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Horseradish peroxidase labeling of growth cones and axons beyond the site of injury in injured rabbit optic nerve axons growing in their own environment
Brain Research, 575 (1992) 1-5 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 17484
1
Research Reports
Horseradish peroxidase labeling of growth cones and axons beyond the site of injury in injured rabbit optic nerve axons growing in their own environment V. Lavie a, A. Solomon b, S. Ben-Bassat b, M. Belkin b and M. Schwartz a aDepartment of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel) and bM. and G. Goldschleger Eye Institute, Sackler School of Medicine, Tel Aviv University, Tel Hashomer (Israel)
(Accepted 15 October 1991) Key words: Growth cone; Optic nerve; Axonal injury
Spontaneous growth of injured axons in the mammalian central nervous system is limited. We have previously shown an apparently regenerative growth of injured optic axons in the adult rabbit, achieved by supplying them with soluble substances originating from growing axons, followed by low energy helium-neon laser irradiation. The growing unmyelinated and thinly myelinated axons were embedded in astrocytes, and some were in the process of remyelination by oligodendrocytes. They were shown to have originated from the retinal ganglion cells. The present study further supports evidence relating to the origin and nature of these axons. Light microscopic analysis of these axons labeled with anterogradely transported horseradish peroxidase revealed that many of these axons have varicosities and bear growth cone-like swellings in their tips. These axons traverse the lesion site and extend into the distal stump in a disorganized pattern. INTRODUCTION Injury to the mammalian optic nerve, a model for the central nervous system (CNS), leads to death of most of the axotomized neurons and a failure of the surviving cells to regrow their axons 8'13. The ability of the mammalian retinal ganglion cells to survive and regenerate their axons after axotomy can, however, be markedly enhanced by modifying the neuronal environment 1'7'9-u'18. Several effectors are likely to be involved in encouraging regeneration of mammalian central neurons. Among them are factors that can enhance neuronal survival and support axonal elongation, and those that can modify the environment, making it more conducive to growth. We have recently shown that treatment of injured adult rabbit optic nerves with medium conditioned by regenerating fish optic nerves (CM), followed by repeated low energy helium-neon (He-Ne) laser irradiation, alters the response of the optic nerve to injury. The CM was shown by us to contain factors affecting astrocytes 4 and oligodendrocytes 5, whereas irradiation with low energy He-Ne laser was effective in slowing down degeneration 2. We have recently observed that the slowing down of degeneration is associated with the presence at the site of injury of astrocytes, whose disappearance we have proposed as a reason for the postinjury growth
arrest (unpublished observations). The growth obtained as a result of the combined treatment was manifested morphologically by the growth of axons within their own, otherwise hostile environment. The growth was shown by electron microscopy to be manifested by the presence of unmyelinated and thinly myelinated axons associated with glial cells. These axons were traversing the site of injury and extending up to 6 m m distal to it. Many or most of these axons represent regenerative sprouts and, indeed, they closely resemble regenerative optic axons described in lower vertebrates 14. For the following reasons our results suggest that the unmyelinated and thinly myelinated axons found distal to the site of the lesion, in association with glial cells, are regenerating axons and not axons spared by the lesion: (i) in transected nerves treated with CM and exposed to irradiation, unmyelinated and thinly myelinated axons, grouped together in a compartment, could be seen only up to a certain distance from the lesion. (ii) In the operated control group, no viable (i.e., unmyelinated or thinly myelinated) axons or growth cones could be detected distal to the lesion site from 6 weeks postoperation onward. We have previously shown that the growing axons originate from the rabbit retinal ganglion cells, since wheat-germ agglutinin ( W G A ) conjugated with horse-
Correspondence: M. Schwartz, Dept. of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
r a d i s h p e r o x i d a s e ( H R P ) i n j e c t e d i n t r a o c u l a r l y was f o u n d to l a b e l t h e g r o w i n g a x o n s 12. T h i s k i n d o f l a b e l i n g failed, however,
to d e m o n s t r a t e ,
at t h e e l e c t r o n m i c r o s c o p e
level, t h e n a t u r e o f g r o w t h f a c i l i t a t e d b y t h e t r e a t m e n t . In the present study we substantiate our previous observations by a complete follow-up of anterograde transport of H R P in t r e a t e d t r a n s e c t e d a d u l t r a b b i t o p t i c n e r v e s .
previously described 16 and the animals returned to their tanks. Eight days later the fish were reanesthetized and the crushed regenerating nerves were dissected out, transferred into serum free medium (DMEM, Gibco) and incubated for 1 h at room temperature. The conditioned medium was then collected and stored at -20°C 16. The protein content of the medium was determined by the Bradford assay 3. The conditioned medium was diluted to 100 #g protein/ml. From this stock solution, 5-10 ~1 were used for each piece of nitrocellulose implanted into an injured nerve 2t22.
Irradiation MATERIALS AND METHODS
Rabbit surgery Adult rabbits (albino, Weizmann Institute Animal House) were deeply anesthetized with xylazine (5 mg/kg) and ketamine (35 mg/ kg). The left optic nerves of 21 rabbits (6 control, 15 combined treatment) were exposed, as previously described 21. The optic nerves were visualized with the aid of a Zeiss operating microscope. At a distance of 5-6 mm from the eyeball, the nerves were transected almost completely using a sharpened dissecting needle2L The meningeal membrane was partially spared intentionally, to ensure continuity of the nerve and to permit placement and retention of a nitrocellulose film, described below. In some cases, this resulted in sparing axons immediately subjacent to the meningeal covering. In the operated control group, nitrocellulose was soaked in serum free medium (DMEM, Gibeo) for 1 h and inserted into the lesion site. In the experimental (combined treatment) group, the nitrocellulose was soaked in CM (100 ~g protein/ml) for 1 h before being inserted into the lesion site.
Preparation of conditioned media Carp (Cyprinus carpio; 800-1200 g) were purchased from Tnuva Israel. The fish were deeply anesthetized with 0.05% tricaine methansulfonate (Sigma). Optic nerves were crushed intraorbitally as
A He-Ne laser (Spectrophysics Instruments) was used to irradiate the nerve through the eye, as described previously 2'~7. The laser beam was directed through the pupil at the optic nerve. Beginning 30 rain after surgery, the rabbits were exposed for 5 min each day for 10 consecutive days to low energy (632.8 mm, 35 mW) laser irradiation.
Horseradish peroxidase labeling Two methods of labeling were: (i) using HRP (Sigma, type V1, 20-30% in distilled water containing 2% DMSO: 10-15/xl were injected into a newly made crush between the globe and the site of injury using a Hamilton syringe); this way of application was used in 4 control injured and 5 injured treated nerves; (ii) using WGAHRP (0.5 mg in 30/~1 phosphate buffer, 0.1 M pH 7.4, Sigma) injected intraocularly using a Hamilton syringe (this was applied to 2 control injured and 10 injured treated nerves; 2 injured treated nerves, which were not injected with HRP or WGA-HRP, were used as control for endogenous peroxidase activity). Forty-eight h after labeling with one of the two methods, the rabbits were perfused through the heart with 500 ml of heparinized (2 × 104U/I; BDH Chemicals Ltd., Poole, U.K.) phosphate buffer, followed by 500 ml of fixative (1% paraformaldehyde, 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Longitudinal cryosections were taken (50 ~m) and HRP was visualized with DAB using the cobalt intensification method.
Fig. 1. Anterograde transport of HRP in injured treated and untreated adult rabbit optic nerves. Longitudinal sections of nerve segments between the optic disc (O.D.) and optic chiasm (O.C.). a: experimental animal, b: control animal. Note in (a) the brownish color of HRPlabeled axons which appeared distal to the site of injury (marked by a thick arrow), while in (b) the brownish color is restricted to the area of HRP application (marked by a thin arrow), which is proximal to the site of lesion. Note also the cross section of the folded nitrocellulose paper. Counter stain - Cresyl violet. Original magnification ×7.
RESULTS Six control and 15 e x p e r i m e n t a l animals were analyzed by longitudinal sectioning of the nerves, which were excised 48 h after H R P application. The pictures shown here r e p r e s e n t results o b t a i n e d from one typical control and one e x p e r i m e n t a l nerve. Fig. 1 shows low magnification ( x 7 ) of a section taken from an animal that was injured and subjected to the c o m b i n e d treatment (Fig. l a ) and of an animal that was injured only (Fig. l b ) . Both animals were labeled with H R P (Sigma, type VI, 10/~l of 20-30% in distilled water containing 2% D M S O ) , 8 weeks postinjury. A s can be seen, in the treated nerve reaction products of H R P were d e t e c t e d up to 3 m m distal to the site of injury (thin arrow points to the site of H R P application; thick arrow, the site of injury). In the control animal, H R P reaction products were found to be restricted to the site of H R P application. In the t r e a t e d animals, in areas proximal to the site of H R P application, longitudinal parallel fascia of labeled axons were seen b e t w e e n the optic disc and the site of application. No such axons could be d e t e c t e d in the control nerve. The site of injury in both treated and control nerves was found to be labeled by dusty-looking reaction products. These might r e p r e s e n t axonal labeling that looks dusty because the application of H R P in-
volved a second nerve lesion leading to the degeneration of the viable axons in these areas. We cannot rule out the possibility that the dusty-looking reaction products r e p r e s e n t a diffusion artifact. Fig. 2 shows a higher magnification of the distal part of the t r e a t e d nerve shown in Fig. l a . A m a r k e d numb e r of axons were found to be labeled by HRP. Many of these axons b e a r varicosities and swellings that look like growth cones at their tips (Figs. 2, 3). The labeled axons in the injured t r e a t e d nerves did not, unlike in the intact nerves, a p p e a r in parallel a r r a n g e m e n t to the longitudinal axis of the nerve, but had a tendency to grow t o w a r d the center. Many thin labeled axons could be seen, but the area was a brownish color that probably represents many more labeled axons too thin to be resolved by the light microscope. Fig. 3 shows c a m e r a lucida compositions of the nerves depicted above, showing that labeling can be detected at areas distal to the site of injury. A r r o w s are pointed at the same axons as in Fig. 2. The c a m e r a lucida drawings show the peculiar m a n n e r in which these axons grow within their own neighborhood. DISCUSSION The present study substantiates our previous conclusions that the newly growing axons observed in injured
Fig. 2. A high power magnification of HRP-labeled axons at a site distal to the site of injury of the injured treated nerve. The picture was taken from an area distal to the site of the injury of the injured treated nerve shown in Fig. la. One thick axon runs parallel to the longitudinal axis of the nerve. Many thin axons show the tendency to grow toward the center of the nerve (axons - thin arrows) and many of them bear growth cone-like swellings at their tips (thick arrow). This picture is a montage constructed from two micrographs taken at two different focal planes. (D) dura. Magnification × 1250.
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/J
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f
3-
lO0/Lm Fig. 3. Camera lucida drawings of HRP-labeled axons in injured treated adult rabbit optic nerve. The presented figure is a superimposition of 4 sequential sections of HRP-labeled axons in an area distal to the site of injury corresponding to Figs. la and 2. N C marks the most distal fold of the nitrocellulose paper, about 1 m m distal to the initial site of injury. (D) represents the dura matter, under which the growing axons are found. The arrows point to the same axons as in Fig. 2. Magnification x65.
adult rabbit optic nerves after treatment originate from retinal ganglion cells. It also shows their special pattern of growth. We induced growth of CNS axons into their own, otherwise hostile environment, using a treatment which consisted of the application of soluble substances originating from regenerating fish optic nerves, followed by daily irradiation with low energy He-Ne laser. The observed growth, shown by electron microscopy, manifested the presence of unmyelinated and thinly myelinated axons and growth cones in injured treated nerves. Using HRP injected intraocularly, we have previously shown that at least some of the growing axons originate from the retinal ganglion cells 12. This procedure, however, failed to provide a complete overview of the whole population of growing axons over their entire length. The present study was designed to overcome these drawbacks. The entire width and length of the optic nerves of both control and treated animals were analyzed using anterograde transport of HRP. In control injured but untreated nerves, no labeled axons could be detected even adjacent to the site of the HRP application (Fig. 1B). The drawing presented here was obtained by camera lucida using 4 serial longitudinal sections in which most of the labeled axons
could be seen. The drawings were then superimposed on each other. In the other sections of the same nerves, a few labeled axons were also observed. The incomplete superimposition of all the sections might explain the apparently discontinuous appearance of some of the labeled axons. The labeled axons had a pattern of appearance which differed from that of intact rabbit optic nerves. In intact nerves, axons were found mainly parallel to the longitudinal axis of the nerve; in the injured treated nerves, axons were found in a disorderly fashion, having the general tendency to grow toward the brain, as well as toward the center of the nerve. These axons might have acquired this pattern of growth if they were seeking their way within the nerve, without the clues necessary for directional guidance. The growing axons reached a distance of 3 mm distal to the site of injury. It appears as if the length of the newly growing axons was greater than 3 mm, the maximal distance where they were observed. This might be due to the fact that the observed axons were not growing straight toward the brain, parallel to the nerve's longitudinal axis. The present results are in line with our previous conclusions 12 that the growing fibers are clustered together
into a special c o m p a r t m e n t subjacent to the dura.
m e n t and thereby makes it conducive for growth. A m o n g
Growth was observed in treated animals, where the di-
the components participating in the alterations are the observed factors cytotoxic to oligodendrocytes5 and glial activating factor 4. Growth within the nerve's own injured
stal stump of the nerve became permissive to growth, unlike the control injured untreated nerve in which the distal stump was hostile to growth. D u e to the incomplete lesion, one might argue that the observed labeled axons might be spared rather than newly growing. This, however, is unlikely as at 3 m m distal to the injury they were not seen any more. If fibers were spared, they would have been seen throughout the entire length of the nerve. Growth of central axons after injury has been demonstrated in peripheral nerve grafts and into fetal CNS tissue 6'19'20'23'24 but the challenge has been to encourage growth within injured CNS tissue. Using our experimental paradigm, growth of the injured rabbit optic nerve has b e e n observed within the injured nerve's own envir o n m e n t . This growth has b e e n permitted probably as a result of the treatment which alters the cellular environ-
REFERENCES 1 Aguayo, A.J., David, S., Richardson, P. and Bray, G., Axonal elongation in peripheral and central nervous system transplantations, Adv. Cell. Neurobiol., 3 (1978) 215-221. 2 Assia, E., Rosner, M., Belkin, M., Solomon, A. and Schwartz, M., Temporal parameters of low energy laser irradiation for optimal delay of posttraumatic degeneration of rat optic nerve, Brain Research, 476 (1989) 205-212. 3 Bradford, M.D., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-284. 4 Cohen, A. and Schwartz, M., Conditioned media of regenerating fish optic nerves modulate laminin levels in glial cells, J. Neurosci. Res., 22 (1989) 269-273. 5 Cohen, A., Sivron, T., Duvdevani, R. and Schwartz, M., Oligodendrocyte cytotoxic factor associated with fish optic nerve regeneration: implication for mammalian CNS regeneration, Brain Res., 537 (1990) 24-32. 6 David, S. and Aguayo, A.J., Axonal elongation into peripheral nervous system 'bridges' after central nervous system injury in adult rats, Science, 214 (1981) 931-933. 7 Davis, G.E., Blaker, S.N., Engvall, E., Varon, S., Manthorpe, M. and Gage, F.M., Human amnion membrane serves as a substratum for growing axons in vitro and in vivo, Science, 236 (1987) 1106-1109 8 Grafstein, B. and Ingoglia, N.A., Intracranial transection of the optic nerve in adult mice: preliminary observations, Exp. Neurol., 76 (1982) 318-330. 9 Hadani, M., Harel, A., Solomon, A., Belkin, M., Lavie, V. and Schwartz, M., Substances originating from optic nerve of neonatal rabbit induce regeneration-association response in the injured optic nerve of adult rabbit, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7965-7969. 10 Kao, C.C., Change, L.W. and Bloodworth, J.M.B., Axonal regeneration across transected mammalian spinal cords. An electron microscopic study of delayed microsurgical nerve grafting, Exp. Neurol., 54 (1977) 591-615. 11 Kramer, L.F., Nerve growth factor treatment after brain injury prevents neuronal death, Science, 235 (1987) 214-216. 12 Lavie, V., Murray, M., Solomon, A., Ben-Bassat, S., Belkin, M., Rumelt, S. and Schwartz, M., Growth of injured rabbit optic axons within their degenerating optic nerve, J. Comp.
e n v i r o n m e n t has recently been demonstrated in adult rat spinal nerves 15. Growth was permitted by the use of antibodies, which neutralized the inhibitory molecules associated with mature oligodendrocytes. In conclusion, the present report further strengthens our previous conclusion that treatment with conditioned m e d i u m followed by He-Ne laser irradiation encourages regrowth of injured m a m m a l i a n axons into their own degenerating e n v i r o n m e n t .
Acknowledgements. This work was supported by the Daniel Heumann Fund for Spinal Cord Injury Research and by the United States-Israel Binational Foundation, given to M.S.M.S. is the incumbent of the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
Neurol., 298 (1990) 293-314. 13 Misantone, L.J., Gershenbaum, M. and Murray, M., Viability of retinal ganglion cells after optic nerve crush in adult rats, J. Neurocytol., 13 (1984) 449-465. 14 Murray, M., A quantitative study of regenerative sprouting by optic axons in goldfish, J. Comp. Neurol., 209 (1982) 352-362. 15 Schnell, L. and Schwab, M.E., Axonal regeneration in the rat spinal cord produced by an antibody against myelin associated neurite growth inhibitors, Nature, 343 (1990) 269-272. 16 Schwartz, M., Belkin, M., Harel, A., Solomon, A., Lavie, V., Hadani, M., Rachailovich, L. and Stein-Izsak, C., Regenerating fish optic nerves trigger regeneration-like response in injured optic nerves of adult rabbits, Science, 228 (1985) 601-603. 17 Schwartz, M., Ehrlich, M., Doron, N., Ben-Bassat, S., Lavie, V. and Belkin, M., The effect of low energy laser irradiation on posttraumatic degeneration of adult rabbit optic nerve, Lasers Surg. Med., 7 (1987) 51-55. 18 Sievers, J., Hausmann, B., Unsicker, K. and Berry, M., Fibroblast growth factors promote the survival of adult rat retinal ganglion cells after transection of the optic nerve, Neurosci. Lett., 76 (1987) 157-162. 19 Smith, G.M., Miller, R.H. and Silver, J., Changing role of forebrain astrocytes during development, regenerative failure and induced regeneration upon transplantation, J Comp. Neurol., 251 (1986) 23-43. 20 So, K.F. and Aguayo, A.J., Length regrowth of cut axons from ganglion cells after peripheral nerve transplantation into the retina of adult rats, Brain Res., 328 (1985) 349-354. 21 Solomon, A., Belkin, M., Hadani, M., Harel, A., Rachailovich, I., Lavie, V. and Schwartz, M., A new transorbital approach to the rabbit's optic nerve, J. Neurosci. Meth., 12 (1985) 259-262. 22 Solomon, A., Lavie, V., Ben-Bassat, S., Belkin, M. and Schwartz, M., A new surgical approach to overcome the inability of injured mammalian axons to grow within their environment, J. Neurol. Transpl., in press. 23 Tessler, A., Himes, B.T., Houle, J.D. and Reier, P.J., Regeneration of adult dorsal root axons into transplants of embryonic spinal cord, J. Comp. Neurol., 270 (1988) 537-548. 24 Vidal-Sanz, M., Bray, M.B., Villegas-P6rez, M.P., Thanos, S. and Aguayo, A.J., Axonal regeneration and synapse formation in superior colliculus by retinal ganglion cells in the adult rat, J. Neurosci., 7 (1987) 2894-2909.
BRES 17484
1
Research Reports
Horseradish peroxidase labeling of growth cones and axons beyond the site of injury in injured rabbit optic nerve axons growing in their own environment V. Lavie a, A. Solomon b, S. Ben-Bassat b, M. Belkin b and M. Schwartz a aDepartment of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel) and bM. and G. Goldschleger Eye Institute, Sackler School of Medicine, Tel Aviv University, Tel Hashomer (Israel)
(Accepted 15 October 1991) Key words: Growth cone; Optic nerve; Axonal injury
Spontaneous growth of injured axons in the mammalian central nervous system is limited. We have previously shown an apparently regenerative growth of injured optic axons in the adult rabbit, achieved by supplying them with soluble substances originating from growing axons, followed by low energy helium-neon laser irradiation. The growing unmyelinated and thinly myelinated axons were embedded in astrocytes, and some were in the process of remyelination by oligodendrocytes. They were shown to have originated from the retinal ganglion cells. The present study further supports evidence relating to the origin and nature of these axons. Light microscopic analysis of these axons labeled with anterogradely transported horseradish peroxidase revealed that many of these axons have varicosities and bear growth cone-like swellings in their tips. These axons traverse the lesion site and extend into the distal stump in a disorganized pattern. INTRODUCTION Injury to the mammalian optic nerve, a model for the central nervous system (CNS), leads to death of most of the axotomized neurons and a failure of the surviving cells to regrow their axons 8'13. The ability of the mammalian retinal ganglion cells to survive and regenerate their axons after axotomy can, however, be markedly enhanced by modifying the neuronal environment 1'7'9-u'18. Several effectors are likely to be involved in encouraging regeneration of mammalian central neurons. Among them are factors that can enhance neuronal survival and support axonal elongation, and those that can modify the environment, making it more conducive to growth. We have recently shown that treatment of injured adult rabbit optic nerves with medium conditioned by regenerating fish optic nerves (CM), followed by repeated low energy helium-neon (He-Ne) laser irradiation, alters the response of the optic nerve to injury. The CM was shown by us to contain factors affecting astrocytes 4 and oligodendrocytes 5, whereas irradiation with low energy He-Ne laser was effective in slowing down degeneration 2. We have recently observed that the slowing down of degeneration is associated with the presence at the site of injury of astrocytes, whose disappearance we have proposed as a reason for the postinjury growth
arrest (unpublished observations). The growth obtained as a result of the combined treatment was manifested morphologically by the growth of axons within their own, otherwise hostile environment. The growth was shown by electron microscopy to be manifested by the presence of unmyelinated and thinly myelinated axons associated with glial cells. These axons were traversing the site of injury and extending up to 6 m m distal to it. Many or most of these axons represent regenerative sprouts and, indeed, they closely resemble regenerative optic axons described in lower vertebrates 14. For the following reasons our results suggest that the unmyelinated and thinly myelinated axons found distal to the site of the lesion, in association with glial cells, are regenerating axons and not axons spared by the lesion: (i) in transected nerves treated with CM and exposed to irradiation, unmyelinated and thinly myelinated axons, grouped together in a compartment, could be seen only up to a certain distance from the lesion. (ii) In the operated control group, no viable (i.e., unmyelinated or thinly myelinated) axons or growth cones could be detected distal to the lesion site from 6 weeks postoperation onward. We have previously shown that the growing axons originate from the rabbit retinal ganglion cells, since wheat-germ agglutinin ( W G A ) conjugated with horse-
Correspondence: M. Schwartz, Dept. of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
r a d i s h p e r o x i d a s e ( H R P ) i n j e c t e d i n t r a o c u l a r l y was f o u n d to l a b e l t h e g r o w i n g a x o n s 12. T h i s k i n d o f l a b e l i n g failed, however,
to d e m o n s t r a t e ,
at t h e e l e c t r o n m i c r o s c o p e
level, t h e n a t u r e o f g r o w t h f a c i l i t a t e d b y t h e t r e a t m e n t . In the present study we substantiate our previous observations by a complete follow-up of anterograde transport of H R P in t r e a t e d t r a n s e c t e d a d u l t r a b b i t o p t i c n e r v e s .
previously described 16 and the animals returned to their tanks. Eight days later the fish were reanesthetized and the crushed regenerating nerves were dissected out, transferred into serum free medium (DMEM, Gibco) and incubated for 1 h at room temperature. The conditioned medium was then collected and stored at -20°C 16. The protein content of the medium was determined by the Bradford assay 3. The conditioned medium was diluted to 100 #g protein/ml. From this stock solution, 5-10 ~1 were used for each piece of nitrocellulose implanted into an injured nerve 2t22.
Irradiation MATERIALS AND METHODS
Rabbit surgery Adult rabbits (albino, Weizmann Institute Animal House) were deeply anesthetized with xylazine (5 mg/kg) and ketamine (35 mg/ kg). The left optic nerves of 21 rabbits (6 control, 15 combined treatment) were exposed, as previously described 21. The optic nerves were visualized with the aid of a Zeiss operating microscope. At a distance of 5-6 mm from the eyeball, the nerves were transected almost completely using a sharpened dissecting needle2L The meningeal membrane was partially spared intentionally, to ensure continuity of the nerve and to permit placement and retention of a nitrocellulose film, described below. In some cases, this resulted in sparing axons immediately subjacent to the meningeal covering. In the operated control group, nitrocellulose was soaked in serum free medium (DMEM, Gibeo) for 1 h and inserted into the lesion site. In the experimental (combined treatment) group, the nitrocellulose was soaked in CM (100 ~g protein/ml) for 1 h before being inserted into the lesion site.
Preparation of conditioned media Carp (Cyprinus carpio; 800-1200 g) were purchased from Tnuva Israel. The fish were deeply anesthetized with 0.05% tricaine methansulfonate (Sigma). Optic nerves were crushed intraorbitally as
A He-Ne laser (Spectrophysics Instruments) was used to irradiate the nerve through the eye, as described previously 2'~7. The laser beam was directed through the pupil at the optic nerve. Beginning 30 rain after surgery, the rabbits were exposed for 5 min each day for 10 consecutive days to low energy (632.8 mm, 35 mW) laser irradiation.
Horseradish peroxidase labeling Two methods of labeling were: (i) using HRP (Sigma, type V1, 20-30% in distilled water containing 2% DMSO: 10-15/xl were injected into a newly made crush between the globe and the site of injury using a Hamilton syringe); this way of application was used in 4 control injured and 5 injured treated nerves; (ii) using WGAHRP (0.5 mg in 30/~1 phosphate buffer, 0.1 M pH 7.4, Sigma) injected intraocularly using a Hamilton syringe (this was applied to 2 control injured and 10 injured treated nerves; 2 injured treated nerves, which were not injected with HRP or WGA-HRP, were used as control for endogenous peroxidase activity). Forty-eight h after labeling with one of the two methods, the rabbits were perfused through the heart with 500 ml of heparinized (2 × 104U/I; BDH Chemicals Ltd., Poole, U.K.) phosphate buffer, followed by 500 ml of fixative (1% paraformaldehyde, 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). Longitudinal cryosections were taken (50 ~m) and HRP was visualized with DAB using the cobalt intensification method.
Fig. 1. Anterograde transport of HRP in injured treated and untreated adult rabbit optic nerves. Longitudinal sections of nerve segments between the optic disc (O.D.) and optic chiasm (O.C.). a: experimental animal, b: control animal. Note in (a) the brownish color of HRPlabeled axons which appeared distal to the site of injury (marked by a thick arrow), while in (b) the brownish color is restricted to the area of HRP application (marked by a thin arrow), which is proximal to the site of lesion. Note also the cross section of the folded nitrocellulose paper. Counter stain - Cresyl violet. Original magnification ×7.
RESULTS Six control and 15 e x p e r i m e n t a l animals were analyzed by longitudinal sectioning of the nerves, which were excised 48 h after H R P application. The pictures shown here r e p r e s e n t results o b t a i n e d from one typical control and one e x p e r i m e n t a l nerve. Fig. 1 shows low magnification ( x 7 ) of a section taken from an animal that was injured and subjected to the c o m b i n e d treatment (Fig. l a ) and of an animal that was injured only (Fig. l b ) . Both animals were labeled with H R P (Sigma, type VI, 10/~l of 20-30% in distilled water containing 2% D M S O ) , 8 weeks postinjury. A s can be seen, in the treated nerve reaction products of H R P were d e t e c t e d up to 3 m m distal to the site of injury (thin arrow points to the site of H R P application; thick arrow, the site of injury). In the control animal, H R P reaction products were found to be restricted to the site of H R P application. In the t r e a t e d animals, in areas proximal to the site of H R P application, longitudinal parallel fascia of labeled axons were seen b e t w e e n the optic disc and the site of application. No such axons could be d e t e c t e d in the control nerve. The site of injury in both treated and control nerves was found to be labeled by dusty-looking reaction products. These might r e p r e s e n t axonal labeling that looks dusty because the application of H R P in-
volved a second nerve lesion leading to the degeneration of the viable axons in these areas. We cannot rule out the possibility that the dusty-looking reaction products r e p r e s e n t a diffusion artifact. Fig. 2 shows a higher magnification of the distal part of the t r e a t e d nerve shown in Fig. l a . A m a r k e d numb e r of axons were found to be labeled by HRP. Many of these axons b e a r varicosities and swellings that look like growth cones at their tips (Figs. 2, 3). The labeled axons in the injured t r e a t e d nerves did not, unlike in the intact nerves, a p p e a r in parallel a r r a n g e m e n t to the longitudinal axis of the nerve, but had a tendency to grow t o w a r d the center. Many thin labeled axons could be seen, but the area was a brownish color that probably represents many more labeled axons too thin to be resolved by the light microscope. Fig. 3 shows c a m e r a lucida compositions of the nerves depicted above, showing that labeling can be detected at areas distal to the site of injury. A r r o w s are pointed at the same axons as in Fig. 2. The c a m e r a lucida drawings show the peculiar m a n n e r in which these axons grow within their own neighborhood. DISCUSSION The present study substantiates our previous conclusions that the newly growing axons observed in injured
Fig. 2. A high power magnification of HRP-labeled axons at a site distal to the site of injury of the injured treated nerve. The picture was taken from an area distal to the site of the injury of the injured treated nerve shown in Fig. la. One thick axon runs parallel to the longitudinal axis of the nerve. Many thin axons show the tendency to grow toward the center of the nerve (axons - thin arrows) and many of them bear growth cone-like swellings at their tips (thick arrow). This picture is a montage constructed from two micrographs taken at two different focal planes. (D) dura. Magnification × 1250.
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lO0/Lm Fig. 3. Camera lucida drawings of HRP-labeled axons in injured treated adult rabbit optic nerve. The presented figure is a superimposition of 4 sequential sections of HRP-labeled axons in an area distal to the site of injury corresponding to Figs. la and 2. N C marks the most distal fold of the nitrocellulose paper, about 1 m m distal to the initial site of injury. (D) represents the dura matter, under which the growing axons are found. The arrows point to the same axons as in Fig. 2. Magnification x65.
adult rabbit optic nerves after treatment originate from retinal ganglion cells. It also shows their special pattern of growth. We induced growth of CNS axons into their own, otherwise hostile environment, using a treatment which consisted of the application of soluble substances originating from regenerating fish optic nerves, followed by daily irradiation with low energy He-Ne laser. The observed growth, shown by electron microscopy, manifested the presence of unmyelinated and thinly myelinated axons and growth cones in injured treated nerves. Using HRP injected intraocularly, we have previously shown that at least some of the growing axons originate from the retinal ganglion cells 12. This procedure, however, failed to provide a complete overview of the whole population of growing axons over their entire length. The present study was designed to overcome these drawbacks. The entire width and length of the optic nerves of both control and treated animals were analyzed using anterograde transport of HRP. In control injured but untreated nerves, no labeled axons could be detected even adjacent to the site of the HRP application (Fig. 1B). The drawing presented here was obtained by camera lucida using 4 serial longitudinal sections in which most of the labeled axons
could be seen. The drawings were then superimposed on each other. In the other sections of the same nerves, a few labeled axons were also observed. The incomplete superimposition of all the sections might explain the apparently discontinuous appearance of some of the labeled axons. The labeled axons had a pattern of appearance which differed from that of intact rabbit optic nerves. In intact nerves, axons were found mainly parallel to the longitudinal axis of the nerve; in the injured treated nerves, axons were found in a disorderly fashion, having the general tendency to grow toward the brain, as well as toward the center of the nerve. These axons might have acquired this pattern of growth if they were seeking their way within the nerve, without the clues necessary for directional guidance. The growing axons reached a distance of 3 mm distal to the site of injury. It appears as if the length of the newly growing axons was greater than 3 mm, the maximal distance where they were observed. This might be due to the fact that the observed axons were not growing straight toward the brain, parallel to the nerve's longitudinal axis. The present results are in line with our previous conclusions 12 that the growing fibers are clustered together
into a special c o m p a r t m e n t subjacent to the dura.
m e n t and thereby makes it conducive for growth. A m o n g
Growth was observed in treated animals, where the di-
the components participating in the alterations are the observed factors cytotoxic to oligodendrocytes5 and glial activating factor 4. Growth within the nerve's own injured
stal stump of the nerve became permissive to growth, unlike the control injured untreated nerve in which the distal stump was hostile to growth. D u e to the incomplete lesion, one might argue that the observed labeled axons might be spared rather than newly growing. This, however, is unlikely as at 3 m m distal to the injury they were not seen any more. If fibers were spared, they would have been seen throughout the entire length of the nerve. Growth of central axons after injury has been demonstrated in peripheral nerve grafts and into fetal CNS tissue 6'19'20'23'24 but the challenge has been to encourage growth within injured CNS tissue. Using our experimental paradigm, growth of the injured rabbit optic nerve has b e e n observed within the injured nerve's own envir o n m e n t . This growth has b e e n permitted probably as a result of the treatment which alters the cellular environ-
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Acknowledgements. This work was supported by the Daniel Heumann Fund for Spinal Cord Injury Research and by the United States-Israel Binational Foundation, given to M.S.M.S. is the incumbent of the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
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