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Regenerative sprouting of retinal ganglion cells of adult hamsters induced by the epineurium of a peripheral nerve1
Brain Research 823 Ž1999. 241–248
Interactive report
Regenerative sprouting of retinal ganglion cells of adult hamsters induced by the epineurium of a peripheral nerve 1 M.Y. Lai, E.Y.P. Cho
)
Department of Anatomy, The Chinese UniÕersity of Hong Kong, Shatin, New Territories, Hong Kong, China Accepted 21 January 1999
Abstract Although it is known that transplantation of a peripheral nerve ŽPN. to the damaged central nervous system ŽCNS. promotes axonal regeneration, the interactions of cellular components of the PN with CNS neurons are still not well defined. Schwann cells in the PN are thought to be the major element involved in supporting CNS regeneration, but very little information exists with regard to whether other PN components also play an active role. Using our previously established model of transplanting a PN segment into the vitreous to stimulate regenerative sprouting of retinal ganglion cells ŽRGCs., we found that the epineurium isolated from a PN which had been pre-injured by transection was able to induce RGC sprouting when implanted intravitreally. Since the epineurium is composed mainly of connective tissue components and is devoid of Schwann cells, our results suggest that other cellular elements of the PN besides Schwann cells may have the potential to support CNS regeneration. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Retinal ganglion cell; Regeneration; Sprouting; Peripheral nerve; Hamster
1. Introduction Previous studies have indicated that grafting of a peripheral nerve ŽPN. to the retina w34x or optic nerve w6,37x induces axonal regeneration of retinal ganglion cells ŽRGCs. into the PN. We have found that intravitreal transplantation of a short PN segment stimulates axotomized RGCs of adult hamsters to form regenerative axon-like processes in the retina w8,9x. In these and other studies, Schwann cells in the PN are believed to be the active component supporting regeneration of RGCs and other neurons of the central nervous system ŽCNS.. This is indicated by the fact that Schwann cells synthesise various neurotrophic factors w1,13,27x, and they have been shown to support axonal regeneration both in co-culture experiments with neurons w2x and when transplanted into the in vivo CNS w21,24,28x. In contrast, relatively little is known how other cellular elements of the PN affect CNS regeneration. In this study, we presented evidence which suggests that the epineurium enclosing the nerve fascicles also plays
a role in promoting RGC sprouting. In the course of our study of the effect of a prior injury of the PN in influencing the sprouting of axon-like processes from RGCs by the intravitreal grafting paradigm, we found that when a desheathed PN Žstripped of epineurium before being implanted into the vitreous. which had been conditioned by a cut injury 1 or 2 weeks prior to intravitreal grafting was used, the number of RGCs which exhibited sprouting was decreased compared to that induced by a normal desheathed PN. However, if the epineurium was not separated from the pre-injured PN and grafted together intravitreally, the number of sprouting RGCs observed was similar to that induced by the normal PN. We further demonstrate that RGC sprouting can be induced by transplanting the isolated epineurium intravitreally. Since the epineurium is devoid of Schwann cells, the results suggest that in addition to Schwann cells, cellular elements of the epineurium may also promote CNS regeneration.
2. Materials and methods ) Corresponding author. Tel: q852-2609-6842; Fax: q852-2603-5031; E-mail: [email protected] 1 Published on the World Wide Web on 16 February 1999.
Female adult Syrian golden hamsters Ž Mesocricetus auratus . 8–10 weeks old were used in all experiments.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 0 2 - 0
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M.Y. Lai, E.Y.P. Cho r Brain Research 823 (1999) 241–248
They were anaesthetised by i.p. injections of 10% chloral hydrate Ž0.4 mlr100 g body weight. in all surgical procedures. All the animals had their right optic nerves ŽONs. crushed intraorbitally at 2 mm behind the eye to axotomize the RGCs and were allocated to 1 of the 3 intravitreal grafting procedures ŽFig. 1.. 2.1. IntraÕitreal grafting 2.1.1. PN without epineurium (desheathed PN, dsPN) A 2 mm segment of common peroneal nerve was removed and the epineurium stripped off. The dsPN was implanted into the vitreous of the ON-crushed eye of the same animal as described previously w8x. In some animals, the PN was transected and allowed to degenerate for 1 or 2 weeks before 2 mm of the distal stump was desheathed and implanted. This group was referred to as pre-injured dsPN. In order to exclude any non-specific stimulus attributed to the grafting procedure w25x, control experiments using non-viable dsPN Žnormal or pre-injured. were per-
formed. The grafts were made non-viable by repetitive freezing and thawing in liquid nitrogen and at room temperature respectively Ž4 cycles. immediately before implantation. 2.1.2. PN with epineurium (PN–Ep) In this group, either a normal 2 mm segment of common peroneal nerve with its epineurium intact was implanted, or the nerve was transected 1 week before the PN together with its epineurium attached was grafted. The latter group was denoted as pre-injured PN–Ep. 2.1.3. Epineurium only (Ep) A 2 mm segment of epineurium was separated from the common peroneal nerve and implanted intravitreally, or the nerve was transected 1 week before the epineurium was removed and implanted Žpre-injured Ep.. In some animals, non-viable normal or pre-injured epineurium was implanted as controls.
Fig. 1. Schematic diagram illustrating the experimental paradigm of grafting different PN components into the vitreous body ŽV. of the hamster eye immediately after crushing the ON. In all 3 groups, the PN component grafted was either derived from a nerve which had not been manipulated Ždenoted as normal. or from one which had been transected 1 week Ždenoted as pre-injured. before grafting was performed. The box at upper left represents the first group in which the epineurium Žrepresented by a profile with fuzzy outline. was removed prior to grafting. Such a graft was known as a desheathed PN ŽdsPN.. In the box at middle left, the PN was grafted with the epineurium still attached to it ŽPN–Ep.. In the box at lower left, the graft consisted of only the epineurium which was dissected from the PN immediately prior to grafting ŽEp.. In all experiments, the graft was introduced via a small hole made at the superior limbus of the eye into the vitreous with the help of a glass micro-pipette.
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Some retinas from the dsPN experiments were stained with an anti-Growth Associated Protein ŽGAP-43. antibody. GAP-43 is a membrane-associated protein which is highly expressed during neuronal development and regeneration w5,17x, and is considered as a marker of regenerative propensity. In the presence of an intravitreal PN, it has been observed that more axotomized RGCs express GAP43 w30x. Thus, the extent of up-regulation of GAP-43 by RGCs after transplantation of either a normal or pre-injured dsPN was compared in order to assess the effect of pre-injury of the PN on stimulating RGC sprouting. Two weeks after intravitreal grafting, animals recruited for the GAP-43 study were perfused with phosphate-buffered saline ŽPBS., after which the retina of the grafted eye was dissected and fixed in 4% paraformaldehyde for 1 h. After washing in PBS and blocking in 20% foetal calf serum, the retina was incubated in a mouse anti-GAP-43 antibody ŽBoehringer, 1:1000 diluted in PBS containing 1% Triton and 0.5% bovine serum albumin. overnight at 48C. This was followed by overnight incubation in a HRP-conjugated anti-mouse secondary antibody ŽJackson Lab.. after which diaminobenzidine staining was used to reveal the GAP-43 positive RGCs. The variations in the mean number of RGCs stained by the silver method or anti-GAP-43 in each experimental group were analysed by one way ANOVA followed by a
Fig. 2. Graphs illustrating the number of axotomized RGCs which sprouted axon-like processes Žas revealed by silver staining, A. or which expressed GAP-43 ŽB. after implantation of either a normal or pre-injured desheathed PN ŽdsPN. into the vitreous. The number of retinas examined in each case is shown in parentheses. Both the number of silver-stained cells and GAP-43 positive cells were reduced as compared to that of a normal dsPN when a pre-injured dsPN was implanted. Implantation of non-viable dsPN Žnormal or pre-injured. induced very little sprouting ŽA..
2.2. Staining and analysis of sprouting RGCs induced by intraÕitreal grafting At 2 weeks after ON crush and intravitreal grafting, the animal was sacrificed by an overdose of chloral hydrate and the right eye enucleated directly into 2% paraformaldehyde Žin 0.1 M phosphate buffer pH 7.4.. The retina was dissected and fixed for 1 h before being processed with a silver staining method Ža modified protocol of Leicester and Stone w20x. to reveal those axotomized RGCs which had sprouted axon-like processes w8x. This method has been shown to reveal the detailed morphology of hamster RGCs which had undergone sprouting whereas non-sprouting RGCs were not stained w8x. The total number of sprouting RGCs in the whole-mount of each experimental animal was counted under the microscope and the mean numberŽ" standard error, SEM. for each group was calculated.
Fig. 3. A comparison between the number of RGCs induced to sprout by intravitreal implantation of a PN with epineurium intact ŽPN–Ep. and a desheathed PN ŽdsPN.. The decrease in sprouting observed after implantation of a pre-injured dsPN was abolished when the epineurium was included as part of the graft. The data from the dsPN group were the same as in Fig. 2A but is included here to facilitate comparison with the PN–Ep group. Number of retinas examined in each case are shown in parentheses.
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Fig. 4. Graph illustrating the effect of intravitreal implantation of an isolated epineurium on RGC sprouting. Epineurium from a PN which had been pre-injured induced RGC sprouting when compared to a normal epineurium. Both normal and pre-injured epineurium rendered non-viable before grafting were without effects. Numbers in parentheses refer to the retinas examined.
post-hoc pair-wise comparison using the Student-Newman–Keuls test, with the level of significance set at p s 0.05.
and B. compared to animals grafted with a normal dsPN Ž p - 0.05.. Intravitreal grafting of a non-viable normal or pre-injured dsPN led to only a small number of RGCs Ž29 " 10, n s 4 and 62 " 22, n s 4 respectively, p - 0.05 when compared to viable normal or pre-injured dsPN. being stained, indicating that induction of RGC sprouting required viable cellular elements in the dsPN and was not the result of a non-specific stimulus of the grafting procedure. The few sprouting RGCs seen after grafting of a non-viable nerve may be due to a small amount of trophic factor originally present in it. When the PN was transected and left for 14 days before the dsPN was being implanted, there was also a significant decrease in the number of sprouting RGCs Ž441 " 123, n s 5; Fig. 2A. compared to the normal dsPN group Ž p - 0.05., but the number of sprouting RGCs between the 7 and 14 days pre-injury dsPN groups were not significantly different Ž p ) 0.1.. The decrease in potential of the pre-injured dsPN to stimulate RGC sprouting was also suggested by the results of anti-GAP-43 staining ŽFig. 2B.. Whereas a mean of 998 " 113 Ž n s 5. GAP-43-positive RGCs was seen after normal dsPN implantation, only 441 " 65 Ž n s 5. and 375 " 111 Ž n s 5. cells were present in the 7 and 14 days pre-injured dsPN groups respectively. The difference in number of GAP-43-positive cells between the normal and both pre-injured groups were statistically significant Ž p 0.05. whereas that between the 7 and 14 days pre-injury groups was not Ž p ) 0.1.. 3.2. PN pre-injury did not reduce RGC sprouting if the PN was implanted with the epineurium intact (Figs. 3 and 5)
3. Results 3.1. A pre-injured desheathed PN induced fewer RGCs to sprout (Figs. 2 and 5) When a dsPN was transplanted intravitreally into an eye inflicted with a concurrent ON crush, silver staining of the whole-mount retina at 2 weeks post-ON crush revealed a total of 1143 " 79 Žmean " SEM, number of retinas Ž n. s 6. RGCs per retina. These silver-stained RGCs had previously been shown to sprout axon-like processes from the somato-dendritic and intra-retinal axonal compartments, presumably under the influence of diffusible trophic factors secreted by the PN w8x. Prior transection of the PN 7 days before implanting the distal stump of the pre-injured dsPN resulted in the presence of fewer silver-stained sprouting RGCs Ž604 " 107, n s 8; see Fig. 2A; Fig. 5A
If the normal uninjured PN was implanted intravitreally with the epineurium intact Žnormal PN–Ep group., the mean number of sprouting RGCs as revealed by silver staining was 1580 " 263 Ž n s 4. which was not significantly different Ž p ) 0.1. from the normal dsPN group. However, when the PN was pre-injured 7 days before the distal stump with epineurium intact was implanted Žpre-injured PN–Ep group., the mean number of 1259 " 138 Ž n s 4. sprouting RGCs induced was significantly higher than the pre-injured dsPN group Ž p - 0.05., but was not appreciably different from the normal PN–Ep group Ž p ) 0.1; Fig. 3; Fig. 5C and D.. Thus, inclusion of the epineurium as part of the graft compensated for the effect of the decrease in sprouting of RGCs due to the pre-injury, leading to similar numbers of sprouting RGCs between the normal and pre-injured PN–Ep groups. However, the stim-
Fig. 5. A–F. Photomicrographs of retinal whole-mounts treated by silver staining to illustrate the differences in the number of sprouting RGCs observed after intravitreal implantation of various PN components. The region shown in all the cases corresponds to the superior nasal quadrant, with the optic disc ŽOD. on the left and the periphery of the retina on the right side of the picture. Examples of silver-stained sprouting RGCs are indicated by arrows. Normal and pre-injured dsPN: 5A and B, respectively; normal and pre-injured PN–Ep: 5C and D; normal and pre-injured Ep: 5E and F. An example of a RGC induced to sprout axon-like processes Žarrowheads. by a pre-injured epineurium is shown in 5G. The scale bar in F also applies from A to G.
M.Y. Lai, E.Y.P. Cho r Brain Research 823 (1999) 241–248
ulatory effect of the epineurium was only observed after pre-injury, since its inclusion in the normal PN did not
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result in an increase in number of sprouting RGCs over that induced by a normal dsPN graft.
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3.3. An isolated pre-injured epineurium induced RGC sprouting (Figs. 4 and 5) In order to ascertain whether the compensatory effect of the epineurium was due to its ability to promote sprouting, the epineurium was isolated from either the normal PN ŽEp group. or the 7 days pre-injured PN Žpre-injured Ep group. and transplanted intravitreally. Implantation of a normal epineurium resulted in the staining of a mean of 119 " 31 RGCs, but although this number was higher than that of animals receiving a non-viable epineurium Žmean s 37 " 17 cells, Fig. 4., the difference did not reach statistical significance Ž p s 0.06.. However, when the epineurium was isolated from a pre-injured PN and implanted, the number of sprouting RGCs was increased by almost 4-fold Ž432 " 53; p - 0.05 when compared to the normal epineurium. ŽFig. 4, Fig. 5E and F.. The effect was abolished if the pre-injured epineurium was rendered nonviable before intravitreal grafting Ž68 " 24 cells, p - 0.05 compared to the viable pre-injured epineurium. ŽFig. 4.. The RGCs induced to sprout by an intravitreal pre-injured epineurium had similar morphologies compared to RGCs which generate axon-like processes under the influence of a desheathed PN w8x: long un-branched processes which looped and extended in different retinal layers were seen to arise from the dendritic tree ŽFig. 5G..
4. Discussion Using the intravitreal grafting paradigm, we show that in addition to Schwann cells, the epineurium of the PN can also induce regenerative sprouting of axotomized RGCs. This finding was first suggested by the observation that whereas a pre-injured dsPN grafted intravitreally exhibited a reduced potency to stimulate RGC sprouting compared to the normal dsPN, as indicated by the number of silverstained sprouting RGCs as well as the number of GAP-43 positive RGCs Žwhich reflects regenerative potential., the inclusion of the epineurium in the graft leads to similar numbers of silver-stained sprouting RGCs between the normal and the pre-injured nerve. The result that a pre-injured dsPN leads to a decrease rather than an increase in the number of sprouting RGCs is rather unexpected, given that in previous studies a PN conditioned by a prior injury was found to be a better substrate for axonal regeneration of both peripheral w15x and CNS axons w3,31x. It is known that after injury of a PN, Schwann cells become ‘activated’ with respect to the increased production of extracellular matrix components, cell adhesion molecules and growth factors like NGF and BDNF w13,16,26,27x, all of which may contribute to the ability of Schwann cells to support axonal regeneration w2x. On the other hand, however, it has been reported that transection of the sciatic nerve leads to a decreased synthesis of ciliary neurotrophic factor ŽCNTF. by Schwann cells in the distal degenerating stump of the
nerve w12,33x. This down regulation of CNTF in the pre-injured PN may provide an explanation as to why a decrease in the number of sprouting RGCs occurs after intravitreal grafting of a pre-injured dsPN, if CNTF Žor other unidentified factors whose expression become down-regulated after injury. is the agent responsible for induction of axon-like processes from RGCs. Our situation may be analogous to that of a previous study of Richardson and Ebendal w32x in which neurite outgrowth from ciliary ganglion neurons was reduced when they were incubated with conditioned medium Žwhich contained CNTF-like activity. derived from a pre-injured PN. Recently, it has been reported that CNTF enhances axonal regeneration of RGCs into a PN graft when they were axotomized at a long distance from the cell body w10x, thus highlighting the important role CNTF may play in the induction of RGC regenerative sprouting. When the epineurium is included in the pre-injured PN graft Žinjured PN–Ep group., more RGCs exhibit sprouting compared to the pre-injured dsPN, whereas inclusion of the epineurium in the normal PN graft Žnormal PN–Ep. does not lead to more RGCs to sprout compared to the normal dsPN. Thus, changes which facilitate sprouting occur in the epineurium after PN injury and, in this regard, it is different from the pattern induced by Schwann cells in the dsPN in which sprouting becomes reduced after a pre-injury. There are at least 2 mechanisms by which the pre-injured epineurium may influence sprouting. The preinjured epineurium may itself be able to stimulate sprouting, or it has to interact synergistically with components of the desheathed PN to induce sprouting. Evidence for the former mechanism is provided by the experiment of transplanting an isolated epineurium intravitreally, the results of which indicate that only epineurium obtained from a preinjured PN is capable of stimulating sprouting of RGCs. Moreover, the number of RGCs induced to sprout in the isolated pre-injured epineurium and the pre-injured dsPN experiments added together is similar to that as stimulated by the PN with epineurium intact group, suggesting that there is no synergistic effect when the dsPN and the epineurium are transplanted together. The changes which occur in the epineurium after injury of the PN and which would be responsible for stimulating RGC sprouting are not clear at present. The components normally present in the epineurium include connective tissue fibres, especially collagen, as well as fibroblasts and small blood vessels w36x. Injury to the PN results in an increase in the number of fibroblasts as well as collagen w29x, while cells in the epineurium of PNs which have been exposed to mechanical vibrations express insulin-like growth factor I w14x. Thus, it is plausible that activation of epineurial fibroblasts may lead to the production of growth factors which induce RGC sprouting. Another potential stimulant is the macrophage of which great numbers could be seen in the epineurium shortly after PN injury w35x. The roles played by macrophages in the regeneration of the PN are multiple, of which the removal of myelin debris w7x,
M.Y. Lai, E.Y.P. Cho r Brain Research 823 (1999) 241–248
and induction of cell division of w4x and synthesis of growth factors w22x by non-neuronal cells of the PN are especially important. Recently, they have also been shown to enhance regeneration in both the peripheral nervous system as well as the CNS when they were experimentally activated or introduced into lesion sites w11,19,23x. Schwartz and colleagues w18x show that blood-borne monocytes which have been pre-activated by PNs become a favourable agent for stimulating axonal regrowth of RGCs when subsequently transplanted to the ON lesion site. This observation may have a relevant bearing to our results, in that a scenario can be envisioned in which the macrophages that have accumulated in the epineurium after PN transection are pre-conditioned by other cellular elements of the PN Že.g. Schwann cells. so that they act as a stimulant for RGC sprouting when they are subsequently implanted intravitreally. Notwithstanding the speculation as to the cellular mechanisms involved, our results indicate that in addition to Schwann cells, the epineurium is another potential candidate that can promote CNS regeneration. Further studies aimed at comparing its action with that of Schwann cells on other neuronal systems may provide clues as to how it influences CNS regeneration.
Acknowledgements This study was supported by a Direct Grant for Research from the Chinese University of Hong Kong.
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Interactive report
Regenerative sprouting of retinal ganglion cells of adult hamsters induced by the epineurium of a peripheral nerve 1 M.Y. Lai, E.Y.P. Cho
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Department of Anatomy, The Chinese UniÕersity of Hong Kong, Shatin, New Territories, Hong Kong, China Accepted 21 January 1999
Abstract Although it is known that transplantation of a peripheral nerve ŽPN. to the damaged central nervous system ŽCNS. promotes axonal regeneration, the interactions of cellular components of the PN with CNS neurons are still not well defined. Schwann cells in the PN are thought to be the major element involved in supporting CNS regeneration, but very little information exists with regard to whether other PN components also play an active role. Using our previously established model of transplanting a PN segment into the vitreous to stimulate regenerative sprouting of retinal ganglion cells ŽRGCs., we found that the epineurium isolated from a PN which had been pre-injured by transection was able to induce RGC sprouting when implanted intravitreally. Since the epineurium is composed mainly of connective tissue components and is devoid of Schwann cells, our results suggest that other cellular elements of the PN besides Schwann cells may have the potential to support CNS regeneration. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Retinal ganglion cell; Regeneration; Sprouting; Peripheral nerve; Hamster
1. Introduction Previous studies have indicated that grafting of a peripheral nerve ŽPN. to the retina w34x or optic nerve w6,37x induces axonal regeneration of retinal ganglion cells ŽRGCs. into the PN. We have found that intravitreal transplantation of a short PN segment stimulates axotomized RGCs of adult hamsters to form regenerative axon-like processes in the retina w8,9x. In these and other studies, Schwann cells in the PN are believed to be the active component supporting regeneration of RGCs and other neurons of the central nervous system ŽCNS.. This is indicated by the fact that Schwann cells synthesise various neurotrophic factors w1,13,27x, and they have been shown to support axonal regeneration both in co-culture experiments with neurons w2x and when transplanted into the in vivo CNS w21,24,28x. In contrast, relatively little is known how other cellular elements of the PN affect CNS regeneration. In this study, we presented evidence which suggests that the epineurium enclosing the nerve fascicles also plays
a role in promoting RGC sprouting. In the course of our study of the effect of a prior injury of the PN in influencing the sprouting of axon-like processes from RGCs by the intravitreal grafting paradigm, we found that when a desheathed PN Žstripped of epineurium before being implanted into the vitreous. which had been conditioned by a cut injury 1 or 2 weeks prior to intravitreal grafting was used, the number of RGCs which exhibited sprouting was decreased compared to that induced by a normal desheathed PN. However, if the epineurium was not separated from the pre-injured PN and grafted together intravitreally, the number of sprouting RGCs observed was similar to that induced by the normal PN. We further demonstrate that RGC sprouting can be induced by transplanting the isolated epineurium intravitreally. Since the epineurium is devoid of Schwann cells, the results suggest that in addition to Schwann cells, cellular elements of the epineurium may also promote CNS regeneration.
2. Materials and methods ) Corresponding author. Tel: q852-2609-6842; Fax: q852-2603-5031; E-mail: [email protected] 1 Published on the World Wide Web on 16 February 1999.
Female adult Syrian golden hamsters Ž Mesocricetus auratus . 8–10 weeks old were used in all experiments.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 2 0 2 - 0
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They were anaesthetised by i.p. injections of 10% chloral hydrate Ž0.4 mlr100 g body weight. in all surgical procedures. All the animals had their right optic nerves ŽONs. crushed intraorbitally at 2 mm behind the eye to axotomize the RGCs and were allocated to 1 of the 3 intravitreal grafting procedures ŽFig. 1.. 2.1. IntraÕitreal grafting 2.1.1. PN without epineurium (desheathed PN, dsPN) A 2 mm segment of common peroneal nerve was removed and the epineurium stripped off. The dsPN was implanted into the vitreous of the ON-crushed eye of the same animal as described previously w8x. In some animals, the PN was transected and allowed to degenerate for 1 or 2 weeks before 2 mm of the distal stump was desheathed and implanted. This group was referred to as pre-injured dsPN. In order to exclude any non-specific stimulus attributed to the grafting procedure w25x, control experiments using non-viable dsPN Žnormal or pre-injured. were per-
formed. The grafts were made non-viable by repetitive freezing and thawing in liquid nitrogen and at room temperature respectively Ž4 cycles. immediately before implantation. 2.1.2. PN with epineurium (PN–Ep) In this group, either a normal 2 mm segment of common peroneal nerve with its epineurium intact was implanted, or the nerve was transected 1 week before the PN together with its epineurium attached was grafted. The latter group was denoted as pre-injured PN–Ep. 2.1.3. Epineurium only (Ep) A 2 mm segment of epineurium was separated from the common peroneal nerve and implanted intravitreally, or the nerve was transected 1 week before the epineurium was removed and implanted Žpre-injured Ep.. In some animals, non-viable normal or pre-injured epineurium was implanted as controls.
Fig. 1. Schematic diagram illustrating the experimental paradigm of grafting different PN components into the vitreous body ŽV. of the hamster eye immediately after crushing the ON. In all 3 groups, the PN component grafted was either derived from a nerve which had not been manipulated Ždenoted as normal. or from one which had been transected 1 week Ždenoted as pre-injured. before grafting was performed. The box at upper left represents the first group in which the epineurium Žrepresented by a profile with fuzzy outline. was removed prior to grafting. Such a graft was known as a desheathed PN ŽdsPN.. In the box at middle left, the PN was grafted with the epineurium still attached to it ŽPN–Ep.. In the box at lower left, the graft consisted of only the epineurium which was dissected from the PN immediately prior to grafting ŽEp.. In all experiments, the graft was introduced via a small hole made at the superior limbus of the eye into the vitreous with the help of a glass micro-pipette.
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Some retinas from the dsPN experiments were stained with an anti-Growth Associated Protein ŽGAP-43. antibody. GAP-43 is a membrane-associated protein which is highly expressed during neuronal development and regeneration w5,17x, and is considered as a marker of regenerative propensity. In the presence of an intravitreal PN, it has been observed that more axotomized RGCs express GAP43 w30x. Thus, the extent of up-regulation of GAP-43 by RGCs after transplantation of either a normal or pre-injured dsPN was compared in order to assess the effect of pre-injury of the PN on stimulating RGC sprouting. Two weeks after intravitreal grafting, animals recruited for the GAP-43 study were perfused with phosphate-buffered saline ŽPBS., after which the retina of the grafted eye was dissected and fixed in 4% paraformaldehyde for 1 h. After washing in PBS and blocking in 20% foetal calf serum, the retina was incubated in a mouse anti-GAP-43 antibody ŽBoehringer, 1:1000 diluted in PBS containing 1% Triton and 0.5% bovine serum albumin. overnight at 48C. This was followed by overnight incubation in a HRP-conjugated anti-mouse secondary antibody ŽJackson Lab.. after which diaminobenzidine staining was used to reveal the GAP-43 positive RGCs. The variations in the mean number of RGCs stained by the silver method or anti-GAP-43 in each experimental group were analysed by one way ANOVA followed by a
Fig. 2. Graphs illustrating the number of axotomized RGCs which sprouted axon-like processes Žas revealed by silver staining, A. or which expressed GAP-43 ŽB. after implantation of either a normal or pre-injured desheathed PN ŽdsPN. into the vitreous. The number of retinas examined in each case is shown in parentheses. Both the number of silver-stained cells and GAP-43 positive cells were reduced as compared to that of a normal dsPN when a pre-injured dsPN was implanted. Implantation of non-viable dsPN Žnormal or pre-injured. induced very little sprouting ŽA..
2.2. Staining and analysis of sprouting RGCs induced by intraÕitreal grafting At 2 weeks after ON crush and intravitreal grafting, the animal was sacrificed by an overdose of chloral hydrate and the right eye enucleated directly into 2% paraformaldehyde Žin 0.1 M phosphate buffer pH 7.4.. The retina was dissected and fixed for 1 h before being processed with a silver staining method Ža modified protocol of Leicester and Stone w20x. to reveal those axotomized RGCs which had sprouted axon-like processes w8x. This method has been shown to reveal the detailed morphology of hamster RGCs which had undergone sprouting whereas non-sprouting RGCs were not stained w8x. The total number of sprouting RGCs in the whole-mount of each experimental animal was counted under the microscope and the mean numberŽ" standard error, SEM. for each group was calculated.
Fig. 3. A comparison between the number of RGCs induced to sprout by intravitreal implantation of a PN with epineurium intact ŽPN–Ep. and a desheathed PN ŽdsPN.. The decrease in sprouting observed after implantation of a pre-injured dsPN was abolished when the epineurium was included as part of the graft. The data from the dsPN group were the same as in Fig. 2A but is included here to facilitate comparison with the PN–Ep group. Number of retinas examined in each case are shown in parentheses.
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Fig. 4. Graph illustrating the effect of intravitreal implantation of an isolated epineurium on RGC sprouting. Epineurium from a PN which had been pre-injured induced RGC sprouting when compared to a normal epineurium. Both normal and pre-injured epineurium rendered non-viable before grafting were without effects. Numbers in parentheses refer to the retinas examined.
post-hoc pair-wise comparison using the Student-Newman–Keuls test, with the level of significance set at p s 0.05.
and B. compared to animals grafted with a normal dsPN Ž p - 0.05.. Intravitreal grafting of a non-viable normal or pre-injured dsPN led to only a small number of RGCs Ž29 " 10, n s 4 and 62 " 22, n s 4 respectively, p - 0.05 when compared to viable normal or pre-injured dsPN. being stained, indicating that induction of RGC sprouting required viable cellular elements in the dsPN and was not the result of a non-specific stimulus of the grafting procedure. The few sprouting RGCs seen after grafting of a non-viable nerve may be due to a small amount of trophic factor originally present in it. When the PN was transected and left for 14 days before the dsPN was being implanted, there was also a significant decrease in the number of sprouting RGCs Ž441 " 123, n s 5; Fig. 2A. compared to the normal dsPN group Ž p - 0.05., but the number of sprouting RGCs between the 7 and 14 days pre-injury dsPN groups were not significantly different Ž p ) 0.1.. The decrease in potential of the pre-injured dsPN to stimulate RGC sprouting was also suggested by the results of anti-GAP-43 staining ŽFig. 2B.. Whereas a mean of 998 " 113 Ž n s 5. GAP-43-positive RGCs was seen after normal dsPN implantation, only 441 " 65 Ž n s 5. and 375 " 111 Ž n s 5. cells were present in the 7 and 14 days pre-injured dsPN groups respectively. The difference in number of GAP-43-positive cells between the normal and both pre-injured groups were statistically significant Ž p 0.05. whereas that between the 7 and 14 days pre-injury groups was not Ž p ) 0.1.. 3.2. PN pre-injury did not reduce RGC sprouting if the PN was implanted with the epineurium intact (Figs. 3 and 5)
3. Results 3.1. A pre-injured desheathed PN induced fewer RGCs to sprout (Figs. 2 and 5) When a dsPN was transplanted intravitreally into an eye inflicted with a concurrent ON crush, silver staining of the whole-mount retina at 2 weeks post-ON crush revealed a total of 1143 " 79 Žmean " SEM, number of retinas Ž n. s 6. RGCs per retina. These silver-stained RGCs had previously been shown to sprout axon-like processes from the somato-dendritic and intra-retinal axonal compartments, presumably under the influence of diffusible trophic factors secreted by the PN w8x. Prior transection of the PN 7 days before implanting the distal stump of the pre-injured dsPN resulted in the presence of fewer silver-stained sprouting RGCs Ž604 " 107, n s 8; see Fig. 2A; Fig. 5A
If the normal uninjured PN was implanted intravitreally with the epineurium intact Žnormal PN–Ep group., the mean number of sprouting RGCs as revealed by silver staining was 1580 " 263 Ž n s 4. which was not significantly different Ž p ) 0.1. from the normal dsPN group. However, when the PN was pre-injured 7 days before the distal stump with epineurium intact was implanted Žpre-injured PN–Ep group., the mean number of 1259 " 138 Ž n s 4. sprouting RGCs induced was significantly higher than the pre-injured dsPN group Ž p - 0.05., but was not appreciably different from the normal PN–Ep group Ž p ) 0.1; Fig. 3; Fig. 5C and D.. Thus, inclusion of the epineurium as part of the graft compensated for the effect of the decrease in sprouting of RGCs due to the pre-injury, leading to similar numbers of sprouting RGCs between the normal and pre-injured PN–Ep groups. However, the stim-
Fig. 5. A–F. Photomicrographs of retinal whole-mounts treated by silver staining to illustrate the differences in the number of sprouting RGCs observed after intravitreal implantation of various PN components. The region shown in all the cases corresponds to the superior nasal quadrant, with the optic disc ŽOD. on the left and the periphery of the retina on the right side of the picture. Examples of silver-stained sprouting RGCs are indicated by arrows. Normal and pre-injured dsPN: 5A and B, respectively; normal and pre-injured PN–Ep: 5C and D; normal and pre-injured Ep: 5E and F. An example of a RGC induced to sprout axon-like processes Žarrowheads. by a pre-injured epineurium is shown in 5G. The scale bar in F also applies from A to G.
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ulatory effect of the epineurium was only observed after pre-injury, since its inclusion in the normal PN did not
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result in an increase in number of sprouting RGCs over that induced by a normal dsPN graft.
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3.3. An isolated pre-injured epineurium induced RGC sprouting (Figs. 4 and 5) In order to ascertain whether the compensatory effect of the epineurium was due to its ability to promote sprouting, the epineurium was isolated from either the normal PN ŽEp group. or the 7 days pre-injured PN Žpre-injured Ep group. and transplanted intravitreally. Implantation of a normal epineurium resulted in the staining of a mean of 119 " 31 RGCs, but although this number was higher than that of animals receiving a non-viable epineurium Žmean s 37 " 17 cells, Fig. 4., the difference did not reach statistical significance Ž p s 0.06.. However, when the epineurium was isolated from a pre-injured PN and implanted, the number of sprouting RGCs was increased by almost 4-fold Ž432 " 53; p - 0.05 when compared to the normal epineurium. ŽFig. 4, Fig. 5E and F.. The effect was abolished if the pre-injured epineurium was rendered nonviable before intravitreal grafting Ž68 " 24 cells, p - 0.05 compared to the viable pre-injured epineurium. ŽFig. 4.. The RGCs induced to sprout by an intravitreal pre-injured epineurium had similar morphologies compared to RGCs which generate axon-like processes under the influence of a desheathed PN w8x: long un-branched processes which looped and extended in different retinal layers were seen to arise from the dendritic tree ŽFig. 5G..
4. Discussion Using the intravitreal grafting paradigm, we show that in addition to Schwann cells, the epineurium of the PN can also induce regenerative sprouting of axotomized RGCs. This finding was first suggested by the observation that whereas a pre-injured dsPN grafted intravitreally exhibited a reduced potency to stimulate RGC sprouting compared to the normal dsPN, as indicated by the number of silverstained sprouting RGCs as well as the number of GAP-43 positive RGCs Žwhich reflects regenerative potential., the inclusion of the epineurium in the graft leads to similar numbers of silver-stained sprouting RGCs between the normal and the pre-injured nerve. The result that a pre-injured dsPN leads to a decrease rather than an increase in the number of sprouting RGCs is rather unexpected, given that in previous studies a PN conditioned by a prior injury was found to be a better substrate for axonal regeneration of both peripheral w15x and CNS axons w3,31x. It is known that after injury of a PN, Schwann cells become ‘activated’ with respect to the increased production of extracellular matrix components, cell adhesion molecules and growth factors like NGF and BDNF w13,16,26,27x, all of which may contribute to the ability of Schwann cells to support axonal regeneration w2x. On the other hand, however, it has been reported that transection of the sciatic nerve leads to a decreased synthesis of ciliary neurotrophic factor ŽCNTF. by Schwann cells in the distal degenerating stump of the
nerve w12,33x. This down regulation of CNTF in the pre-injured PN may provide an explanation as to why a decrease in the number of sprouting RGCs occurs after intravitreal grafting of a pre-injured dsPN, if CNTF Žor other unidentified factors whose expression become down-regulated after injury. is the agent responsible for induction of axon-like processes from RGCs. Our situation may be analogous to that of a previous study of Richardson and Ebendal w32x in which neurite outgrowth from ciliary ganglion neurons was reduced when they were incubated with conditioned medium Žwhich contained CNTF-like activity. derived from a pre-injured PN. Recently, it has been reported that CNTF enhances axonal regeneration of RGCs into a PN graft when they were axotomized at a long distance from the cell body w10x, thus highlighting the important role CNTF may play in the induction of RGC regenerative sprouting. When the epineurium is included in the pre-injured PN graft Žinjured PN–Ep group., more RGCs exhibit sprouting compared to the pre-injured dsPN, whereas inclusion of the epineurium in the normal PN graft Žnormal PN–Ep. does not lead to more RGCs to sprout compared to the normal dsPN. Thus, changes which facilitate sprouting occur in the epineurium after PN injury and, in this regard, it is different from the pattern induced by Schwann cells in the dsPN in which sprouting becomes reduced after a pre-injury. There are at least 2 mechanisms by which the pre-injured epineurium may influence sprouting. The preinjured epineurium may itself be able to stimulate sprouting, or it has to interact synergistically with components of the desheathed PN to induce sprouting. Evidence for the former mechanism is provided by the experiment of transplanting an isolated epineurium intravitreally, the results of which indicate that only epineurium obtained from a preinjured PN is capable of stimulating sprouting of RGCs. Moreover, the number of RGCs induced to sprout in the isolated pre-injured epineurium and the pre-injured dsPN experiments added together is similar to that as stimulated by the PN with epineurium intact group, suggesting that there is no synergistic effect when the dsPN and the epineurium are transplanted together. The changes which occur in the epineurium after injury of the PN and which would be responsible for stimulating RGC sprouting are not clear at present. The components normally present in the epineurium include connective tissue fibres, especially collagen, as well as fibroblasts and small blood vessels w36x. Injury to the PN results in an increase in the number of fibroblasts as well as collagen w29x, while cells in the epineurium of PNs which have been exposed to mechanical vibrations express insulin-like growth factor I w14x. Thus, it is plausible that activation of epineurial fibroblasts may lead to the production of growth factors which induce RGC sprouting. Another potential stimulant is the macrophage of which great numbers could be seen in the epineurium shortly after PN injury w35x. The roles played by macrophages in the regeneration of the PN are multiple, of which the removal of myelin debris w7x,
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and induction of cell division of w4x and synthesis of growth factors w22x by non-neuronal cells of the PN are especially important. Recently, they have also been shown to enhance regeneration in both the peripheral nervous system as well as the CNS when they were experimentally activated or introduced into lesion sites w11,19,23x. Schwartz and colleagues w18x show that blood-borne monocytes which have been pre-activated by PNs become a favourable agent for stimulating axonal regrowth of RGCs when subsequently transplanted to the ON lesion site. This observation may have a relevant bearing to our results, in that a scenario can be envisioned in which the macrophages that have accumulated in the epineurium after PN transection are pre-conditioned by other cellular elements of the PN Že.g. Schwann cells. so that they act as a stimulant for RGC sprouting when they are subsequently implanted intravitreally. Notwithstanding the speculation as to the cellular mechanisms involved, our results indicate that in addition to Schwann cells, the epineurium is another potential candidate that can promote CNS regeneration. Further studies aimed at comparing its action with that of Schwann cells on other neuronal systems may provide clues as to how it influences CNS regeneration.
Acknowledgements This study was supported by a Direct Grant for Research from the Chinese University of Hong Kong.
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