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Prior Collateral Sprouting of Sensory Axons Delays Recovery of Pain Sensitivity after Subsequent Nerve Crush
EXPERIMENTAL NEUROLOGY ARTICLE NO.
141, 207–213 (1996)
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Prior Collateral Sprouting of Sensory Axons Delays Recovery of Pain Sensitivity after Subsequent Nerve Crush FAJKO BAJROVIC´
AND
JANEZ SKETELJ
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
Regeneration of motor axons is enhanced if they have sprouted prior to nerve injury. We examined whether sensory axon regeneration and recovery of pain response was affected by previous collateral sprouting. In the experimental group of rats, the right saphenous, tibial, and sural nerves were transected and ligated. The peroneal nerve was left to sprout into the adjacent denervated skin. Two months later, the axons of the peroneal nerve were crushed in the sciatic nerve. In the control group, the right sciatic nerve was crushed at the same time that the saphenous, tibial, and sural nerves were transected. Recovery of pain response in the foot was determined by the skin pinch test. Sensory axon elongation rate was measured by the nerve pinch test. The number of myelinated axons was determined in nerve cross sections stained by Azur blue. Recovery of pain sensitivity in the animals of the experimental group was delayed for 2–3 weeks in comparison to the control group. Moreover, the spatial pattern of pain response in the experimental group was irregular, displaying residual regions of insensitive skin which were not present in controls. The elongation rate of regenerating sensory axons in the experimental group was not decreased, and the number of myelinated axons in the peroneal nerves was even about 10% higher than in the control group. Therefore, we assume that the terminal arborization of the neurilemmal tubes pertaining to the former axon sprouts delayed regrowth of sensory axon terminals in the skin. r 1996 Academic Press, Inc.
INTRODUCTION
At the beginning of regeneration following peripheral nerve injury, axon elongation is preceded by morphological and functional changes in the nerve cell bodies, shifting the metabolism of neurons toward a growth supporting state (12). Experimental studies have shown that a prior, so-called conditioning, nerve lesion enhances regeneration of both motor and sensory axons after a subsequent test lesion of the same nerve. Conditioning lesion reduced the initial delay before the onset of axon elongation and accelerated the growth rate of the majority of regenerating axons (14, 15). The
growth rate of the fastest growing axons did not change appreciably, but the results of some other studies differ in this respect (3, 18). Studies of function recovery in experiments with conditioning nerve lesion are few. It has been reported that a conditioning lesion accelerated both the regeneration of unmyelinated sudomotor axons and the recovery of sweating (18). In contrast, de Medinaceli (17) did not observe any beneficial effects of a conditioning lesion on motor function recovery. Although the mechanism of the conditioning lesion effect is not known in detail, it is generally attributed to switching on the growth supporting metabolic state of the neuronal cell body, which can then respond faster to the next lesion of its axon (9). After peripheral nerve injury, sensory function in the denervated skin may gradually recover due to either regeneration of severed axons or extension of axonal sprouts from the nerve endings of the adjacent noninjured nerves, which is called collateral sprouting (14, 27). If the injured peripheral nerve axons are prevented from regenerating, collateral sprouting of the adjacent nerve axons into the denervated territory is the only possibility for recovery of function. Previous studies have demonstrated that the capacity of axons to undergo collateral sprouting depends on the age of experimental animals, type of axons and, eventually, prior lesioning of the axons so that they will sprout after regeneration (4, 8, 11, 27). In the latter case, the adjacent nerves were crushed in parallel with a permanent injury of one nerve. The regenerating axons of the adjacent nerves first reinnervated their own territory and then spread into the ‘‘foreign’’ skin, for which the term expansive regenerative reinnervation has been proposed (28). The authors observed that the area reinnervated after expansive regenerative reinnervation was larger than the area occupied by the axons sprouting from intact nerves. It has been shown that prior sprouting of uninjured motor axons could accelerate the outgrowth of these axons regenerating after subsequent nerve crush (25). However, we are not aware of any study examining the possible influence of prior sprouting on the regenerative capacity of the sensory axons. Collateral sprouting is a kind of axon growth and requires activation of
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0014-4886/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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growth machinery in the cell body, like regeneration after nerve lesion (26). Also, NGF production becomes increased in the denervated skin, which seems to be important for the induction of sprouting (5). Sprouting sensory neurons are, therefore, probably preloaded with NGF. This may enhance their regeneration capacity. Namely, NGF expression is greatly increased also in the distal stump of the injured peripheral nerve and is believed to have a favorable influence on sensory axon regeneration and/or neuron survival (2, 7, 13). Therefore, it can be hypothesized that (a) prior sprouting may enhance the regenerative capacity of sensory axons, and (b) established neurilemmal tubes of previous sprouts in the denervated skin may also accelerate subsequent axon regeneration and functional recovery in comparison to usual recovery during which the regenerating axons must invade the denervated skin territory de novo. In order to check this hypothesis, the effect of prior collateral sprouting on sensory axon regeneration and recovery of nociception in the skin of the foot after crush lesion of the rat sciatic nerve was examined. Contrary to expectations, we found no acceleration of elongation rate of the regenerating sensory axons which had sprouted prior to the crush, and the functional recovery of skin nociception was in fact delayed and more irregular than that following lesion of control nonsprouting nerves. MATERIALS AND METHODS
Surgical Procedures Male albino rats (Wistar), weighing 220–240 g at the beginning of the experiment, were used. All surgical procedures were performed on animals under deep anesthesia with dihydrothiazine and ketamine cocktail (Rompun, Bayer, Levercusen, 5 mg/kg; Ketalar, ParkeDavis and Co., Berlin, 90 mg/kg, ip). The right saphenous, tibial, and sural nerves, but not the peroneal nerve, were transected and ligated in 21 animals. The animals were divided into three groups: In group I, the peroneal nerve was left intact and collateral sprouting of its axons into the denervated skin in the foot was monitored for 2 months. In group II, axonotmesis of the peroneal nerve axons was performed by crushing the right sciatic nerve in the thigh with a 3-mm-wide nonserrated hemostat concomitantly with transections of the saphenous, tibial, and sural nerves. In group III, the peroneal nerve was left intact and its axons were allowed to sprout for 2 months after the right saphenous, tibial, and sural nerves had been transected and ligated. The sciatic nerve was then crushed in the thigh as described above to study subsequent sensory axon regeneration in the peroneal
nerve and sensory function recovery in the foot skin. The animals of groups II and III were of the same age when the nerve crush was performed. After the nerve injury, the transected thigh muscles were sutured, the skin wound was closed, and the animals were left to recover. In the experiments designed for studying axon elongation rate, the same surgical procedures were performed as above, except that (a) the peroneal nerve axons were allowed to sprout either 2 weeks or 2 months prior to crushing the sciatic nerve, and (b) the crush site on the sciatic nerve was marked by an epineurial suture from which the distances reached by the regenerating axons could be measured. Assessment of Axon Elongation Rate Sensory axon elongation rate was determined using the nerve pinch test (16, 30) on Days 2, 4, and 6 after the sciatic nerve crush. The animals were anesthetised by a low dose of pentobarbital (25 mg/kg ip, Nembutal, Abbott Labs, IL), supported by ether inhalation. Ether anesthesia was discontinued as soon as the sciatic and peroneal nerves were exposed. A series of light pinches was then delivered to the peroneal nerve with finetipped watchmaker forceps, proceeding in millimeter steps in a distal to proximal direction. The spot on the nerve where a pinch first elicited the animal’s response was recorded and its distance from the epineurial suture depicting the proximal limit of the crush site was measured. Recovery of Skin Sensitivity to Pain A nociceptive pinch-test (4) was used to estimate the extent of recovery of sensitivity of the foot skin to painful stimuli. The test was performed in a blind fashion so that the examiner was not acquainted with the treatment of tested animals. Rats were lightly anesthetized by pentobarbital as described above. The skin of the sole and instep of the foot was pinched by a fine forceps in 1-mm intervals from the toes to the ankle. Reflex withdrawal response on the treated side was compared to that elicited by pinching the corresponding spot on the nontreated foot and was recorded as positive in the case of identical response. The area of identical response was depicted in the schematic drawing of the rat foot (of both plantar and dorsal surfaces). These drawings were then used to estimate the percentage of reinnervated skin surface by computer-based planimetry. Pain sensitivity of the foot was first tested every week and later every second week until the end of the experiment 4 months after initial nerve injury. Counts of Regenerated Axons in the Injured Nerves Six days after axonotmesis, regenerating axons in the injured nerves were visualized by the immunohisto-
COLLATERAL SPROUTING AND AXON REGENERATION
chemical reaction against neurofilament polypeptides. Peroneal nerve segments extending from the crush site to the positive pinch site were excised and fixed in buffered paraformaldehyde (pH 7.4) for 24 h. The samples were then taken from the mid region, dehydrated, and embedded in paraffin. Cross sections, 2–5 µm thick, were cut at the halfway point between the crush site and the pinch site. Monoclonal mouse antineurofilament polipeptide antibody was used in primary antiserum, diluted 1:100 (Dakopatts, Denmark). A commercial avidin–biotin–peroxidase staining procedure was applied according to the manufacturer’s instructions (Vectastain ABC kit, Vector Lab). Three animals with sciatic nerve permanently transected for 6 days were used as controls to check for possible binding of antibodies to residual neurofilaments in degenerating axons. Regenerated myelinated axons in the injured nerves 2 months after axonotmesis were visualized by staining the myelin sheaths with Azur blue. Small segments of the peroneal nerve lying about 20 mm distal to the crush site were excised and fixed in 2% glutaraldehyde and 2% paraformaldehyde in veronal-acetate buffer, pH 7.4, for 12 h at 4°C. The nerve samples were then dehydrated and embedded in Epon. Semithin cross sections, 1.5–2 µm thick, were prepared from the middle portion and stained with Azur blue. The total number of neurofilament positive profiles or myelin sheaths was determined in whole nerve cross sections under a Zeiss-Opton light microscope (1003 objective). A computerized image analysis system, Horizon, developed at our institute, was used for counting. Statistical Analysis Statistical significance of the difference between several samples was estimated by analysis of variance. Bonferroni’s correction was taken into account when individual pairs of samples were tested for the significance of the difference between them by the Student t test. RESULTS
Rate of Axon Elongation after Nerve Crush The rate of elongation of the fastest regenerating sensory axons under different experimental conditions was determined by the nerve pinch test (Fig. 1). In control animals (crush only), the most distal point from which the defense response could be elicited moved away from the crush site at the rate of 3.4 mm/day (slope of the regresion line). Initial delay was about 1 day. The distances between the crush site and the positive pinch site in the nerves which had sprouted for either 2 weeks (Fig. 1A) or 2 months (Fig. 1B) before axonotmesis were virtually the same as those in control
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FIG. 1. Distances reached by the fastest growing regenerating sensory axons of the peroneal nerve, determined by the nerve-pinch test at different times after nerve crush. (W) Control crushed nerves; (N) nerves which had sprouted before the crush for either 2 weeks (A) or 2 months (B). Data are shown as means and SEM. Slope of regression lines represents rate of axon elongation.
injured nerves; small differences were not statistically significant. Therefore, both the initial delay and the elongation rate of the fastest sensory axons in the experimental groups were about the same as in the control group. Axon Counts The number of regenerating axons in the peroneal nerves 6 days after axonotmesis was estimated by counting all the neurofilament-positive profiles in whole cross sections taken at the middistance from the crush site to the positive pinch test site. In the control experiments on permanently transected nerves we found that, after 6 days, a few neurofilament-positive profiles could still be observed in spite of the absence of regenerating axons in the distal stump of permanently transected nerves, probably due to residual antigenic material from degenerating axons. Their number, however, was less than 5% of total axon counts in regenerating nerves, so they could be neglected. Neurofilamentpositive profiles observed in crushed nerves, therefore, were taken to represent regenerating axons. A small difference observed between the number of neurofilament-positive profiles in control regenerating nerves and in the nerves crushed after 2 weeks of sprouting was not statistically significant (Fig. 2A). To examine the long-term effect of prior sprouting on the number of regenerated axons in the crushed peroneal nerves, the number of myelinated axons in the whole cross sections of the peroneal nerves was determined 2 months after nerve crush (Fig. 2B). Cross sections of the nerves were cut about 20 mm distal to the crush site. The number of myelinated axons in the peroneal nerve in the control group (crush only) was not significantly different from that in normal peroneal nerves and in the peroneal nerves which had just been left to sprout for 2 months. The number of myelinated axons in the peroneal nerves in which the axons had collaterally sprouted for 2 months prior to crush was
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FIG. 2. Axon counts in whole cross sections of peroneal nerves as determined by (A) the immunohistochemical reaction against neurofilament polypeptides 6 days after the nerve crush, and (B) staining the myelin sheaths with Azur blue 2 months after nerve crush. (a) Normal peroneal nerves, (b) regenerating peroneal nerve distally to the crush, (c) regenerating peroneal nerves which had sprouted for 2 months before crushing, and (d) intact peroneal nerves which were left to sprout for 2 months. Data are presented as mean and SEM. A small difference between the number of neurofilament positive profiles in control regenerating nerves (A, column b) and in the nerves crushed after two weeks of sprouting (A, column c) is not statistically significant. The number of myelinated axons in the peroneal nerves in which the axons had collaterally sprouted for 2 months prior to crush (B, column c) was 12% higher than that in the nonsprouting crushed nerves (B, b); the difference was statistically significant (P , 0.05).
12% higher than that in the control crushed nerves. The difference was statistically significant (P , 0.05). Recovery of Pain Sensitivity of the Foot Skin after Sciatic Nerve Crush (and Transection of the Saphenous, Tibial, and Sural Nerves) In order to induce collateral sprouting of the skin sensory axons of the peroneal nerve, the adjacent saphenous, tibial, and sural nerves were cut and ligated at the start of the experiment. Thereafter, the animals were divided into three groups. In group I, the peroneal nerve was not injured and was just left to sprout. In group II, the peroneal nerve was crushed concomitantly with transection of the other nerves so that axon regeneration preceded their sprouting into ‘‘foreign’’ denervated skin. In group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter, so that sprouting occurred prior to axon regeneration. Typical patterns of recovery of pain sensitivity of the foot skin in these three groups are shown in Fig. 3. Recovery due to simple collateral sprouting of the peroneal nerve (group I) started already during the first 2 weeks after transection of the other nerves. In contrast, recovery was delayed for about 2 weeks in the animals in which the peroneal nerve had been crushed concomitantly with transection of the other nerves (group II). Pain sensitivity to pinch in these animals reappeared on the lateral aspect of the ankle during the third week after crush injury and thereafter spread continuously to the rest of the skin of the foot. The pattern of spreading of pain sensitivity across the foot
skin was about the same as that observed in group I animals in which the intact peroneal nerve had just been left to sprout after injury of the adjacent nerves. The rate of recovery of pain sensitivity in the two groups of animals was very similar too (Fig. 4). At the end of the experiment, the pain-sensitive areas observed in groups I and II were the same. In the animals in which collateral sprouting of the peroneal nerve occurred prior to its regeneration (group III), the onset of recovery of pain sensitivity in the foot skin was very slow so that sensitivity of 50% of the foot surface was recovered about 2–3 weeks later than in group II animals in which sprouting occurred after crush-induced regeneration (Figs. 4A and 4B). Differences between the two groups in the size of the painsensitive area of the foot surface were statistically significant in the fifth, seventh, and ninth weeks after injury (P , 0.01). Moreover, the spatial pattern of recovery of the pain response in the animals in which the peroneal nerve had been left to sprout prior to regeneration (group III) was highly irregular. Residual regions of insensitive skin were observed in contrast to contiguous recovery of pain sensitivity in group II (Figs. 3A and 3B). DISCUSSION
It is generally recognized that in addition to neuron cell body reaction, trophic factors produced by proliferating Schwann cells in the degenerating distal nerve stump and a suitable growth substratum are the most important factors supporting axon regeneration after
COLLATERAL SPROUTING AND AXON REGENERATION
FIG. 3. Typical patterns of recovery of skin sensitivity to pinch in the sole (A) and instep (B) of the right hind foot in rats after the saphenous, tibial, and sural nerves were transected. Group I, peroneal nerve was left intact and alowed to sprout into adjacent skin; group II, peroneal nerve had been crushed concomitantly with transection of the other nerves so that its axons had to regenerate before sprouting into adjacent skin; group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter. Shaded area represents normal sensitivity to pinch indistinguishable from that on the contralateral leg.
nerve injury (6, 7, 21, 24). Our hypothesis that collateral sprouting of sensory axons prior to nerve crush lesion may enhance subsequent regeneration of these axons and, therefore, functional recovery, was based on three arguments related to the above-mentioned factors: (a) sprouting activates the growth supporting
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state in the neuron cell bodies (26); (b) sprouting sensory axons are exposed to a high level of NGF in denervated skin (5); and (c) neurilemmal tubes of sprouts may serve as a ready-for-use growth substratum for regenerating axons. Sprouting of sensory axons of the peroneal nerve into the adjacent denervated skin region could be elicited by transecting all the peripheral nerves except the peroneal to the rat hindpaw. Accordingly, we found that such partial denervation was followed by gradual expansion of pain sensitivity into the previously insensitive skin areas. Earlier morphological studies demonstrated that such an expansion of pain sensitivity was based on growth of collateral sprouts of the uninjured sensory axons into the denervated skin (10, 11, 19, 27). However, contrary to our expectations, we could find no acceleration of elongation rate of regenerating sensory axons which had sprouted prior to the crush, and the functional recovery after the crush lesion of the sprouting nerve was in fact delayed and more irregular than that following the lesion of control nonsprouting nerves. Therefore, the above-mentioned regenerationenhancing factors putatively associated with sprouting either were not influential enough to cause measurable effects under our conditions or were outweighed by some hitherto unknown adverse factors related to prior sprouting. It is possible that we could demonstrate no significant effect of prior sprouting on the elongation rate of regenerating sensory axons because we missed the right period during which sprouting stimulates the neurons and when axon growth acceleration after crush can be observed. However, this is not very probable because we examined the effect both at the time when intensive sprouting was initiated (2 weeks after adjacent skin denervation), and later when sprouting approached its completion 2 months after the lesion of the adjacent nerves. The rate of the fastest growing axons as determined by the pinch test was not enhanced by previous sprouting. This is in accordance with the results describing the effects of prior sprouting on motor axon regeneration (25), as well as with results of related experiments with conditioning lesion (14, 15). If, instead, the rate of elongation of the majority of axons had been accelerated by previous sprouting, we would find more axons in these peroneal nerves at a certain distance from the crush lesion than in the nonsprouting nerves, but this was not the case. This is at variance with the effect of a conditioning lesion on regeneration (14, 15) and the effect of previous sprouting on motor axon regeneration (25). It is possible that by counting the regenerating axons in a mixed peroneal nerve we were not able to detect above the level of statistical significance the increased number of axons pertaining to a limited population of sensory neurons involved in previous sprouting, or perhaps sensory
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FIG. 4. Time course of recovery of skin sensitivity to pinch in the sole (A) and instep (B) of the right hind foot in the rats after the saphenous, tibial, and sural nerves were transected. (W) Group I, peroneal nerve was left intact and alowed to sprout into adjacent skin; (X) group II, peroneal nerve had been crushed concomitantly with transection of the other nerves so that its axons had to regenerate before sprouting into adjacent skin; (M) group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter. Data are shown as means and SEM, broken lines show average percentages of sole and instep surface corresponding to the maximal innervation field of the peroneal nerve.
neurons behave differently in this respect than motor neurons examined in an earlier study (25). The effect of prior sprouting on functional recovery of pain sensitivity of the skin after nerve lesion was also studied. We found that prior sprouting of the peroneal nerve significantly delayed functional recovery after crush injury of its axons. There are several possible reasons for this delay, such as: (a) slower elongation rate of the regenerating axons that had sprouted before crush lesion, in comparison to the nonsprouting axons; (b) progressive death of previously sprouting neurons during crush-induced regeneration, presumably due to increased sensitivity of the sprouting neurons to lesion of their axons in comparison to the nonsprouting neurons; (c) hindrances in establishment of the terminal arborization of the regenerating nerve endings in the skin itself. The first possibility can be ruled out because we showed that the rate of elongation of regenerating axons was not significantly affected by previous sprouting, and, therefore, could not have caused the observed delay in recovery. Second, it is well documented that a variable population of sensory neurons dies after transection or crush of their axons, the exact percentage depending on many
factors such as lesion site, age of animals, availability of trophic support in the distal nerve stump, and ability to reinnervate the target tissue (1, 2, 20, 22, 23, 29). If previous sprouting increased susceptibility of sensory neurons to lesion-induced cell death, this could explain spatially irregular and delayed functional recovery of pain sensitivity in the animals with prior axon sprouting. However, after functional regeneration had been largely completed, the number of myelinated axons in the peroneal nerves of the animals with prior axon sprouting was in fact about 10% higher than in the animals with nonsprouting neurons. This practically rules out the possibility that a major neuron loss was responsible for compromised function recovery observed after the previously sprouting peroneal nerves had been crushed. Therefore, it seems that the untoward effect of prior sprouting can best be explained by assuming that the existing net of neurilemmal tubes established by axon sprouts during the sprouting period hinders the subsequent regeneration of terminal axon extensions in the skin. It is possible that, because of many new ramifications produced by previous axon sprouting in the terminal portion of the sensory axons, many regenerating nerve endings would be misdirected on reaching this terminal arborization. Reinnervation may, therefore, be more haphazard than after expansive regenerative reinnervation, which would explain the irregular-
COLLATERAL SPROUTING AND AXON REGENERATION
ity of recovery of pain sensitivity after lesion of previously sprouting nerves. This misrouting of growth cones of regenerating axons might finally be responsible also for the delay of function recovery due to its irregular progress. In conclusion, the results indicate that previous axon sprouting probably causes unfavorable local conditions in the terminal axon arborization for those axons regenerating after the subsequent crush lesion. These conditions far outweigh any possible enhancing effect that previous sprouting might have on axon regeneration, thereby causing delay and irregularity of functional recovery of pain sensitivity after nerve crush, in comparison to the animals whose neurons had not sprouted before their axons were injured.
12.
13.
14. 15.
16.
ACKNOWLEDGMENTS 17. The authors thank Dr. M. Bresjanac for valuable discussions, Mrs. ˇ ucˇek and Mrs. A. Kljun for help with histological analysis of D. C nerves, and Mr. B. Pecˇenko for preparation of the figures. This work was supported by a grant from the Ministry of Science and Technology of the Republic of Slovenia.
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REFERENCES 1.
2.
3.
4.
5.
6. 7.
8.
9.
10.
11.
ARVIDSON, J., J. YGGE, AND G. GRANT. 1986. Cell loss in lumbal dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in the rat. Brain Res. 373: 15–21. BAJROVIC´ , F., M. BRESJANAC, AND J. SKETELJ. 1994. Long-term effects of deprivation of cell support in the distal stump on peripheral nerve regeneration. J. Neurosci. Res. 39: 23–30. BISBY, M. A. 1985. Enhancement of the conditioning lesion effect in rat sciatic motoraxons after superimposition of conditioning and test lesion. Exp. Neurol. 90: 385–394. DEVOR, M., D. SCHONFELD, Z. SELTZER, AND P. D. WALL. 1979. Two modes of cutaneous reinnervation following peripheral nerve injury. J. Comp. Neurol. 185: 211–220. DIAMOND, J., M. COUGHLIN, L. MACINTYRE, M. HOLMES, AND B. VISHEAU. 1987. Evidence that endogenous nerve growth factor is responsible for collateral sprouting, but not the regeneration, of nociceptive axons in adult rats. Proc. Natl. Acad. Sci. USA 84: 6596–6600. FAWCETT, J. W., AND R. J. KEYNES. 1990. Peripheral nerve regeneration. Annu. Rev. Neurosci. 13: 43–60. HEUMANN, R., S. KORSCHING, C. BANDTLOW, AND H. THOENEN. 1987. Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection. J. Cell. Biol. 104: 1623–1631. JACKSON, P. C., AND J. DIAMOND. 1984. Temporal and spatial constraints of the collateral sprouting of low-threshold mechanosensory nerves in the skin of rats. J. Comp. Neurol. 226: 336–345. JENQ, C.-B., L.-L. JENQ, H. M. BEAR, AND R. E. COGGESHALL. 1988. Conditioning lesions of peripheral nerves change regenerated axon numbers. Brain Res. 457: 63–69. KINNMAN, E., AND H. ALDSKOGIUS. 1986. Collateral sprouting of sensory axons in the glabrous skin of the hindpaw after chronic sciatic nerve lesion in adult and neonatal rats: A morphological study. Brain Res. 377: 73–82. KINNMAN, E., H. ALDSKOGIUS, O. JOHANSSON, AND Z. WIESENFELD-
20.
21.
22.
23. 24.
25. 26. 27.
28.
29.
30.
213
HALLIN. 1992. Collateral reinnervation and expansive regenerative reinnervation by sensory axons into foreign denervated skin: Immunohistochemical study in the rat. Exp. Brain Res. 91: 61–72. LIEBERMAN, A. R. 1974. Some factors affecting retrograde neuronal responses to axonal lesions. In Essay on the Nervous System (R. Bellairs and E. G. Gray, Eds.), pp. 71–105. Clarendon, Oxford. LINDHOLM, D., R. HEUMANN, M. MEYER, AND H. THOENEN. 1987. Changes of nerve growth factor synthesis in non-neuronal cells of rat sciatic nerve. Nature 330: 658–659. MCQUARRIE, I. G. 1987. The effects of a conditioning lesion on the regeneration of motoraxons. Brain Res. 152: 597–602. MCQUARRIE, I. G., AND B. GRAFSTEIN. 1981. Effect of a conditioning lesion on optic nerve regeneration in goldfish. Brain Res. 216: 253–264. MCQUARRIE, I. G., B. GRAFSTEIN, AND M. D. GERSHON. 1977. Axonal regeneration in the rat sciatic nerve: Effect of a conditioning lesion and of dbcAMP. Brain Res. 132: 443–453. DE MEDINACELI, L. 1988. Functional consequences of experimental nerve lesions: Effects of reinnervation blend. Exp. Neurol. 100: 166–178. NAVARRO, X., AND W. R. KENNEDY. 1990. The effect of a conditioning lesion on sudomotor axon regeneration. Brain Res. 509: 232–236. NIXON, B. J., R. DOUCETTE, P. C. JACKSON, AND J. DIAMOND. 1984. Impulse activity evokes precocious sprouting of nociceptive nerves into denervated skin. Somatosens. Res. 2: 97–126. OTTO, D., K. UNSICKER, AND C. GROTHE. 1987. Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia. Neurosci. Lett. 83: 156–160. RAIVICH, G., AND G. W. KREUTZBERG. 1993. Peripheral nerve regeneration: Role of growth factors and their receptors. J. Dev. Neurosci. 11: 311–324. RICH, K. M., J. R. LUSCZYNSKY, P. A. OSBORNE, AND E. M. JOHNSON, JR. 1987. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J. Neurocytol. 16: 261–268. SCHMALBRUCH, H. 1987. Loss of sensory neurons after sciatic nerve section in the rat. Anat. Rec. 219: 323–329. SKETELJ, J., M. BRESJANAC, AND M. POPOVIC´ . 1989. Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells. J. Neurosci. Res. 24: 153–162. SPARROW, J. R., AND B. GRAFSTEIN. 1983. Prior collateral sprouting enhances axonal regeneration. Brain Res. 269: 133–136. WATSON, W. E. 1973. Some responses of dorsal root ganglia to axotomy. J. Physiol. 231: 41–42. WEDDELL, G., L. GUTTMANN, AND E. GUTMANN. 1941. The local extension of nerve fibers into denervated areas of skin. J. Neurol. Psych. 4: 206–225. WIESENFELD-HALLIN, Z., E. KINNMAN, AND H. ALDSKOGIUS. 1989. Expansion of innervation territory by afferents involved in plasma extravasation after nerve regeneration in adult and neonatal rats. Exp. Brain Res. 76: 88–96. YGGE, J. H., AND H. ALDSKOGIUS. 1984. Intercostal nerve transection and its effect on the dorsal root ganglion. A quantitative study on thoracic ganglion cell numbers and sizes in the rat. Exp. Brain Res. 55: 402–408. YOUNG, J. Z., AND P. B. MEDAWAR. 1940. Fibrin suture of peripheral nerves. Measurement of the rate of regeneration. Lancet II: 126–128.
141, 207–213 (1996)
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Prior Collateral Sprouting of Sensory Axons Delays Recovery of Pain Sensitivity after Subsequent Nerve Crush FAJKO BAJROVIC´
AND
JANEZ SKETELJ
Institute of Pathophysiology, School of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia
Regeneration of motor axons is enhanced if they have sprouted prior to nerve injury. We examined whether sensory axon regeneration and recovery of pain response was affected by previous collateral sprouting. In the experimental group of rats, the right saphenous, tibial, and sural nerves were transected and ligated. The peroneal nerve was left to sprout into the adjacent denervated skin. Two months later, the axons of the peroneal nerve were crushed in the sciatic nerve. In the control group, the right sciatic nerve was crushed at the same time that the saphenous, tibial, and sural nerves were transected. Recovery of pain response in the foot was determined by the skin pinch test. Sensory axon elongation rate was measured by the nerve pinch test. The number of myelinated axons was determined in nerve cross sections stained by Azur blue. Recovery of pain sensitivity in the animals of the experimental group was delayed for 2–3 weeks in comparison to the control group. Moreover, the spatial pattern of pain response in the experimental group was irregular, displaying residual regions of insensitive skin which were not present in controls. The elongation rate of regenerating sensory axons in the experimental group was not decreased, and the number of myelinated axons in the peroneal nerves was even about 10% higher than in the control group. Therefore, we assume that the terminal arborization of the neurilemmal tubes pertaining to the former axon sprouts delayed regrowth of sensory axon terminals in the skin. r 1996 Academic Press, Inc.
INTRODUCTION
At the beginning of regeneration following peripheral nerve injury, axon elongation is preceded by morphological and functional changes in the nerve cell bodies, shifting the metabolism of neurons toward a growth supporting state (12). Experimental studies have shown that a prior, so-called conditioning, nerve lesion enhances regeneration of both motor and sensory axons after a subsequent test lesion of the same nerve. Conditioning lesion reduced the initial delay before the onset of axon elongation and accelerated the growth rate of the majority of regenerating axons (14, 15). The
growth rate of the fastest growing axons did not change appreciably, but the results of some other studies differ in this respect (3, 18). Studies of function recovery in experiments with conditioning nerve lesion are few. It has been reported that a conditioning lesion accelerated both the regeneration of unmyelinated sudomotor axons and the recovery of sweating (18). In contrast, de Medinaceli (17) did not observe any beneficial effects of a conditioning lesion on motor function recovery. Although the mechanism of the conditioning lesion effect is not known in detail, it is generally attributed to switching on the growth supporting metabolic state of the neuronal cell body, which can then respond faster to the next lesion of its axon (9). After peripheral nerve injury, sensory function in the denervated skin may gradually recover due to either regeneration of severed axons or extension of axonal sprouts from the nerve endings of the adjacent noninjured nerves, which is called collateral sprouting (14, 27). If the injured peripheral nerve axons are prevented from regenerating, collateral sprouting of the adjacent nerve axons into the denervated territory is the only possibility for recovery of function. Previous studies have demonstrated that the capacity of axons to undergo collateral sprouting depends on the age of experimental animals, type of axons and, eventually, prior lesioning of the axons so that they will sprout after regeneration (4, 8, 11, 27). In the latter case, the adjacent nerves were crushed in parallel with a permanent injury of one nerve. The regenerating axons of the adjacent nerves first reinnervated their own territory and then spread into the ‘‘foreign’’ skin, for which the term expansive regenerative reinnervation has been proposed (28). The authors observed that the area reinnervated after expansive regenerative reinnervation was larger than the area occupied by the axons sprouting from intact nerves. It has been shown that prior sprouting of uninjured motor axons could accelerate the outgrowth of these axons regenerating after subsequent nerve crush (25). However, we are not aware of any study examining the possible influence of prior sprouting on the regenerative capacity of the sensory axons. Collateral sprouting is a kind of axon growth and requires activation of
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growth machinery in the cell body, like regeneration after nerve lesion (26). Also, NGF production becomes increased in the denervated skin, which seems to be important for the induction of sprouting (5). Sprouting sensory neurons are, therefore, probably preloaded with NGF. This may enhance their regeneration capacity. Namely, NGF expression is greatly increased also in the distal stump of the injured peripheral nerve and is believed to have a favorable influence on sensory axon regeneration and/or neuron survival (2, 7, 13). Therefore, it can be hypothesized that (a) prior sprouting may enhance the regenerative capacity of sensory axons, and (b) established neurilemmal tubes of previous sprouts in the denervated skin may also accelerate subsequent axon regeneration and functional recovery in comparison to usual recovery during which the regenerating axons must invade the denervated skin territory de novo. In order to check this hypothesis, the effect of prior collateral sprouting on sensory axon regeneration and recovery of nociception in the skin of the foot after crush lesion of the rat sciatic nerve was examined. Contrary to expectations, we found no acceleration of elongation rate of the regenerating sensory axons which had sprouted prior to the crush, and the functional recovery of skin nociception was in fact delayed and more irregular than that following lesion of control nonsprouting nerves. MATERIALS AND METHODS
Surgical Procedures Male albino rats (Wistar), weighing 220–240 g at the beginning of the experiment, were used. All surgical procedures were performed on animals under deep anesthesia with dihydrothiazine and ketamine cocktail (Rompun, Bayer, Levercusen, 5 mg/kg; Ketalar, ParkeDavis and Co., Berlin, 90 mg/kg, ip). The right saphenous, tibial, and sural nerves, but not the peroneal nerve, were transected and ligated in 21 animals. The animals were divided into three groups: In group I, the peroneal nerve was left intact and collateral sprouting of its axons into the denervated skin in the foot was monitored for 2 months. In group II, axonotmesis of the peroneal nerve axons was performed by crushing the right sciatic nerve in the thigh with a 3-mm-wide nonserrated hemostat concomitantly with transections of the saphenous, tibial, and sural nerves. In group III, the peroneal nerve was left intact and its axons were allowed to sprout for 2 months after the right saphenous, tibial, and sural nerves had been transected and ligated. The sciatic nerve was then crushed in the thigh as described above to study subsequent sensory axon regeneration in the peroneal
nerve and sensory function recovery in the foot skin. The animals of groups II and III were of the same age when the nerve crush was performed. After the nerve injury, the transected thigh muscles were sutured, the skin wound was closed, and the animals were left to recover. In the experiments designed for studying axon elongation rate, the same surgical procedures were performed as above, except that (a) the peroneal nerve axons were allowed to sprout either 2 weeks or 2 months prior to crushing the sciatic nerve, and (b) the crush site on the sciatic nerve was marked by an epineurial suture from which the distances reached by the regenerating axons could be measured. Assessment of Axon Elongation Rate Sensory axon elongation rate was determined using the nerve pinch test (16, 30) on Days 2, 4, and 6 after the sciatic nerve crush. The animals were anesthetised by a low dose of pentobarbital (25 mg/kg ip, Nembutal, Abbott Labs, IL), supported by ether inhalation. Ether anesthesia was discontinued as soon as the sciatic and peroneal nerves were exposed. A series of light pinches was then delivered to the peroneal nerve with finetipped watchmaker forceps, proceeding in millimeter steps in a distal to proximal direction. The spot on the nerve where a pinch first elicited the animal’s response was recorded and its distance from the epineurial suture depicting the proximal limit of the crush site was measured. Recovery of Skin Sensitivity to Pain A nociceptive pinch-test (4) was used to estimate the extent of recovery of sensitivity of the foot skin to painful stimuli. The test was performed in a blind fashion so that the examiner was not acquainted with the treatment of tested animals. Rats were lightly anesthetized by pentobarbital as described above. The skin of the sole and instep of the foot was pinched by a fine forceps in 1-mm intervals from the toes to the ankle. Reflex withdrawal response on the treated side was compared to that elicited by pinching the corresponding spot on the nontreated foot and was recorded as positive in the case of identical response. The area of identical response was depicted in the schematic drawing of the rat foot (of both plantar and dorsal surfaces). These drawings were then used to estimate the percentage of reinnervated skin surface by computer-based planimetry. Pain sensitivity of the foot was first tested every week and later every second week until the end of the experiment 4 months after initial nerve injury. Counts of Regenerated Axons in the Injured Nerves Six days after axonotmesis, regenerating axons in the injured nerves were visualized by the immunohisto-
COLLATERAL SPROUTING AND AXON REGENERATION
chemical reaction against neurofilament polypeptides. Peroneal nerve segments extending from the crush site to the positive pinch site were excised and fixed in buffered paraformaldehyde (pH 7.4) for 24 h. The samples were then taken from the mid region, dehydrated, and embedded in paraffin. Cross sections, 2–5 µm thick, were cut at the halfway point between the crush site and the pinch site. Monoclonal mouse antineurofilament polipeptide antibody was used in primary antiserum, diluted 1:100 (Dakopatts, Denmark). A commercial avidin–biotin–peroxidase staining procedure was applied according to the manufacturer’s instructions (Vectastain ABC kit, Vector Lab). Three animals with sciatic nerve permanently transected for 6 days were used as controls to check for possible binding of antibodies to residual neurofilaments in degenerating axons. Regenerated myelinated axons in the injured nerves 2 months after axonotmesis were visualized by staining the myelin sheaths with Azur blue. Small segments of the peroneal nerve lying about 20 mm distal to the crush site were excised and fixed in 2% glutaraldehyde and 2% paraformaldehyde in veronal-acetate buffer, pH 7.4, for 12 h at 4°C. The nerve samples were then dehydrated and embedded in Epon. Semithin cross sections, 1.5–2 µm thick, were prepared from the middle portion and stained with Azur blue. The total number of neurofilament positive profiles or myelin sheaths was determined in whole nerve cross sections under a Zeiss-Opton light microscope (1003 objective). A computerized image analysis system, Horizon, developed at our institute, was used for counting. Statistical Analysis Statistical significance of the difference between several samples was estimated by analysis of variance. Bonferroni’s correction was taken into account when individual pairs of samples were tested for the significance of the difference between them by the Student t test. RESULTS
Rate of Axon Elongation after Nerve Crush The rate of elongation of the fastest regenerating sensory axons under different experimental conditions was determined by the nerve pinch test (Fig. 1). In control animals (crush only), the most distal point from which the defense response could be elicited moved away from the crush site at the rate of 3.4 mm/day (slope of the regresion line). Initial delay was about 1 day. The distances between the crush site and the positive pinch site in the nerves which had sprouted for either 2 weeks (Fig. 1A) or 2 months (Fig. 1B) before axonotmesis were virtually the same as those in control
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FIG. 1. Distances reached by the fastest growing regenerating sensory axons of the peroneal nerve, determined by the nerve-pinch test at different times after nerve crush. (W) Control crushed nerves; (N) nerves which had sprouted before the crush for either 2 weeks (A) or 2 months (B). Data are shown as means and SEM. Slope of regression lines represents rate of axon elongation.
injured nerves; small differences were not statistically significant. Therefore, both the initial delay and the elongation rate of the fastest sensory axons in the experimental groups were about the same as in the control group. Axon Counts The number of regenerating axons in the peroneal nerves 6 days after axonotmesis was estimated by counting all the neurofilament-positive profiles in whole cross sections taken at the middistance from the crush site to the positive pinch test site. In the control experiments on permanently transected nerves we found that, after 6 days, a few neurofilament-positive profiles could still be observed in spite of the absence of regenerating axons in the distal stump of permanently transected nerves, probably due to residual antigenic material from degenerating axons. Their number, however, was less than 5% of total axon counts in regenerating nerves, so they could be neglected. Neurofilamentpositive profiles observed in crushed nerves, therefore, were taken to represent regenerating axons. A small difference observed between the number of neurofilament-positive profiles in control regenerating nerves and in the nerves crushed after 2 weeks of sprouting was not statistically significant (Fig. 2A). To examine the long-term effect of prior sprouting on the number of regenerated axons in the crushed peroneal nerves, the number of myelinated axons in the whole cross sections of the peroneal nerves was determined 2 months after nerve crush (Fig. 2B). Cross sections of the nerves were cut about 20 mm distal to the crush site. The number of myelinated axons in the peroneal nerve in the control group (crush only) was not significantly different from that in normal peroneal nerves and in the peroneal nerves which had just been left to sprout for 2 months. The number of myelinated axons in the peroneal nerves in which the axons had collaterally sprouted for 2 months prior to crush was
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FIG. 2. Axon counts in whole cross sections of peroneal nerves as determined by (A) the immunohistochemical reaction against neurofilament polypeptides 6 days after the nerve crush, and (B) staining the myelin sheaths with Azur blue 2 months after nerve crush. (a) Normal peroneal nerves, (b) regenerating peroneal nerve distally to the crush, (c) regenerating peroneal nerves which had sprouted for 2 months before crushing, and (d) intact peroneal nerves which were left to sprout for 2 months. Data are presented as mean and SEM. A small difference between the number of neurofilament positive profiles in control regenerating nerves (A, column b) and in the nerves crushed after two weeks of sprouting (A, column c) is not statistically significant. The number of myelinated axons in the peroneal nerves in which the axons had collaterally sprouted for 2 months prior to crush (B, column c) was 12% higher than that in the nonsprouting crushed nerves (B, b); the difference was statistically significant (P , 0.05).
12% higher than that in the control crushed nerves. The difference was statistically significant (P , 0.05). Recovery of Pain Sensitivity of the Foot Skin after Sciatic Nerve Crush (and Transection of the Saphenous, Tibial, and Sural Nerves) In order to induce collateral sprouting of the skin sensory axons of the peroneal nerve, the adjacent saphenous, tibial, and sural nerves were cut and ligated at the start of the experiment. Thereafter, the animals were divided into three groups. In group I, the peroneal nerve was not injured and was just left to sprout. In group II, the peroneal nerve was crushed concomitantly with transection of the other nerves so that axon regeneration preceded their sprouting into ‘‘foreign’’ denervated skin. In group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter, so that sprouting occurred prior to axon regeneration. Typical patterns of recovery of pain sensitivity of the foot skin in these three groups are shown in Fig. 3. Recovery due to simple collateral sprouting of the peroneal nerve (group I) started already during the first 2 weeks after transection of the other nerves. In contrast, recovery was delayed for about 2 weeks in the animals in which the peroneal nerve had been crushed concomitantly with transection of the other nerves (group II). Pain sensitivity to pinch in these animals reappeared on the lateral aspect of the ankle during the third week after crush injury and thereafter spread continuously to the rest of the skin of the foot. The pattern of spreading of pain sensitivity across the foot
skin was about the same as that observed in group I animals in which the intact peroneal nerve had just been left to sprout after injury of the adjacent nerves. The rate of recovery of pain sensitivity in the two groups of animals was very similar too (Fig. 4). At the end of the experiment, the pain-sensitive areas observed in groups I and II were the same. In the animals in which collateral sprouting of the peroneal nerve occurred prior to its regeneration (group III), the onset of recovery of pain sensitivity in the foot skin was very slow so that sensitivity of 50% of the foot surface was recovered about 2–3 weeks later than in group II animals in which sprouting occurred after crush-induced regeneration (Figs. 4A and 4B). Differences between the two groups in the size of the painsensitive area of the foot surface were statistically significant in the fifth, seventh, and ninth weeks after injury (P , 0.01). Moreover, the spatial pattern of recovery of the pain response in the animals in which the peroneal nerve had been left to sprout prior to regeneration (group III) was highly irregular. Residual regions of insensitive skin were observed in contrast to contiguous recovery of pain sensitivity in group II (Figs. 3A and 3B). DISCUSSION
It is generally recognized that in addition to neuron cell body reaction, trophic factors produced by proliferating Schwann cells in the degenerating distal nerve stump and a suitable growth substratum are the most important factors supporting axon regeneration after
COLLATERAL SPROUTING AND AXON REGENERATION
FIG. 3. Typical patterns of recovery of skin sensitivity to pinch in the sole (A) and instep (B) of the right hind foot in rats after the saphenous, tibial, and sural nerves were transected. Group I, peroneal nerve was left intact and alowed to sprout into adjacent skin; group II, peroneal nerve had been crushed concomitantly with transection of the other nerves so that its axons had to regenerate before sprouting into adjacent skin; group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter. Shaded area represents normal sensitivity to pinch indistinguishable from that on the contralateral leg.
nerve injury (6, 7, 21, 24). Our hypothesis that collateral sprouting of sensory axons prior to nerve crush lesion may enhance subsequent regeneration of these axons and, therefore, functional recovery, was based on three arguments related to the above-mentioned factors: (a) sprouting activates the growth supporting
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state in the neuron cell bodies (26); (b) sprouting sensory axons are exposed to a high level of NGF in denervated skin (5); and (c) neurilemmal tubes of sprouts may serve as a ready-for-use growth substratum for regenerating axons. Sprouting of sensory axons of the peroneal nerve into the adjacent denervated skin region could be elicited by transecting all the peripheral nerves except the peroneal to the rat hindpaw. Accordingly, we found that such partial denervation was followed by gradual expansion of pain sensitivity into the previously insensitive skin areas. Earlier morphological studies demonstrated that such an expansion of pain sensitivity was based on growth of collateral sprouts of the uninjured sensory axons into the denervated skin (10, 11, 19, 27). However, contrary to our expectations, we could find no acceleration of elongation rate of regenerating sensory axons which had sprouted prior to the crush, and the functional recovery after the crush lesion of the sprouting nerve was in fact delayed and more irregular than that following the lesion of control nonsprouting nerves. Therefore, the above-mentioned regenerationenhancing factors putatively associated with sprouting either were not influential enough to cause measurable effects under our conditions or were outweighed by some hitherto unknown adverse factors related to prior sprouting. It is possible that we could demonstrate no significant effect of prior sprouting on the elongation rate of regenerating sensory axons because we missed the right period during which sprouting stimulates the neurons and when axon growth acceleration after crush can be observed. However, this is not very probable because we examined the effect both at the time when intensive sprouting was initiated (2 weeks after adjacent skin denervation), and later when sprouting approached its completion 2 months after the lesion of the adjacent nerves. The rate of the fastest growing axons as determined by the pinch test was not enhanced by previous sprouting. This is in accordance with the results describing the effects of prior sprouting on motor axon regeneration (25), as well as with results of related experiments with conditioning lesion (14, 15). If, instead, the rate of elongation of the majority of axons had been accelerated by previous sprouting, we would find more axons in these peroneal nerves at a certain distance from the crush lesion than in the nonsprouting nerves, but this was not the case. This is at variance with the effect of a conditioning lesion on regeneration (14, 15) and the effect of previous sprouting on motor axon regeneration (25). It is possible that by counting the regenerating axons in a mixed peroneal nerve we were not able to detect above the level of statistical significance the increased number of axons pertaining to a limited population of sensory neurons involved in previous sprouting, or perhaps sensory
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FIG. 4. Time course of recovery of skin sensitivity to pinch in the sole (A) and instep (B) of the right hind foot in the rats after the saphenous, tibial, and sural nerves were transected. (W) Group I, peroneal nerve was left intact and alowed to sprout into adjacent skin; (X) group II, peroneal nerve had been crushed concomitantly with transection of the other nerves so that its axons had to regenerate before sprouting into adjacent skin; (M) group III, the peroneal nerve had first been left to sprout for 2 months and was crushed thereafter. Data are shown as means and SEM, broken lines show average percentages of sole and instep surface corresponding to the maximal innervation field of the peroneal nerve.
neurons behave differently in this respect than motor neurons examined in an earlier study (25). The effect of prior sprouting on functional recovery of pain sensitivity of the skin after nerve lesion was also studied. We found that prior sprouting of the peroneal nerve significantly delayed functional recovery after crush injury of its axons. There are several possible reasons for this delay, such as: (a) slower elongation rate of the regenerating axons that had sprouted before crush lesion, in comparison to the nonsprouting axons; (b) progressive death of previously sprouting neurons during crush-induced regeneration, presumably due to increased sensitivity of the sprouting neurons to lesion of their axons in comparison to the nonsprouting neurons; (c) hindrances in establishment of the terminal arborization of the regenerating nerve endings in the skin itself. The first possibility can be ruled out because we showed that the rate of elongation of regenerating axons was not significantly affected by previous sprouting, and, therefore, could not have caused the observed delay in recovery. Second, it is well documented that a variable population of sensory neurons dies after transection or crush of their axons, the exact percentage depending on many
factors such as lesion site, age of animals, availability of trophic support in the distal nerve stump, and ability to reinnervate the target tissue (1, 2, 20, 22, 23, 29). If previous sprouting increased susceptibility of sensory neurons to lesion-induced cell death, this could explain spatially irregular and delayed functional recovery of pain sensitivity in the animals with prior axon sprouting. However, after functional regeneration had been largely completed, the number of myelinated axons in the peroneal nerves of the animals with prior axon sprouting was in fact about 10% higher than in the animals with nonsprouting neurons. This practically rules out the possibility that a major neuron loss was responsible for compromised function recovery observed after the previously sprouting peroneal nerves had been crushed. Therefore, it seems that the untoward effect of prior sprouting can best be explained by assuming that the existing net of neurilemmal tubes established by axon sprouts during the sprouting period hinders the subsequent regeneration of terminal axon extensions in the skin. It is possible that, because of many new ramifications produced by previous axon sprouting in the terminal portion of the sensory axons, many regenerating nerve endings would be misdirected on reaching this terminal arborization. Reinnervation may, therefore, be more haphazard than after expansive regenerative reinnervation, which would explain the irregular-
COLLATERAL SPROUTING AND AXON REGENERATION
ity of recovery of pain sensitivity after lesion of previously sprouting nerves. This misrouting of growth cones of regenerating axons might finally be responsible also for the delay of function recovery due to its irregular progress. In conclusion, the results indicate that previous axon sprouting probably causes unfavorable local conditions in the terminal axon arborization for those axons regenerating after the subsequent crush lesion. These conditions far outweigh any possible enhancing effect that previous sprouting might have on axon regeneration, thereby causing delay and irregularity of functional recovery of pain sensitivity after nerve crush, in comparison to the animals whose neurons had not sprouted before their axons were injured.
12.
13.
14. 15.
16.
ACKNOWLEDGMENTS 17. The authors thank Dr. M. Bresjanac for valuable discussions, Mrs. ˇ ucˇek and Mrs. A. Kljun for help with histological analysis of D. C nerves, and Mr. B. Pecˇenko for preparation of the figures. This work was supported by a grant from the Ministry of Science and Technology of the Republic of Slovenia.
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REFERENCES 1.
2.
3.
4.
5.
6. 7.
8.
9.
10.
11.
ARVIDSON, J., J. YGGE, AND G. GRANT. 1986. Cell loss in lumbal dorsal root ganglia and transganglionic degeneration after sciatic nerve resection in the rat. Brain Res. 373: 15–21. BAJROVIC´ , F., M. BRESJANAC, AND J. SKETELJ. 1994. Long-term effects of deprivation of cell support in the distal stump on peripheral nerve regeneration. J. Neurosci. Res. 39: 23–30. BISBY, M. A. 1985. Enhancement of the conditioning lesion effect in rat sciatic motoraxons after superimposition of conditioning and test lesion. Exp. Neurol. 90: 385–394. DEVOR, M., D. SCHONFELD, Z. SELTZER, AND P. D. WALL. 1979. Two modes of cutaneous reinnervation following peripheral nerve injury. J. Comp. Neurol. 185: 211–220. DIAMOND, J., M. COUGHLIN, L. MACINTYRE, M. HOLMES, AND B. VISHEAU. 1987. Evidence that endogenous nerve growth factor is responsible for collateral sprouting, but not the regeneration, of nociceptive axons in adult rats. Proc. Natl. Acad. Sci. USA 84: 6596–6600. FAWCETT, J. W., AND R. J. KEYNES. 1990. Peripheral nerve regeneration. Annu. Rev. Neurosci. 13: 43–60. HEUMANN, R., S. KORSCHING, C. BANDTLOW, AND H. THOENEN. 1987. Changes of nerve growth factor synthesis in non-neuronal cells in response to sciatic nerve transection. J. Cell. Biol. 104: 1623–1631. JACKSON, P. C., AND J. DIAMOND. 1984. Temporal and spatial constraints of the collateral sprouting of low-threshold mechanosensory nerves in the skin of rats. J. Comp. Neurol. 226: 336–345. JENQ, C.-B., L.-L. JENQ, H. M. BEAR, AND R. E. COGGESHALL. 1988. Conditioning lesions of peripheral nerves change regenerated axon numbers. Brain Res. 457: 63–69. KINNMAN, E., AND H. ALDSKOGIUS. 1986. Collateral sprouting of sensory axons in the glabrous skin of the hindpaw after chronic sciatic nerve lesion in adult and neonatal rats: A morphological study. Brain Res. 377: 73–82. KINNMAN, E., H. ALDSKOGIUS, O. JOHANSSON, AND Z. WIESENFELD-
20.
21.
22.
23. 24.
25. 26. 27.
28.
29.
30.
213
HALLIN. 1992. Collateral reinnervation and expansive regenerative reinnervation by sensory axons into foreign denervated skin: Immunohistochemical study in the rat. Exp. Brain Res. 91: 61–72. LIEBERMAN, A. R. 1974. Some factors affecting retrograde neuronal responses to axonal lesions. In Essay on the Nervous System (R. Bellairs and E. G. Gray, Eds.), pp. 71–105. Clarendon, Oxford. LINDHOLM, D., R. HEUMANN, M. MEYER, AND H. THOENEN. 1987. Changes of nerve growth factor synthesis in non-neuronal cells of rat sciatic nerve. Nature 330: 658–659. MCQUARRIE, I. G. 1987. The effects of a conditioning lesion on the regeneration of motoraxons. Brain Res. 152: 597–602. MCQUARRIE, I. G., AND B. GRAFSTEIN. 1981. Effect of a conditioning lesion on optic nerve regeneration in goldfish. Brain Res. 216: 253–264. MCQUARRIE, I. G., B. GRAFSTEIN, AND M. D. GERSHON. 1977. Axonal regeneration in the rat sciatic nerve: Effect of a conditioning lesion and of dbcAMP. Brain Res. 132: 443–453. DE MEDINACELI, L. 1988. Functional consequences of experimental nerve lesions: Effects of reinnervation blend. Exp. Neurol. 100: 166–178. NAVARRO, X., AND W. R. KENNEDY. 1990. The effect of a conditioning lesion on sudomotor axon regeneration. Brain Res. 509: 232–236. NIXON, B. J., R. DOUCETTE, P. C. JACKSON, AND J. DIAMOND. 1984. Impulse activity evokes precocious sprouting of nociceptive nerves into denervated skin. Somatosens. Res. 2: 97–126. OTTO, D., K. UNSICKER, AND C. GROTHE. 1987. Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia. Neurosci. Lett. 83: 156–160. RAIVICH, G., AND G. W. KREUTZBERG. 1993. Peripheral nerve regeneration: Role of growth factors and their receptors. J. Dev. Neurosci. 11: 311–324. RICH, K. M., J. R. LUSCZYNSKY, P. A. OSBORNE, AND E. M. JOHNSON, JR. 1987. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J. Neurocytol. 16: 261–268. SCHMALBRUCH, H. 1987. Loss of sensory neurons after sciatic nerve section in the rat. Anat. Rec. 219: 323–329. SKETELJ, J., M. BRESJANAC, AND M. POPOVIC´ . 1989. Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells. J. Neurosci. Res. 24: 153–162. SPARROW, J. R., AND B. GRAFSTEIN. 1983. Prior collateral sprouting enhances axonal regeneration. Brain Res. 269: 133–136. WATSON, W. E. 1973. Some responses of dorsal root ganglia to axotomy. J. Physiol. 231: 41–42. WEDDELL, G., L. GUTTMANN, AND E. GUTMANN. 1941. The local extension of nerve fibers into denervated areas of skin. J. Neurol. Psych. 4: 206–225. WIESENFELD-HALLIN, Z., E. KINNMAN, AND H. ALDSKOGIUS. 1989. Expansion of innervation territory by afferents involved in plasma extravasation after nerve regeneration in adult and neonatal rats. Exp. Brain Res. 76: 88–96. YGGE, J. H., AND H. ALDSKOGIUS. 1984. Intercostal nerve transection and its effect on the dorsal root ganglion. A quantitative study on thoracic ganglion cell numbers and sizes in the rat. Exp. Brain Res. 55: 402–408. YOUNG, J. Z., AND P. B. MEDAWAR. 1940. Fibrin suture of peripheral nerves. Measurement of the rate of regeneration. Lancet II: 126–128.