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Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve
Brain Research, 530 (1990) 167-169
167
Elsevier BRES 24341
Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve Jacob Sjrberg and Martin Kanje Department of Zoophysiology, University of Lund, Lund (Sweden) (Accepted 10 July 1990)
Key words: Nerve regeneration; Conditioning lesion effect; Schwann cell; Rat sciatic nerve; Thymidine
The effect of repetitive conditioning lesions was tested on regeneration of the rat sciatic nerve. The nerve was conditioned by crush lesions one, two or three times with an interval of 2 or 4 days between each successive lesion. Axonal elongation was measured 3 days after a final test crush lesion. Two conditioning lesions stimulated axonal elongation more than one, while a third conditioning lesion had no further effect on axonal outgrowth. However, if the number of conditioning lesions were varied within a constant conditioning interval, outgrowth after the test lesion was the same. This suggests that the conditioning interval and not the number of conditioning lesions determined the outgrowth after a test lesion. When the conditioning lesion(s) and the test lesion were made at the same place, outgrowth was longer than if the lesions were spatially separated. Incorporation of [3H]thymidine in the regenerated nerve segment showed that proliferation of non-neuronal cells was initiated by each lesion. By counting the number of cell nuclei this proliferation was shown to correspond to an increase of cells in the regenerating nerve. It is therefore possible that the greater number of non-neuronal cells in the distal nerve segment accounts for the enhanced conditioning lesion effect in nerves where the conditioning and test lesions are made at the same place. That nerve cells have the ability to regenerate m o r e rapidly after a second injury has been d e m o n s t r a t e d in m a m m a l i a n 3'4'1°'12'16'19 as well as goldfish 8'15'18 and amphibian nerves 6'21. This is known as the conditioning lesion effect ( C L E ) . The mechanism underlying this C L E is not clear, but it has been ascribed to changes in the cell b o d y 11'18. H o w e v e r , Schwann and o t h e r non-neuronal cells in the distal nerve segment into which the axons grow might also be i m p o r t a n t for the expression of the CLE 3'5'7"15'24. In this study we have e x a m i n e d if outgrowth of nerve fibers could be further enhanced by successive conditioning lesions, and if conditioning affects proliferation of non-neuronal cells in the sciatic nerve. F e m a l e S p r a q u e - D a w l e y rats ( A L A B , Sweden) weighing a p p r o x i m a t e l y 200 g were used. Two o r three animals were kept in each cage at a r o o m t e m p e r a t u r e of 21 °C and 55% humidity, and given water and food ad libitum. The rats were anesthetized by intraperitoneal injection of 0.25 ml of a N e m b u t a l (50 m g / m l ) - V a l i u m (5 m g / m l ) - s a l i n e mixture (1:2:1 v/v). The sciatic nerve was e x p o s e d and crushed with specially designed pliers either at the mid-thigh or 20 m m distally. The crush site was labelled by attaching a 9-0 suture to the epineurium. A f t e r an interval of 2, 4 or 12 days the nerve was re-exposed and a test crush lesion was m a d e at the mid-thigh. In some nerves the conditioning crush was
r e p e a t e d once or twice with intervals of 2 o r 4 days. Control nerves were only e x p o s e d to a test crush lesion. The regeneration distance of the sensory neurons was evaluated with the pinch test, as previously described z3, 3 days after the test lesion. Results o b t a i n e d with the pinch test agree with regeneration distances o b t a i n e d by immunocytochemical staining for n e u r o f i l a m e n t protein in the regenerating neurites, as previously described 22'23 I n c o r p o r a t i o n of [3H]thymidine was m e a s u r e d 3 days after the test lesion. In these experiments a 30-mm segment of the sciatic nerve was r e m o v e d , d e s h e a t h e d and incubated for two hours in vials containing 50/~Ci of [3H]thymidine (91 Ci/mmol) in 1 ml m o d i f i e d Ringer solution, as previously described 23. A f t e r thorough washing with ice-cold Ringer solution the nerves were cut in 2-mm pieces and extracted for 60 min in ice-cold 10% trichloroacetic acid ( T C A ) . T C A - i n s o l u b l e material was solubilized in 200 ktl Soluene 350 (Packard). Radioactivity of T C A - s o l u b l e and insoluble fractions were d e t e r m i n e d by liquid scintillation counting. I n c o ~ o r a t i o n was expressed as T C A - i n s o l u b l e radioactivity in percent of total radioactivity in 2-mm nerve segment. The incorporation into the r e g e n e r a t e d nerve - - consisting of the test crush and a n o t h e r 6 distal segments - was calculated by summation of radioactivity in the individual segments. The n u m b e r of n o n - n e u r o n a l cell nuclei in the regenerating nerve was c o u n t e d 3 days after the test lesion. A
Correspondence: J. SjOberg, Department of Zoophysiology, University of Lund, Helgonav~igen 3B, S-223 62 Lund, Sweden. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
168 400" 141
1CTD contr~
12
~t T-C
E 300" aS
E
g
o o
T-C
== 20~
=.
"6 CT
.=E 10C 0
4
2+2
4*4
T
4*4*4
Conditioning interval (days) , .
Fig. 1. Regeneration distances 3 days after a test crush lesion in nerves subjected to different numbers of conditioning lesions. The total conditioning interval was 4, 8 or 12 days. CT = the conditioning crush lesion was at the same place as the test lesion; T-C = the conditioning crush lesion was placed distal to the test lesion; control = test crush only. Mean values + S.E.M., n = 5. 30-ram segment of the nerve was removed and sections with a thickness of 5 / t i n were made on a cryostat. After fixation with acid ethanol for 1 min, the sections were stained for 15 min with acridine orange, diluted 1:40,000. After thorough washing in phosphate-buffered saline (PBS) 0.01 M pH 7.2, the sections were mounted in PBS. The cell nuclei were visualized in a fluorescence microscope and counted within 0.4 m m 2 at every m m along the nerve. Nerves subjected to a single test lesion regenerated 6.3 mm during 3 days. The regeneration distance increased by 48% with a distal conditioning lesion (T-C nerves) (Fig. 1). If the conditioning lesion was at the same place
• CT
=~
t~ r-c
@
0'
2*2
4
4+4
4+ 4 +4
12
Conditioning interval (days)
Fig. 2. Incorporation of [3H]thymidine in nerve segments 3 days after the test crush lesion with different numbers of conditioning lesions. The conditioning interval was 2, 4 or 12 days. The incorporation was measured in the nerve section 1 mm proximal to the test crush and as far as 4 mm distal to the measured regeneration distance. CT = the conditioning crush lesion was at the same place as the test lesion; T-C = the conditioning crush lesion was placed distal to the test lesion. Mean values + S.E.M., n = 3.
. . . .
-4
, , , .
-2
. . . . . .
T ~ c'r
,
2
,
,
4
.
,
.
,
.
,
6
.
,
.
,
.
8
,
.
,
.
,
.
10
,
.
.
.
,
12
.
,
.
,
.
,
14
.
,
.
,
.
16
,
.
.
.
18
20
22
t
r-c
Distance from test crush (mm)
Fig. 3. The number of non-neuronal cell nuclei along the nerve 3 days after the test lesion. The cell nuclei were stained with acridine orange and counted within 0.4 mm2 at every mm along the nerve. The conditioning interval was 4 days. T = test lesion only. CT = the conditioning crush lesion was at the same place as the test lesion. T-C = the conditioning crush lesion was placed distal to the test lesion. Control nerves were not lesioned. Arrows indicate position of the respective conditioning lesion. Mean values, n = 3.
as the test crush (CT nerves), the effect on axonal outgrowth was more p r o n o u n c e d , which has previously been observed 3"7'24, and the regeneration distance increased by 68%. In nerves subjected to two conditioning lesions the regeneration distance increased an additional 10% in T-C nerves and by 11% in CT nerves. A third successive conditioning lesion had only a small but not significant effect on the regeneration distance. This enhanced regeneration after a second conditioning lesion could imply that several conditioning lesions stimulate regeneration more than a single one, but it could also be due to the total conditioning interval. The latter possibility was first tested by performing a single conditioning lesion and increasing the conditioning interval to 12 days (Fig. 1). Outgrowth after the test lesion was similar to that of nerves subjected to 3 successive conditioning lesions - - with an interval of 4 days between each crush. Furthermore, two conditioning lesions with an interval of 2 days was no more effective than a single conditioning lesion with a conditioning interval of 4 days (Fig. 1). Thus, the conditioning interval and not the number of conditioning lesions determine the rate of axonal elongation after a test crush lesion. The present results indicated that the optimal conditioning interval is 8-12 days, a figure which agrees with earlier reports 2"4'8A°. Local cell proliferation after one or more crush lesions was measured as [3H]thymidine incorporation in the nerve (Fig. 2). The incorporation of thymidine was lower in the CT nerves with one conditioning lesion than in the
169 corresponding T-C nerves. With each successive conditioning lesion, the thymidine incorporation decreased in the C T nerves while no significant changes could be observed in the T-C nerves. The conditioning interval did not affect thymidine incorporation since incorporation after a 12-day interval between conditioning and test lesions was not significantly different from that after a 4-day interval. These results show that the proliferation of non-
also previous observations that repeated crush injuries produce an increased n u m b e r of Schwann cells 1"25. It is possible that the n u m b e r of n o n - n e u r o n a l cells in the distal segment could affect the rate of axonal elongation. This notion is supported by the observation that outgrowth is increased when axons are allowed to grow into a nerve graft containing 8 times more Schwann cells than normal 9, and that regeneration is retarded
n e u r o n a l cells is initiated by each successive crush lesion, although fewer cells appear to be activated each time. The latter notion is also supported by the observation of a smaller increase in n o n - n e u r o n a l D N A after a second injury than after the first 2°. The repeated initiation of the cell proliferation suggests that the total n u m b e r of
when the Schwann cell proliferation is inhibited 13. The n u m b e r of cell nuclei in the segment distal to the test
n o n - n e u r o n a l cells in the area surrounding the injury increase with each successive lesion. The counting of cell
made at the same place as the conditioned lesion(s) and
nuclei in nerves subjected to a conditioning crush 4 days before the test lesion showed that this was true (Fig. 3). The n u m b e r of cell nuclei was significantly increased in the area surrounding the test crush in the C T nerves
lesion was nearly doubled in the C T nerves compared with the T-C and u n c o n d i t i o n e d nerves, This increased n u m b e r of n o n - n e u r o n a l cells could thus account for the difference of the C L E between nerves with the test crush nerves with spatially separated lesions.
compared with T-C or unconditioned nerves. There are
This study was supported by grants from the Swedish Natural Science Research Council and Hierta-Retzius Stiftelse. We wish to thank Marie Adlher-Maihofer for her skillful technical assistance and Marianne Andersson for excellent artwork.
1 Abercrombie, M. and Santler, J., An analysis of growth in nuclear population during Wallerian degeneration, J. Cell. Comp. Physiol., 50 (1957) 429-450. 2 Arntz, C., Kanje, M. and Lundborg, G., Regeneration of the rat sciatic nerve after different conditioning lesions: effects of the conditioning interval, Microsurgery, 10 (1989) 118-121. 3 Bisby, M.A. and Pollock, B., Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon, J. Neurobiol., 14 (1983) 467-472. 4 Bondoux-Jahan, M. and Sebille, A., Electrophysiological study of conditioning lesion effect on rat sciatic nerve regeneration following either prior section or freeze. I. Intensity and time course, Brain Research, 382 (1986) 39-45. 5 Brown, M.C. and Hopkins, W.G., Role of degeneration axon pathways in regeneration of mouse soleus motor axons, J. Physiol. (Lond.), 318 (1981) 365-373. 6 Carlsen, R.C., Delayed induction of the cell body response and enhancement of regeneration following a condition/test lesion of frog peripheral nerve at 15 °C, Brain Research, 279 (1983) 9-18. 7 Edbladh, M. and Edstr6m, A., Environmental factors contribute to the enhanced regeneration of frog sciatic sensory axons by a conditioning lesion, Acta Physiol. Scand., 135 (1989) 169-172. 8 Edwards, D.L., Alpert, R.M. and Grafstein, B., Recovery of vision in regeneration of goldfish optic axons: enhancement of axonal outgrowth by a conditioning lesion, Exp. Neurol., 72 (1981) 672-686. 9 Ferzaz, B., Koenig, H. and Ressouches, A., Axonal regeneration in Trembler mouse, a Schwann cell mutant, C.R. Acad. Sci. Paris, 309 (1989) 377-382. 10 Forman, D.S., McQuarrie, I.G., Labore, F.W., Wood, D.K., Stone, L.S., Braddock, C.H. and Fuchs, D.A., Time course of the conditioning effect on axonal regeneration, Brain Research, 182 (1980) 180-185. 11 Grafstein, B. and McQuarrie, I.G., Role of the nerve cell body in axonal regeneration. In W.C. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 155-195. 12 Gutmann, E., Gutmann, L., Medawar, P.B. and Young, J.Z., The rate of regeneration of nerve/Regeneration, J. Exp. Biol., 19 (1942) 14-44. 13 Hall, S.M., The effect of inhibiting Schwann cell mitosis on the
re-innervation of acellular autografts in the peripheral nervous system of the mouse, Neuropath. Appl. Neurobiol., 12 (1986) 401-414. 14 Lanners, H.N. and Grafstein, B., Effect of a conditioning lesion on regeneration of goldfish optic axons: ultrastructural evidence of enhanced outgrowth and pinocytosis, Brain Research, 196 (1980) 547-553. 15 Lubinska, L., The influence of the state of peripheral stump on the early stages of nerve regeneration, Acta Biol. Exp., 16 (1952) 55-63. 16 McQuarrie, I.G. and Grafstein, B., Axon outgrowth enhanced by a previous nerve injury, Arch. Neurol., 29 (1973) 53-55. 17 McQuarrie, I.G. and Grafstein, B., Effect of a conditioning lesion of optic nerve regeneration in goldfish, Brain Research, 216 (1981) 253-264. 18 McQuarrie, I.G., Effect of a conditioning lesion on axonal transport during regeneration: the role of slow transport. In J.S. Elam and P. Cancalon (Eds.), Axonal Transport in Neuronal Growth and Regeneration, Plenum, New York, 1984, pp. 185-209. 19 McQuarrie, I.G., Grafstein, B., Dreyfys, C.E and Gershon, M.D., Regeneration of adrenergic axons in rat sciatic nerve: effect of a conditioning lesion, Brain Research, 141 (1978) 21-34. 20 Oderfeld-Nowak, B. and Niemierko, S., Synthesis of nucleic acids in the Schwann cells as the early cellular response to nerve injury, J. Neurochem., 16 (1969) 235-248. 21 Perry, G.W., Krayanek, S.R. and Wilson, D.L., Effects of a conditioning lesion on bullfrog sciatic nerve regeneration: analysis of fast axonally transported proteins, Brain Research, 423 (1987) 1-12. 22 Sj6berg, J., Kanje, M. and Edstr6m, A., Influence of nonneuronal cells on regeneration of the rat sciatic nerve, Brain Research, 453 (1988) 221-226. 23 Sj6berg, J. and Kanje, M., Insulin-like growth factor I (IGF-I) as a stimulator of regeneration in the freeze-injured rat sciatic nerve, Brain Research, 485 (1989) 102-108. 24 Sj6berg, J. and Kanje, M., The initial period of peripheral nerve regeneration and the importance of the local environmentfor the conditioning lesion effect, Brain Research, in press. 25 Thomas, P.K., The cellular response to nerve injury, J. Anat., 106 (1970) 463-470.
167
Elsevier BRES 24341
Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve Jacob Sjrberg and Martin Kanje Department of Zoophysiology, University of Lund, Lund (Sweden) (Accepted 10 July 1990)
Key words: Nerve regeneration; Conditioning lesion effect; Schwann cell; Rat sciatic nerve; Thymidine
The effect of repetitive conditioning lesions was tested on regeneration of the rat sciatic nerve. The nerve was conditioned by crush lesions one, two or three times with an interval of 2 or 4 days between each successive lesion. Axonal elongation was measured 3 days after a final test crush lesion. Two conditioning lesions stimulated axonal elongation more than one, while a third conditioning lesion had no further effect on axonal outgrowth. However, if the number of conditioning lesions were varied within a constant conditioning interval, outgrowth after the test lesion was the same. This suggests that the conditioning interval and not the number of conditioning lesions determined the outgrowth after a test lesion. When the conditioning lesion(s) and the test lesion were made at the same place, outgrowth was longer than if the lesions were spatially separated. Incorporation of [3H]thymidine in the regenerated nerve segment showed that proliferation of non-neuronal cells was initiated by each lesion. By counting the number of cell nuclei this proliferation was shown to correspond to an increase of cells in the regenerating nerve. It is therefore possible that the greater number of non-neuronal cells in the distal nerve segment accounts for the enhanced conditioning lesion effect in nerves where the conditioning and test lesions are made at the same place. That nerve cells have the ability to regenerate m o r e rapidly after a second injury has been d e m o n s t r a t e d in m a m m a l i a n 3'4'1°'12'16'19 as well as goldfish 8'15'18 and amphibian nerves 6'21. This is known as the conditioning lesion effect ( C L E ) . The mechanism underlying this C L E is not clear, but it has been ascribed to changes in the cell b o d y 11'18. H o w e v e r , Schwann and o t h e r non-neuronal cells in the distal nerve segment into which the axons grow might also be i m p o r t a n t for the expression of the CLE 3'5'7"15'24. In this study we have e x a m i n e d if outgrowth of nerve fibers could be further enhanced by successive conditioning lesions, and if conditioning affects proliferation of non-neuronal cells in the sciatic nerve. F e m a l e S p r a q u e - D a w l e y rats ( A L A B , Sweden) weighing a p p r o x i m a t e l y 200 g were used. Two o r three animals were kept in each cage at a r o o m t e m p e r a t u r e of 21 °C and 55% humidity, and given water and food ad libitum. The rats were anesthetized by intraperitoneal injection of 0.25 ml of a N e m b u t a l (50 m g / m l ) - V a l i u m (5 m g / m l ) - s a l i n e mixture (1:2:1 v/v). The sciatic nerve was e x p o s e d and crushed with specially designed pliers either at the mid-thigh or 20 m m distally. The crush site was labelled by attaching a 9-0 suture to the epineurium. A f t e r an interval of 2, 4 or 12 days the nerve was re-exposed and a test crush lesion was m a d e at the mid-thigh. In some nerves the conditioning crush was
r e p e a t e d once or twice with intervals of 2 o r 4 days. Control nerves were only e x p o s e d to a test crush lesion. The regeneration distance of the sensory neurons was evaluated with the pinch test, as previously described z3, 3 days after the test lesion. Results o b t a i n e d with the pinch test agree with regeneration distances o b t a i n e d by immunocytochemical staining for n e u r o f i l a m e n t protein in the regenerating neurites, as previously described 22'23 I n c o r p o r a t i o n of [3H]thymidine was m e a s u r e d 3 days after the test lesion. In these experiments a 30-mm segment of the sciatic nerve was r e m o v e d , d e s h e a t h e d and incubated for two hours in vials containing 50/~Ci of [3H]thymidine (91 Ci/mmol) in 1 ml m o d i f i e d Ringer solution, as previously described 23. A f t e r thorough washing with ice-cold Ringer solution the nerves were cut in 2-mm pieces and extracted for 60 min in ice-cold 10% trichloroacetic acid ( T C A ) . T C A - i n s o l u b l e material was solubilized in 200 ktl Soluene 350 (Packard). Radioactivity of T C A - s o l u b l e and insoluble fractions were d e t e r m i n e d by liquid scintillation counting. I n c o ~ o r a t i o n was expressed as T C A - i n s o l u b l e radioactivity in percent of total radioactivity in 2-mm nerve segment. The incorporation into the r e g e n e r a t e d nerve - - consisting of the test crush and a n o t h e r 6 distal segments - was calculated by summation of radioactivity in the individual segments. The n u m b e r of n o n - n e u r o n a l cell nuclei in the regenerating nerve was c o u n t e d 3 days after the test lesion. A
Correspondence: J. SjOberg, Department of Zoophysiology, University of Lund, Helgonav~igen 3B, S-223 62 Lund, Sweden. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
168 400" 141
1CTD contr~
12
~t T-C
E 300" aS
E
g
o o
T-C
== 20~
=.
"6 CT
.=E 10C 0
4
2+2
4*4
T
4*4*4
Conditioning interval (days) , .
Fig. 1. Regeneration distances 3 days after a test crush lesion in nerves subjected to different numbers of conditioning lesions. The total conditioning interval was 4, 8 or 12 days. CT = the conditioning crush lesion was at the same place as the test lesion; T-C = the conditioning crush lesion was placed distal to the test lesion; control = test crush only. Mean values + S.E.M., n = 5. 30-ram segment of the nerve was removed and sections with a thickness of 5 / t i n were made on a cryostat. After fixation with acid ethanol for 1 min, the sections were stained for 15 min with acridine orange, diluted 1:40,000. After thorough washing in phosphate-buffered saline (PBS) 0.01 M pH 7.2, the sections were mounted in PBS. The cell nuclei were visualized in a fluorescence microscope and counted within 0.4 m m 2 at every m m along the nerve. Nerves subjected to a single test lesion regenerated 6.3 mm during 3 days. The regeneration distance increased by 48% with a distal conditioning lesion (T-C nerves) (Fig. 1). If the conditioning lesion was at the same place
• CT
=~
t~ r-c
@
0'
2*2
4
4+4
4+ 4 +4
12
Conditioning interval (days)
Fig. 2. Incorporation of [3H]thymidine in nerve segments 3 days after the test crush lesion with different numbers of conditioning lesions. The conditioning interval was 2, 4 or 12 days. The incorporation was measured in the nerve section 1 mm proximal to the test crush and as far as 4 mm distal to the measured regeneration distance. CT = the conditioning crush lesion was at the same place as the test lesion; T-C = the conditioning crush lesion was placed distal to the test lesion. Mean values + S.E.M., n = 3.
. . . .
-4
, , , .
-2
. . . . . .
T ~ c'r
,
2
,
,
4
.
,
.
,
.
,
6
.
,
.
,
.
8
,
.
,
.
,
.
10
,
.
.
.
,
12
.
,
.
,
.
,
14
.
,
.
,
.
16
,
.
.
.
18
20
22
t
r-c
Distance from test crush (mm)
Fig. 3. The number of non-neuronal cell nuclei along the nerve 3 days after the test lesion. The cell nuclei were stained with acridine orange and counted within 0.4 mm2 at every mm along the nerve. The conditioning interval was 4 days. T = test lesion only. CT = the conditioning crush lesion was at the same place as the test lesion. T-C = the conditioning crush lesion was placed distal to the test lesion. Control nerves were not lesioned. Arrows indicate position of the respective conditioning lesion. Mean values, n = 3.
as the test crush (CT nerves), the effect on axonal outgrowth was more p r o n o u n c e d , which has previously been observed 3"7'24, and the regeneration distance increased by 68%. In nerves subjected to two conditioning lesions the regeneration distance increased an additional 10% in T-C nerves and by 11% in CT nerves. A third successive conditioning lesion had only a small but not significant effect on the regeneration distance. This enhanced regeneration after a second conditioning lesion could imply that several conditioning lesions stimulate regeneration more than a single one, but it could also be due to the total conditioning interval. The latter possibility was first tested by performing a single conditioning lesion and increasing the conditioning interval to 12 days (Fig. 1). Outgrowth after the test lesion was similar to that of nerves subjected to 3 successive conditioning lesions - - with an interval of 4 days between each crush. Furthermore, two conditioning lesions with an interval of 2 days was no more effective than a single conditioning lesion with a conditioning interval of 4 days (Fig. 1). Thus, the conditioning interval and not the number of conditioning lesions determine the rate of axonal elongation after a test crush lesion. The present results indicated that the optimal conditioning interval is 8-12 days, a figure which agrees with earlier reports 2"4'8A°. Local cell proliferation after one or more crush lesions was measured as [3H]thymidine incorporation in the nerve (Fig. 2). The incorporation of thymidine was lower in the CT nerves with one conditioning lesion than in the
169 corresponding T-C nerves. With each successive conditioning lesion, the thymidine incorporation decreased in the C T nerves while no significant changes could be observed in the T-C nerves. The conditioning interval did not affect thymidine incorporation since incorporation after a 12-day interval between conditioning and test lesions was not significantly different from that after a 4-day interval. These results show that the proliferation of non-
also previous observations that repeated crush injuries produce an increased n u m b e r of Schwann cells 1"25. It is possible that the n u m b e r of n o n - n e u r o n a l cells in the distal segment could affect the rate of axonal elongation. This notion is supported by the observation that outgrowth is increased when axons are allowed to grow into a nerve graft containing 8 times more Schwann cells than normal 9, and that regeneration is retarded
n e u r o n a l cells is initiated by each successive crush lesion, although fewer cells appear to be activated each time. The latter notion is also supported by the observation of a smaller increase in n o n - n e u r o n a l D N A after a second injury than after the first 2°. The repeated initiation of the cell proliferation suggests that the total n u m b e r of
when the Schwann cell proliferation is inhibited 13. The n u m b e r of cell nuclei in the segment distal to the test
n o n - n e u r o n a l cells in the area surrounding the injury increase with each successive lesion. The counting of cell
made at the same place as the conditioned lesion(s) and
nuclei in nerves subjected to a conditioning crush 4 days before the test lesion showed that this was true (Fig. 3). The n u m b e r of cell nuclei was significantly increased in the area surrounding the test crush in the C T nerves
lesion was nearly doubled in the C T nerves compared with the T-C and u n c o n d i t i o n e d nerves, This increased n u m b e r of n o n - n e u r o n a l cells could thus account for the difference of the C L E between nerves with the test crush nerves with spatially separated lesions.
compared with T-C or unconditioned nerves. There are
This study was supported by grants from the Swedish Natural Science Research Council and Hierta-Retzius Stiftelse. We wish to thank Marie Adlher-Maihofer for her skillful technical assistance and Marianne Andersson for excellent artwork.
1 Abercrombie, M. and Santler, J., An analysis of growth in nuclear population during Wallerian degeneration, J. Cell. Comp. Physiol., 50 (1957) 429-450. 2 Arntz, C., Kanje, M. and Lundborg, G., Regeneration of the rat sciatic nerve after different conditioning lesions: effects of the conditioning interval, Microsurgery, 10 (1989) 118-121. 3 Bisby, M.A. and Pollock, B., Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon, J. Neurobiol., 14 (1983) 467-472. 4 Bondoux-Jahan, M. and Sebille, A., Electrophysiological study of conditioning lesion effect on rat sciatic nerve regeneration following either prior section or freeze. I. Intensity and time course, Brain Research, 382 (1986) 39-45. 5 Brown, M.C. and Hopkins, W.G., Role of degeneration axon pathways in regeneration of mouse soleus motor axons, J. Physiol. (Lond.), 318 (1981) 365-373. 6 Carlsen, R.C., Delayed induction of the cell body response and enhancement of regeneration following a condition/test lesion of frog peripheral nerve at 15 °C, Brain Research, 279 (1983) 9-18. 7 Edbladh, M. and Edstr6m, A., Environmental factors contribute to the enhanced regeneration of frog sciatic sensory axons by a conditioning lesion, Acta Physiol. Scand., 135 (1989) 169-172. 8 Edwards, D.L., Alpert, R.M. and Grafstein, B., Recovery of vision in regeneration of goldfish optic axons: enhancement of axonal outgrowth by a conditioning lesion, Exp. Neurol., 72 (1981) 672-686. 9 Ferzaz, B., Koenig, H. and Ressouches, A., Axonal regeneration in Trembler mouse, a Schwann cell mutant, C.R. Acad. Sci. Paris, 309 (1989) 377-382. 10 Forman, D.S., McQuarrie, I.G., Labore, F.W., Wood, D.K., Stone, L.S., Braddock, C.H. and Fuchs, D.A., Time course of the conditioning effect on axonal regeneration, Brain Research, 182 (1980) 180-185. 11 Grafstein, B. and McQuarrie, I.G., Role of the nerve cell body in axonal regeneration. In W.C. Cotman (Ed.), Neuronal Plasticity, Raven, New York, 1978, pp. 155-195. 12 Gutmann, E., Gutmann, L., Medawar, P.B. and Young, J.Z., The rate of regeneration of nerve/Regeneration, J. Exp. Biol., 19 (1942) 14-44. 13 Hall, S.M., The effect of inhibiting Schwann cell mitosis on the
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