-107. 4 Chung, J.M., Lee, K.H., Hori, Y., Endo, K. and Willis, W.D., Factors influencing peripheral nerve stimulation produced inhibition of primate spinothalamic tract cells, Pain, (1984) 277-293. 5 Devor, M., The pathophysiology and anatomy of damaged nerve. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, Churchill Livingstone, New York, 1984, pp. 49~a4. 6 Dyck, P.J., Lambert, E.H. and O'Brien, P.C., Pain in peripheral neuropathy related to rate and kind of fiber degeneration, Neurology, 26 (1976) 466-471. 7 Gautron, M., Jazat, F., Ratinahirana, H., Hauw, J.J. and Guilbaud, G., Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of
92 abnormal small myelinated fibres and pain-related behavior, Neurosci. Lett., 111 (1990)28-33. 8 Govrin-Lippmann, R. and Devor, M., Ongoing activity in severed nerves: source and variation with time, Brain Res., 159 (1978) 406410. 9 Kajander, K.C., Sahara, Y., ladarola, M.J. and Bennett, G.J., Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy, Peptides, 11 (1990) 719-728.
10 Melzack, R. and Wall, P.D., Pain mechanisms: a new theory, Science, 150 (1965) 971-979. 11 Payer, A.F., An ultrastructural study of Schwann cell response to axonal degeneration, J. Comp. Neurol., 183 (1979) 365-384. 12 Sunderland, S., Nerves and nerve injuries, 2nd edn., Churchill Livingstone, New York, 1988, pp. 188-193. 13 Willis, W.D., The Pain System, Karger, New York, 1985, pp. 145 206.