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Time-dependent regenerative influence of predegenerated nerve grafts on hippocampus
0361-9230/92 $5.00 + .OO
Brain Research Bulletin, Vol. 29, pp. 831-835, 1992
Copyright0 1992Pergamon Press Ltd.
Printed in the USA. All rights reserved.
Time-Dependent Regenerative Influence of Predegenerated Nerve Grafts on Hippocampus JOANNA
LEWIN-KOWALIK,’ ALEKSANDER JAROSLAW-JERZY BARSKI
L. SIERON, MIECZYSLLAW AND DARIUSZ G6RKA
KRAUSE,
Department of Physiology, Silesian Medical School, 40-762 Katowice, Medykbw 18, Poland
Received
19 August
199 1; Accepted
16 April
1992
LEWIN-KOWALIK, J., A. L. SIERON, M. KRAUSE, J.-J. BARSKI AND D. GGRKA. Time-dependent regenerative influence ofpredegenerated nerve grafts on hippocampus. BRAIN RES BULL 29(6) 83 l-835, 1992;-Our previous studies have revealed that the predegeneration facilitated the neurite outgrowth from hippocampus following the peripheral nerve grafts implantation. The aim of the present work is to find whether the stimulative power of peripheral nerve grafts depends on the time lapse after the transection. Autologous predegenereted distal stumps of the rat sciatic nerves were implanted into the hippocampus on the 7th, 14th, 28th, and 35th day following the transection. Six weeks later, horseradish peroxidase conjugated with fluoresceine isothiocyanate was injected into the graft and frozen sections of brains were made. Fluorescence microscope examination has shown that PITC-HRP labeled cells were present among the hippocampal neurons in all the brains under examination, excluding these grafted with 14-day predegenarated peripheral nerves. The RTC-HRP labeled neurons were particularly numerous when the 7- and 35-day-old predegenerated stumps were used as grafts. Degeneration
Sciatic nerve isografis
Neurite outgrowth
NEURONS in the peripheral nervous system of adult mammals successfully regenerate after nerve injury and elongate their axons over long distances. In contrast, a great majority of neurons in the central nervous system (CNS) does not regenerate after injury. Their axons do not elongate, although they exhibit initial sprouting (2,12). Recently, successful regeneration was obtained over considerable distances through grafts consisting of peripheral nerve implanted into the different regions of CNS (28). However, the role played by nonneuronal cells in the guidance or promotion of axonal outgrowth is poorly understood. There are indications that the distal stump of transected mammalian peripheral nerve secretes diffusible factors which are able to support axonal regeneration in vivo (6,17,21). The putative source of such factors seems to be the Schwann cells (2 1). In a transected peripheral nerve the Schwann cells divide frequently, and they represent the predominant cell type within the sciatic nerve grafts. The intensity of cellular divisions and the metabolic activity in nonneuronal components of the transected peripheral nerve are not constant, but depend on the time lapse (6,2 1). Our previous studies have revealed that predegenerated peripheral nerve grafts facilitated the neurite outgrowth of damaged hippocampal neurons, whereas nonpredegenerated ones are, in this respect, less effective (11).
Hippocampus
The aim of the present work is to find whether the outgrowth of damaged central neurites is time dependent or not when the injured peripheral nerve has been used for grafting. METHOD Experiments were carried out on 25 adult male Wistar C rats. Animals were divided into five equal groups. During the whole experiment animals received standard diet and water ad lib. Surgery
The sciatic nerve in animals from the first experimental group (D7) was cut near the hip joint under intraperitoneal chloral hydrate anesthesia (420 mg per kg b.wt.). Seven days later, the animals were reanesthetised and a IO-mm long fragment of the distal stump of sciatic nerve was dissected. It was subsequently inserted into a glass cannula (1 mm internal and 2 mm external diameter) containing the Ringer’s solution for mammals. In the other groups, fragments of predegenerated sciatic nerves, prepared in the same way, were taken at the 14th, 28th, and 35th day after injury (groups D14, D28, D35, respectively). In the
’ To whom requests for reprints should be addressed.
831
832
control (C) group. peripheral nerve segment was dissected immediately after the transection and was, thus, nonpredegenerated. The peripheral nerve segments were implanted into the hippocampus according to the method described by Lewin-Kowalik at al. Prior to the implantation, a 3 mm deep brain tissue injury was made. The caudal tip of the grafts was laid over the skull bone, tied with 4-O surgical silk, and attached to the skull by means of Tissucoll Kit (Immuno AG Wien). Six weeks after surgery all animals were anaesthetized as described above. Ten ~1 ofOS% FITC-HRP (horseradish peroxidase conjugated with fluoresceine isothiocyanate) solution was injected into the free end of each graft. The FITC-HRP is retrogradely transported into the cell body. Twenty-four h later the rats were perfused transcardially with 200 ml 5% sucrose in phosphate buffer of pH 7.2 and, subsequently, with fixative solution of 2.4% formaldehyde buffered as before. tJislolo~id Prowdurc
ILEWIN-KQWALIK
F’I 41..
prepared. The slices (IO pm thick) were treated by hematoxylineosin (H-E) staining, examined in fluorescence (Univar ReichertJung) and light microscopes. and photographed. The H-E staining was used in order to verify the anatomical positions of the grafts (Fig. 1). Five animals were examined in each group. Labeled cells were present in the neighbourhood of the graft’s tips only. For this reason, we decided to take under consideration, out of the lot. only 10 sections derived from the above area. Labeled cells were counted in these slices in areas of 0.5 X 0.9 mm around the tip of the graft. This space contained practically all traced cells. To avoid double counting, cells were counted in every second section. Cells have been counted by means of a picture computer analyzer Quantimet 720 type 30 (Cambridge Instruments), and an HP 9825 A computer was used. The cells were indicated with the help of a light pen. The results were subjected to statistical analysis according to the Student’s I-test and nonparametric Wilcoxon test. Statistical significance was set at 17< 0.05. RESLJLl S
Whole grafted brains were dissected from the skull, and frontal frozen sections (Frigocut Mod. 2700, Reichert-Jung) were
Fluorescence microscope examination has showed that FITCHRP labeled cells were present among the hippocampal neurons in the examined brains derived from C, D7, D28, and D35 groups. However, the number of these cells was different in each group (Figs. 2. 3, Table 1). Horseradish peroxidase labeled neurons were found to be particularly numerous in slices derived from the brains treated with 7 days predegenerated distal stumps (Fig. 3, Table 1). Cell bodies containing the marker were regularly dispersed around the tip of the graft. The average number of labeled cells was 241 t I 13.2, and it was about six times higher than in slices obtained from brains treated with freshly transected nerves (Fig. 2). Statistical analysis has shown that such an increase was statistically significant (Table I). In the brain slices from the D 14 group no FITC-HRP labeled cells were found in the neighbourhood of the graft’s tip in three cases, but only single ones in two cases. In comparison to the control group, such a decrease was significant (Table 1). Horseradish peroxidase labeled cells appeared again in D28 and D35 slices. Their average number was 70.2 + 8.98 and 78.4 + 6.94, respectively (Table 1). These repeated increases in D28 as well as in D35 groups were statistically significant as compared to controls (Table I).
1
TABLE
THE NUMBER OF RTC-HRP LABELLED ESTABLISHED IN DIFFERENT EXPERIMENTAL GROUPS
Group
C
D7 D14 D28 D35 FIG. 1. Placement of the nonpredegenerated PN-graft (G) in the hippocampus (H) 6 weeks after grafting. Hematoxylin-eosin stained section. Bar = 800 Wm.
CELLS
Mean + SD
39.4 241.0 0.8 70.2 78.4
t 9.44 f 113.2 f I .09 t 8.98 f 6.94
N = 5 in each case. * Nonparametric Wilcoxon
t Student’s t-test.
test.
p < 0.005* p < 0.005 * p < 0.0008 t pi0.00008t
TIME-DEPENDENT
PREDEGENERATION
833
INFLUENCE
FIG. 2. Fluorescence microscope dark field picture of the control brain, Magnification 630X. Arrows indicate FITC-
HRP positive cells. Bar = 30 pm.
DISCUSSION
The mechanism of peripheral nerve grafts influence on central neurites regeneration is still an open question ( 19). TWO of the
The present study shows that the growth of host axons into the predegenerated peripheral nerve grafts depends on the time lapse after the sciatic nerve transection. This stimulative influence on hippocampal neurites regrowth was particularly effective in the 7-day predegenerated grafts, but existed in the 28- and 35 day predegenerated ones as well. It is worthwhile to note that Windebank and Poduslo (21) showed, in vitro, that permanently transected adult rat sciatic nerves exhibited in culture biphasic neurite-promoting activity. The first phase lasts 7 days , then decreases and reaches a minimum at the 14th day after the transection. The second phase of increased neurite promoting activity appears in the 28-, 35-, 4 l- and 5 1-day posttransection tissue. The early phase was partially inhibited by antisera to NGF. This does not apply to the late phase. Similar biphasic effects were observed in our experiments. The influence of predegenerated grafts implantation on injured hippocampal neurites was especially significant when the 7-, 28-. and 35-day old grafts were used, and practically disappeared after the grafting 1Cday old sciatic nerve segments. In a normal nerve, the rate of Schwann cell division is very low but increases rapidly after transection, reaching a maximum at 3-4 days and returning to baseline by 14 days. Schwann cells number reaches a maximum plateau at 14 days and then remains steady through at least 28 days posttransection (2 1). These cells became increasingly metabolically active in terms of secreting soluble proteins. Maximal secretory activity was reached on the 35th day (14). Parallel experiments carried out in our laboratory have revealed different time-dependent changes of protein contents and composition in the distal stumps of rat sciatic nerve following transection (16).
most prominent components of peripheral nerve trunks, the extracellular matrix (ECM) and the Schwann cells, have been implicated as the agents in promoting regeneration (3,4,9,10,20). The role of the Schwann cells seems to be particularly important and complex (2,4). These cells produce some extracellular matrix components, e.g., laminin, which have been found to stimulate axonal growth both in vivo and in vitro (5,7,13,15). The prevailing opinion is that in vivo regenerating axons need viable Schwann cells, and the presence of the basal lamina components alone is not sufficient (2,8). On the other hand, it is also suggested that the undestroyed biochemical integrity of acellular basal lamina provides favourable conditions for the elongation of regenerating sensory and motor axons ( 18). It is also possible that the Schwann cell surface molecules are essential for the support of some central neurites regrowth into a peripheral nerve environment ( 1, IO). Another possibility is that nonneuronal components of peripheral nerve grafts, first of all Schwann cells, can synthesize substances with neurotrophic activity (17,2 1). Ferguson at al. (6) revealed that the Schwann cells in the rat sciatic nerve in culture synthesise both NGF and non-NGF trophic factors. This neurotrophic activity of tissue culture medium begins within the first 24 h and is maintained throughout 6 days These observations in vitro agree with our results in vivo because 7-day predegenerated grafts seem to be most effective in stimulation of hippocampal neurites regrowth. Thus, on the basis of the above facts, we suppose that Schwann cells in the predegenerated peripheral nerve graft can synthesise neuronotrophic molecules, which seem to be different from the NGF factor. The intensity of this process changes with the lapse of time following the transection.
I.EWIN-KOWALIK
FIG. 3. The site of implantation of the ‘I-day predegenerated PN-graft 6 weeks after grafting. (a) Fluorescence microscope dark field picture. Numerous FITC-HRP labeled cells (arrows) and tibres (asterisks) are visualized in the brain tissue (H) around the tip of the graft (G). Bar = 100 Nrn. (b) Fragment of the above picture in a greater magnification. Bar = 30 Wm.Individual labeled cells are marked by arrows.
El
AI
TIME-DEPENDENT
PREDEGENERATION
835
INFLUENCE REFERENCES
1. Aebischer, P.; Salessiotis, A. N.; Winn, S. R. Basic fibroblast growth factor released from synthetic guidance channels facilitates peripheral nerve regeneration across long nerve gaps. J. Neurosci. Res. 23:282289; 1989. 2. Aguayo, A. J. Axonal regeneration from injured neurons in the adult mammalian central nervous system. In: Cotman, C. W., ed. Synaptic plasticity. chap. IS. New York: The Guilford Press; 1985:457-484. 3. Carbonetto. S.: &chard. F. In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system. J. Physiol. (Paris) 82:258-270; 1987. 4. Carey, D. J.; Todd, M. S. Schwann cell myelination in a chemically defined medium; demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation. Dev. Brain Res. 32:95-102; 1987. 5. David, S. Neurite outgrowth from mammalian CNS neurons on astrocytes in vitro may be mediated primarily by laminin. J. Neurocytol. 17:131-144; 1988. 6. Ferguson, I. A.; Williams, R.; Rush, R. A. Chicken NGF and nonNGF trophic factor synthesis and release by sciatic nerves in vitro. J. Neurosci. Res. 22:408-417; 1989. 7. Giftochristos, N.; David, S. Laminin and heparan sulphate proteoglycan in the lesioned adult mammalian central nervous system and their possible relationship to axonal sprouting. J. Neurocytol. 17: 385-397; 1988. 8. Gulati, A. G. Evaulation of acellular and cellular nerve grafts in repair of rat peripheral nerve. J. Neurosurg. 68: 117- 123; 1988. 9. Kleitman, N.; Simon, D. K.; Schachner, M.; Bunge, R. P. Growth of embryonic neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to Ll. Exp. Neurol. 102:298-306; 1988. 10. KIeitman, N.; Wood, P.; Johnson, M. I.; Bunge, R. P. Schwan cell surfaces but not the extracellular matrix organized by Schwann cells support neurite ougrowth from embryonic rat retina. J. Neurosci. 8(2):653-663; 1988.
Il. Lewin-Kowalik, J.; Sierod, A. L.; Krause, M.; Kwiek, S. Predegenerated peripheral nerve grafts facilitate neurite outgrowth from the hippocampus. Brain Res. Bull. 25:669-673; 1990. 12. Lundborg, G. Nerve regeneration and repair. Acta Orthop. Stand. 58:154-169; 1987. 13. Muller, H. Further evaluation of laminin, testosterone, ganglioside GM 1, and catalase on early growth in rat nerve regeneration chambers. Exp. Neurol. 101:228-233; 1988. 14. Poduslo, J. F.; Dyck, P. J.; Berg, C. T. Regulation of myelination: Schwann cell transition from myelin-maintaining state to a quiescent state after permanent nerve transection. J. Neurochem. 44388-400; 1985. 15. Salonen, V.; Peltonen, J.; Roytta, M.; Virtanen, I. Laminin in traumatized peripheral nerve: Basement membrane changes during degeneration and regeneration. J. Neurocytol. 16:713-720; 1987. 16. Sieron, A. L.; Lewin-Kowalik, J.; Krause, M.; Fertala, A. Time dependent dynamic changes of protein contents and composition in the submicrosomal fraction from the rat sciatic nerve following transection. Ann. Acad. Med. Siles. 23:81-89; 1992. 17. Singer, P. A.; Mehler, S.; Femandez, H. Effect of extract of injured nerve on initiating the regenerative response in the hypoglossal nucleus in the rat. Neurosci. Len. 84:155-160; 1988. 18. Sketelj, J.; Bresjanac, M.; Popovic, M. Rapid growth of regenerating axons across the segments of sciatic nerve devoid of Schwann cells. J. Neurosci. Res. 24; 153-162;1989. 19. Valentini, L. R.; Aebischer, P.; Galletti, P. M. Collagen- and laminincontaining gels impede peripheral nerve regeneration through semipermeable guidance channels. Exp. Neural. 98:350-356; 1987. 20. Williams, L. R.; Danielsen, hr.; Muller, H.; Varon, S. Influence of the acellular fibrin matrix on nerve regeneration success within the silicone chamber model. In: The current status of peripheral nerve regeneration. New York: Alan R. Liss, Inc.; 1988:111-122. 21. Windebank, A. J.; Poduslo, J. F. Neuronal growth factors produced by adult peripheral nerve after injury. Brain Res. 385:197-200; 1986.
Brain Research Bulletin, Vol. 29, pp. 831-835, 1992
Copyright0 1992Pergamon Press Ltd.
Printed in the USA. All rights reserved.
Time-Dependent Regenerative Influence of Predegenerated Nerve Grafts on Hippocampus JOANNA
LEWIN-KOWALIK,’ ALEKSANDER JAROSLAW-JERZY BARSKI
L. SIERON, MIECZYSLLAW AND DARIUSZ G6RKA
KRAUSE,
Department of Physiology, Silesian Medical School, 40-762 Katowice, Medykbw 18, Poland
Received
19 August
199 1; Accepted
16 April
1992
LEWIN-KOWALIK, J., A. L. SIERON, M. KRAUSE, J.-J. BARSKI AND D. GGRKA. Time-dependent regenerative influence ofpredegenerated nerve grafts on hippocampus. BRAIN RES BULL 29(6) 83 l-835, 1992;-Our previous studies have revealed that the predegeneration facilitated the neurite outgrowth from hippocampus following the peripheral nerve grafts implantation. The aim of the present work is to find whether the stimulative power of peripheral nerve grafts depends on the time lapse after the transection. Autologous predegenereted distal stumps of the rat sciatic nerves were implanted into the hippocampus on the 7th, 14th, 28th, and 35th day following the transection. Six weeks later, horseradish peroxidase conjugated with fluoresceine isothiocyanate was injected into the graft and frozen sections of brains were made. Fluorescence microscope examination has shown that PITC-HRP labeled cells were present among the hippocampal neurons in all the brains under examination, excluding these grafted with 14-day predegenarated peripheral nerves. The RTC-HRP labeled neurons were particularly numerous when the 7- and 35-day-old predegenerated stumps were used as grafts. Degeneration
Sciatic nerve isografis
Neurite outgrowth
NEURONS in the peripheral nervous system of adult mammals successfully regenerate after nerve injury and elongate their axons over long distances. In contrast, a great majority of neurons in the central nervous system (CNS) does not regenerate after injury. Their axons do not elongate, although they exhibit initial sprouting (2,12). Recently, successful regeneration was obtained over considerable distances through grafts consisting of peripheral nerve implanted into the different regions of CNS (28). However, the role played by nonneuronal cells in the guidance or promotion of axonal outgrowth is poorly understood. There are indications that the distal stump of transected mammalian peripheral nerve secretes diffusible factors which are able to support axonal regeneration in vivo (6,17,21). The putative source of such factors seems to be the Schwann cells (2 1). In a transected peripheral nerve the Schwann cells divide frequently, and they represent the predominant cell type within the sciatic nerve grafts. The intensity of cellular divisions and the metabolic activity in nonneuronal components of the transected peripheral nerve are not constant, but depend on the time lapse (6,2 1). Our previous studies have revealed that predegenerated peripheral nerve grafts facilitated the neurite outgrowth of damaged hippocampal neurons, whereas nonpredegenerated ones are, in this respect, less effective (11).
Hippocampus
The aim of the present work is to find whether the outgrowth of damaged central neurites is time dependent or not when the injured peripheral nerve has been used for grafting. METHOD Experiments were carried out on 25 adult male Wistar C rats. Animals were divided into five equal groups. During the whole experiment animals received standard diet and water ad lib. Surgery
The sciatic nerve in animals from the first experimental group (D7) was cut near the hip joint under intraperitoneal chloral hydrate anesthesia (420 mg per kg b.wt.). Seven days later, the animals were reanesthetised and a IO-mm long fragment of the distal stump of sciatic nerve was dissected. It was subsequently inserted into a glass cannula (1 mm internal and 2 mm external diameter) containing the Ringer’s solution for mammals. In the other groups, fragments of predegenerated sciatic nerves, prepared in the same way, were taken at the 14th, 28th, and 35th day after injury (groups D14, D28, D35, respectively). In the
’ To whom requests for reprints should be addressed.
831
832
control (C) group. peripheral nerve segment was dissected immediately after the transection and was, thus, nonpredegenerated. The peripheral nerve segments were implanted into the hippocampus according to the method described by Lewin-Kowalik at al. Prior to the implantation, a 3 mm deep brain tissue injury was made. The caudal tip of the grafts was laid over the skull bone, tied with 4-O surgical silk, and attached to the skull by means of Tissucoll Kit (Immuno AG Wien). Six weeks after surgery all animals were anaesthetized as described above. Ten ~1 ofOS% FITC-HRP (horseradish peroxidase conjugated with fluoresceine isothiocyanate) solution was injected into the free end of each graft. The FITC-HRP is retrogradely transported into the cell body. Twenty-four h later the rats were perfused transcardially with 200 ml 5% sucrose in phosphate buffer of pH 7.2 and, subsequently, with fixative solution of 2.4% formaldehyde buffered as before. tJislolo~id Prowdurc
ILEWIN-KQWALIK
F’I 41..
prepared. The slices (IO pm thick) were treated by hematoxylineosin (H-E) staining, examined in fluorescence (Univar ReichertJung) and light microscopes. and photographed. The H-E staining was used in order to verify the anatomical positions of the grafts (Fig. 1). Five animals were examined in each group. Labeled cells were present in the neighbourhood of the graft’s tips only. For this reason, we decided to take under consideration, out of the lot. only 10 sections derived from the above area. Labeled cells were counted in these slices in areas of 0.5 X 0.9 mm around the tip of the graft. This space contained practically all traced cells. To avoid double counting, cells were counted in every second section. Cells have been counted by means of a picture computer analyzer Quantimet 720 type 30 (Cambridge Instruments), and an HP 9825 A computer was used. The cells were indicated with the help of a light pen. The results were subjected to statistical analysis according to the Student’s I-test and nonparametric Wilcoxon test. Statistical significance was set at 17< 0.05. RESLJLl S
Whole grafted brains were dissected from the skull, and frontal frozen sections (Frigocut Mod. 2700, Reichert-Jung) were
Fluorescence microscope examination has showed that FITCHRP labeled cells were present among the hippocampal neurons in the examined brains derived from C, D7, D28, and D35 groups. However, the number of these cells was different in each group (Figs. 2. 3, Table 1). Horseradish peroxidase labeled neurons were found to be particularly numerous in slices derived from the brains treated with 7 days predegenerated distal stumps (Fig. 3, Table 1). Cell bodies containing the marker were regularly dispersed around the tip of the graft. The average number of labeled cells was 241 t I 13.2, and it was about six times higher than in slices obtained from brains treated with freshly transected nerves (Fig. 2). Statistical analysis has shown that such an increase was statistically significant (Table I). In the brain slices from the D 14 group no FITC-HRP labeled cells were found in the neighbourhood of the graft’s tip in three cases, but only single ones in two cases. In comparison to the control group, such a decrease was significant (Table 1). Horseradish peroxidase labeled cells appeared again in D28 and D35 slices. Their average number was 70.2 + 8.98 and 78.4 + 6.94, respectively (Table 1). These repeated increases in D28 as well as in D35 groups were statistically significant as compared to controls (Table I).
1
TABLE
THE NUMBER OF RTC-HRP LABELLED ESTABLISHED IN DIFFERENT EXPERIMENTAL GROUPS
Group
C
D7 D14 D28 D35 FIG. 1. Placement of the nonpredegenerated PN-graft (G) in the hippocampus (H) 6 weeks after grafting. Hematoxylin-eosin stained section. Bar = 800 Wm.
CELLS
Mean + SD
39.4 241.0 0.8 70.2 78.4
t 9.44 f 113.2 f I .09 t 8.98 f 6.94
N = 5 in each case. * Nonparametric Wilcoxon
t Student’s t-test.
test.
p < 0.005* p < 0.005 * p < 0.0008 t pi0.00008t
TIME-DEPENDENT
PREDEGENERATION
833
INFLUENCE
FIG. 2. Fluorescence microscope dark field picture of the control brain, Magnification 630X. Arrows indicate FITC-
HRP positive cells. Bar = 30 pm.
DISCUSSION
The mechanism of peripheral nerve grafts influence on central neurites regeneration is still an open question ( 19). TWO of the
The present study shows that the growth of host axons into the predegenerated peripheral nerve grafts depends on the time lapse after the sciatic nerve transection. This stimulative influence on hippocampal neurites regrowth was particularly effective in the 7-day predegenerated grafts, but existed in the 28- and 35 day predegenerated ones as well. It is worthwhile to note that Windebank and Poduslo (21) showed, in vitro, that permanently transected adult rat sciatic nerves exhibited in culture biphasic neurite-promoting activity. The first phase lasts 7 days , then decreases and reaches a minimum at the 14th day after the transection. The second phase of increased neurite promoting activity appears in the 28-, 35-, 4 l- and 5 1-day posttransection tissue. The early phase was partially inhibited by antisera to NGF. This does not apply to the late phase. Similar biphasic effects were observed in our experiments. The influence of predegenerated grafts implantation on injured hippocampal neurites was especially significant when the 7-, 28-. and 35-day old grafts were used, and practically disappeared after the grafting 1Cday old sciatic nerve segments. In a normal nerve, the rate of Schwann cell division is very low but increases rapidly after transection, reaching a maximum at 3-4 days and returning to baseline by 14 days. Schwann cells number reaches a maximum plateau at 14 days and then remains steady through at least 28 days posttransection (2 1). These cells became increasingly metabolically active in terms of secreting soluble proteins. Maximal secretory activity was reached on the 35th day (14). Parallel experiments carried out in our laboratory have revealed different time-dependent changes of protein contents and composition in the distal stumps of rat sciatic nerve following transection (16).
most prominent components of peripheral nerve trunks, the extracellular matrix (ECM) and the Schwann cells, have been implicated as the agents in promoting regeneration (3,4,9,10,20). The role of the Schwann cells seems to be particularly important and complex (2,4). These cells produce some extracellular matrix components, e.g., laminin, which have been found to stimulate axonal growth both in vivo and in vitro (5,7,13,15). The prevailing opinion is that in vivo regenerating axons need viable Schwann cells, and the presence of the basal lamina components alone is not sufficient (2,8). On the other hand, it is also suggested that the undestroyed biochemical integrity of acellular basal lamina provides favourable conditions for the elongation of regenerating sensory and motor axons ( 18). It is also possible that the Schwann cell surface molecules are essential for the support of some central neurites regrowth into a peripheral nerve environment ( 1, IO). Another possibility is that nonneuronal components of peripheral nerve grafts, first of all Schwann cells, can synthesize substances with neurotrophic activity (17,2 1). Ferguson at al. (6) revealed that the Schwann cells in the rat sciatic nerve in culture synthesise both NGF and non-NGF trophic factors. This neurotrophic activity of tissue culture medium begins within the first 24 h and is maintained throughout 6 days These observations in vitro agree with our results in vivo because 7-day predegenerated grafts seem to be most effective in stimulation of hippocampal neurites regrowth. Thus, on the basis of the above facts, we suppose that Schwann cells in the predegenerated peripheral nerve graft can synthesise neuronotrophic molecules, which seem to be different from the NGF factor. The intensity of this process changes with the lapse of time following the transection.
I.EWIN-KOWALIK
FIG. 3. The site of implantation of the ‘I-day predegenerated PN-graft 6 weeks after grafting. (a) Fluorescence microscope dark field picture. Numerous FITC-HRP labeled cells (arrows) and tibres (asterisks) are visualized in the brain tissue (H) around the tip of the graft (G). Bar = 100 Nrn. (b) Fragment of the above picture in a greater magnification. Bar = 30 Wm.Individual labeled cells are marked by arrows.
El
AI
TIME-DEPENDENT
PREDEGENERATION
835
INFLUENCE REFERENCES
1. Aebischer, P.; Salessiotis, A. N.; Winn, S. R. Basic fibroblast growth factor released from synthetic guidance channels facilitates peripheral nerve regeneration across long nerve gaps. J. Neurosci. Res. 23:282289; 1989. 2. Aguayo, A. J. Axonal regeneration from injured neurons in the adult mammalian central nervous system. In: Cotman, C. W., ed. Synaptic plasticity. chap. IS. New York: The Guilford Press; 1985:457-484. 3. Carbonetto. S.: &chard. F. In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system. J. Physiol. (Paris) 82:258-270; 1987. 4. Carey, D. J.; Todd, M. S. Schwann cell myelination in a chemically defined medium; demonstration of a requirement for additives that promote Schwann cell extracellular matrix formation. Dev. Brain Res. 32:95-102; 1987. 5. David, S. Neurite outgrowth from mammalian CNS neurons on astrocytes in vitro may be mediated primarily by laminin. J. Neurocytol. 17:131-144; 1988. 6. Ferguson, I. A.; Williams, R.; Rush, R. A. Chicken NGF and nonNGF trophic factor synthesis and release by sciatic nerves in vitro. J. Neurosci. Res. 22:408-417; 1989. 7. Giftochristos, N.; David, S. Laminin and heparan sulphate proteoglycan in the lesioned adult mammalian central nervous system and their possible relationship to axonal sprouting. J. Neurocytol. 17: 385-397; 1988. 8. Gulati, A. G. Evaulation of acellular and cellular nerve grafts in repair of rat peripheral nerve. J. Neurosurg. 68: 117- 123; 1988. 9. Kleitman, N.; Simon, D. K.; Schachner, M.; Bunge, R. P. Growth of embryonic neurites elicited by contact with Schwann cell surfaces is blocked by antibodies to Ll. Exp. Neurol. 102:298-306; 1988. 10. KIeitman, N.; Wood, P.; Johnson, M. I.; Bunge, R. P. Schwan cell surfaces but not the extracellular matrix organized by Schwann cells support neurite ougrowth from embryonic rat retina. J. Neurosci. 8(2):653-663; 1988.
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