334
Brain Research, 545 (1991) 334-338 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939124603J
BRES 24603
Tumor necrosis factor facilitates regeneration of injured central nervous system axons M. Schwartz 1, A. Solomon 2, V. Lavie ~, S. Ben-Bassat 2, M.
Belkin 2 and A. Cohen 1
1Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100 (Israel) and 2Goldschleger Eye Research Institute, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Hashomer (Israel)
(Accepted 8 January 1991) Key words: Central nervous system; Regeneration; Mammal; Tumor necrosis factor
The results of this study attribute to tumor necrosis factor (TNF) a role in regeneration of injured mammalian central nervous system (CNS) axons which grow into their own degenerating environment. This is the first time that a specific factor involved in axonal regeneration has been identified. The axonal environment is occupied mostly by glia cells, i.e., astrocytes and oligodendrocytes. Previous studies have shown that mature oligodendrocytes are inhibitory to axonal growth. Therefore, it seemed likely that application of a factor such as TNF, which has been shown to be cytotoxic to oligodendrocytes, would contribute to the creation of permissive conditions for axonal regeneration. In the present work, injured adult rabbit optic nerves were treated with human recombinant TNF (rhTNF). As a result, abundant newly growing axons (circa 9000, about 4% of the total estimated number of axons in an intact adult rabbit) were observed traversing the site of injury. M a m m a l i a n CNS neurons have a negligible capacity to r e g e n e r a t e after lesions 8'~2'15. In contrast, central neurons of lower vertebrates reliably regenerate after axotomy 9'12'16'18. The prevailing hypothesis that axotomized neurons, m a m m a l i a n as well as n o n - m a m m a l i a n , are capable of regeneration after injury TMis s u p p o r t e d by the fact that axotomized m a m m a l i a n central neurons can regenerate their axons over long distances, if special conditions are provided, such as replacement of a severed central nerve by a segment of a peripheral autologous nerve 1"29. Thus, the success or failure of a regenerative process d e p e n d s upon the response of the non-neuronal cells (astrocytes, oligodendrocytes, microglia, macrophages) in CNS or Schwann in PNS to the axonal injury. As a result of neuronal injury in mammals, astrocytes form glial scar, which is non-supportive to axonal growth 19. M a t u r e oligodendrocytes, which are present in the adult m a m m a l i a n system, have been shown in vitro to inhibit axonal growth 4'22'23. The inhibitory effect of the oligodendrocytes can be circumvented by antibodies specific to the observed inhibitors, as was shown lately 21. We have previously shown that the CNS of lower vertebrates, specifically the regenerating fish optic nerve, is a source of factors which, when applied to injured adult m a m m a l i a n optic nerves, can support regenerative axonal growth 1°'13"j4. For example, in injured adult rabbit optic nerves which were treated with soluble substances derived from regenerating fish optic nerves, along with
low energy H e - N e laser irradiation, which has been shown to delay the d e g e n e r a t i o n process 2, r e m a r k a b l e growth was observed TM. Thus, a b u n d a n t newly growing axons, in a typical CNS environment, were observed traversing the site of injury and extending into the distal stump of nerves, up to a distance of 6.5 mm, 8 weeks postsurgery 14. The observed axons, organized into a special compartment, were indeed newly growing axons, as was evident by the facts that they were either unmyelinated or thinly myelinated and appeared together with abundant growth cones. The total number of unmyelinated axons was greater than in normal unoperated nerves. Anterograde transport of horseradish peroxidase injected intraoculady provided evidence that these newly growing axons were emerging from the retinal ganglion cells. In control injured but untreated nerves, no viable axons or growth cones were observed distal to the site of the injury from 4 - 6 weeks postinjury and beyond. The effect of the applied soluble substances was attributed to the presence of molecules which have been shown to have a cytotoxic effect on oligodendrocytes in vitro 6'26, and to molecules which activate astrocytes to p r o d u c e high levels of laminin 5. This brought to light the intriguing possibility that substitution of the soluble substances with a factor which has b e e n suggested to have a lethal effect on m a t u r e oligodendrocytes, such as T N F 2°'25, might facilitate axonal regeneration across the site of injury and distally.
Correspondence: M. Schwartz, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.
335 Adult rabbits (albinos, from the Weizmann Institute Animal House) were deeply anesthetized with xylazine (5 mg/kg) and ketamine (35 mg/kg). The left optic nerves were exposed, as previously described 2a, and transected almost completely, except for the meningeal membrane, at a distance of 5-6 mm from the eyeball, using a sharpened needle. In all operated nerves, a piece of nitrocellulose, 2-4 mm long and 1 mm wide, was inserted at the site of the injury. In the experimental group, the nitrocellulose was soaked with rhTNF (100 U/nerve; 6 x 107 U/mg); in the control groups it was soaked in medium, free of any active substances. The meningeal membrane was partially spared to ensure continuity of the injured nerve and retention of the applied nitrocellulose. Animals of the experimental group and one control group were also treated daily with low energy He-Ne laser irradiation (10 days, 5 min/day, 35 mW) 14. Six and 8 weeks postsurgery, the experimental and control injured nerves were examined qualitatively and quantitatively by transmission electron microscopy, which allows distinction between newly growing axons and spared ones. Quantitative analyses were carded out as have been described previously 14. Excised experimental and control nerves (6 and 8 weeks postoperatively), which were processed for electron microscopy, were closely analyzed. Characteristics of growth (distal to the site of injury) were observed only in the experimental but not control nerves. Both nerves were systematically examined. Fig. 1 shows some characteristics of the experimental injured nerve after treatment with TNF and laser irradiation. The electron micrographs were taken from cross-sections of experimental nerves, from an area within the second millimeter distal to the site of injury. This area was chosen because in the control nerve it was free of any non-myelinated axons or any other reminiscence of growth. The pictures depict characteristics of newly growing axons, including abundant unmyelinated and thinly myelinated axons embedded in a typical astrocytic environment (Fig. 1A). Among these axons, structures resembling growth c o n e s 14'27, w e r e also observed, one of which is shown in Fig. lB. Some axons were seen in early stages of myelination, as they were surrounded by oligodendrocyte dark cytoplasm (Fig. 1C). In control-injured but untreated nerves, into which nitrocellulose free of active substances was applied, the area corresponding to 2 mm distal to the site of injury was in a state of degeneration (Fig. 1D), containing astrocytic processes and degenerating axons. In a few cases, both in control and in treated operated animal groups, the lesion was less extensive, and there were regions throughout the entire length of the nerve in which viable axons showed the organization, density and thickness of myelin sheath characteristic of
the intact uninjured nerves. Such axons were considered to be spared axons and these animals were not included in our studies. That those spared axons kept their myelin in the presence of TNF, the putative oligodendrocyte cytotoxin, could be due either to the fact that they were not directly exposed to the applied TNF or that they have already undergone a process of remyelination, 6 weeks after the exposure to T N E This issue should be further investigated. In order to evaluate the extent of growth in the experimental treated nerves and to compare it to the situation in the injured non-treated animals, we used the sampling method 14 and we counted the number of unmyelinated and myelinated axons in 4 regions along the nerve: 1 mm proximal to the site of injury, within the second, third and fifth mm distal to the injury, and deep within the distal stump (see Table I). In the treated animals, viable, unmyelinated and myelinated, axons were abundant at areas proximal to the site of injury; their number decreases upon increase in the distance from the globe and the site of injury. In the treated nerves, the maximal number (8466) of unmyelinated axons is seen within the second m m distal to the site of injury. In operated control nerves which were treated with nitrocellulose soaked with medium only, the degeneration process was evident and showed that by 6 and 8 weeks postinjury, no unmyelinated axons were observed at 2 mm to the site of injury. At 8 weeks post-operation only a very small number of myelinated axons were found in the area 2 m m distal to the site of injury (Table I). This small number (127) might be even an overestimation of the concrete number, since it was calculated by the sampling method in which very few axons were actually counted. The fact that no viable axons were observed in the control animals from 2 mm on distal to the site of injury, rules out the possibility that the high number of surviving axons found in the section taken from the area proximal to the site of injury were spared axons, possibly resulting from an incomplete transection but that they were injured ones which would eventually degenerate retrogradely (Table I). The percentage of unmyelinated axons out of the total number of viable axons in the two groups is shown in Table I. The most striking result is the high percentage (93%) of unmyelinated axons in the experimental nerves, 2 mm distal to the site of injury. In the experimental nerve, the area 2 m m distal to the site of injury, in which 93% of the viable axons were unmyelinated, probably represents the growth frontier. The apparent increasing number of myelinated axons in the area located 3 mm distal to the site of injury, relative to the area located 2 mm distal to the site of the lesion, might be due to myelination of some of the growing unmyelinated axons seen in the former (Table I). Such
336
Fig. 1. Characteristics of newly growing axons in TNF-treated nerves. At a distance of 5-6 mm from the eyeball, the nerves were transected except for part of the meningeal membrane26. A piece of nitrocellulose (NC), 3 mm long by 1 mm wide, was inserted at the site of injury; the NC was soaked in rhTNF (100 U, 6 x 107 U/mg) for 1 h prior to the insertion. Beginning within 30 rain after surgery, the rabbits were transocularly irradiated for 5 min each day, for 10 consecutive days, with low energy (6328 nm, 35 roW) He-Ne laser14. Six weeks after the injury, the nerve was removed, fixed by immersion overnight with 1% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer 0.1 M pH 7.4 containing 2.5 mM CaCI2, and then cut serially into 1 mm segments from the retina to the chiasm, which were further processed for electron microscopy (see ref. 14). The electron micrographs shown in this figure were taken from the area of the second millimeter distally to the site of injury. Note in (A) abundant unmyelinated axons (Ax) grouped together surrounded by astrocytic processes (Ap); (B) a typical growth cone (gc) in an astrocytic environment; (C) unmyelinated axons in an early process of myelination by dark cytoplasm of oligodendrocytes (arrows); and (D) control injured untreated rabbit optic nerve, excised 6 weeks postinjury at a distance of 2 mm distal to the site injury, showing only interdigitated astrocytic processes, representing glial sear (gs), and degenerating axonal profiles. Bar represents 1/~m. (A, B, C the same magnification.) myelination is carried out by oligodendrocytes (Fig. 1C), p r e s u m a b l y those which spared the cytotoxic effect of TNF. Alternatively, these myelinated axons at the distal region might represent axons which were injured but in which the degenerative process had not yet reached the e x a m i n e d a r e a (4 m m from the injury). Three-dimensional reconstruction of the treated nerve (Fig. 2) shows that the area occupied by the viable unmyelinated and m y e l i n a t e d axons, as well as their total number, decreases upon distance from the site of injury, emphasizing that the observed unmyelinated axons are, indeed, newly growing axons, rather than spared axons that have lost their myelin sheaths.
Summarizing, we have seen regenerative growth of injured CNS axons into their own injured environment, achieved, for the first time, by a t r e a t m e n t which consisted of the application of a single factor - - T N E T N F was the factor of choice, because it has been shown in vitro by us (M. Lotan, A . C o h e n and M. Schwartz, in p r e p a r a t i o n ) and by others 2°, that rhTNF, in a crossspecies m a n n e r , is active as a cytotoxic factor on oligodendrocytes. The conclusion that the unmyelinated and thinly myelinated axons o b s e r v e d distal to the site of injury are regenerating axons, and not axons spared by the lesion, is based on the following observations: (1) in o p e r a t e d controls, no u n m y e l i n a t e d axons could be
337
mAx IAx ~
GIs+dAx
1mmt__.~,. 1mm Fig. 2. Three-dimensional reconstruction of an injured nerve treated with TNF and laser irradiation, 6 weeks postinjury. The injured rabbit optic nerve was operated and treated as described in Fig. 1. Six weeks later, the animal was anesthetized and the nerve excised. Thin sections were cut from each 1 mm segment and were mounted on grids, photographed at x 140. Compartments identified in each cross-section contained the following: unmyelinated, presumably growing axons (Ax) and myelinated axons (mAx); nitrocellulose (NC); and glial scar and degenerating axons (Gls + dAx). Arrow indicates first section in which nitrocellulose is seen. TABLE I
Quantitative analysis of operated nerves Animals from the experimental and operated control groups were quantitatively analyzed using the sampling method. Further, 6 experimental and 10 operated control animals were qualitatively analyzed. Experimental nerves which contained an area with the organization, and density and thickness of myelin sheath characteristic of intact uninjured nerves, and in which this area ran the entire length of the nerve, were not included in the analysis14,since they were considered to overcome an incomplete injury, In the experimental nerves, the site of injury is between 6 and 7 mm distal to the globe, whereas the last region in which nitrocellulosecould be observed, was 10 mm distal to the globe. In control operated nerves examined 6 weeks post-injury, no viable axons, myelinated or unmyelinated, could be observed at any of the examined regions. The table intentionally gives an example of an operated control nerve, excised 8 weeks post-injury, in which a few unmyelinated axons were observed, but only proximally to the site of the injury.
Nerve region
Number of counted axons (% of total viable axons) Experimental Unmyelinated
1 mm 7530 (19%) proximal to injury
Operated control Myelinated Unmyelinated Myelinated 31357
195 (11%)
1666
2 mm distal to injury
8466 (93%)
591
0
127
3 mm distal to injury
200 (9%)
2272
0
0
5 mm distal to injury Deep distal
0
200
0
0
detected distal to the site of injury; (2) in the TNFtreated nerves, the n u m b e r of viable axons decreases with increasing distance from the site of injury (Table I). If u n m y e l i n a t e d axons were axons spared by the injury, and demyelinated by the TNF, survival of these axons, either as u n m y e l i n a t e d or myelinated, at more distal locations and throughout the entire length of the nerve would be seen; and (3) along with the u n m y e l i n a t e d axons, a b u n d a n t growth cones do appear. These newly growing axons could be sprouts from the cell bodies of the injured axons, or sprouts from the cut tip. Whatever the nature of these sprouts, they were absent from sites distal to the injury in all operated control animals. The newly growing axons observed 6 weeks postsurgery traversed the site of injury. The lack of further growth might be a reflection of (a) the limited time elapsed postinjury before the nerves were examined; (b) the limited a m o u n t of T N F provided by the single application; (c) the limited accessibility to TNF, provided by the nitrocellulose up to a distance of 4 m m distal to the lesion; and (d) the need for additional factor(s), like those which activate the astrocytes and are present among the soluble substances in regenerating fish optic nerves 5. In the absence of such activating factors, these astrocytes eventually develop into non-supportive scar° forming cells, and elongation of the growing axons will be prevented. U n d e r normal physiological conditions, T N F may be a product of macrophages, which invade nerves after injury 17. If that is the case, one would expect T N F to
338 facilitate axonal growth and regeneration, provided that the invasion of macrophages is in temporal coordination with the injury-induced activation of the cell bodies of the injured axons. A temporal shift of the local invasion of macrophages after injury might lead to absence of T N F at the critical period. It is also possible that T N F is not the product of macrophages but of other cells, such as activated astrocytes 2°, which, in the m a m m a l i a n CNS, might not be activated or, at least, not activated at the
thus supporting their regeneration. A t this stage, we cannot rule out the possibility that the applied T N F might have additional effects on additional cells, such as astrocytes, resident microglia, invading macrophages and/or lymphocytes. Such an effect might be primarily manifested by changes in IL-1 and IL-2 levels 7 and, consequently, possibly on neurotrophic factors 11.
shortage of TNF, possibly helping to eliminate inhibitors from the e n v i r o n m e n t surrounding injured axons and
We thank Prof. D. Wallach and Dr. H. Engelman, for the fruitful discussions and for their generosity in giving us rhTNF and anti-TNF antibodies. This work is supported by Farmitalia (Milan) and by the Daniel Heumann Fund for Spinal Cord Injury Research. M. Schwartz is an incumbent of the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
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 Anardi, D.G. and Sperry, R.W., Preferential selection of central pathways by regenerating optic nerve, Exp. Neurol., 7 (1963) 46-64. 4 Caroni, P. and Schwab, M.E., Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading, J. Cell Biol., 196 (1989) 1281-1288. 5 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. 6 Cohen, A., Sivron, T., Duvdevani, R. and Schwartz, M., Oligodendrocyte cytotoxic factor associated with fish optic nerve regeneration: implications for mammalian CNS regeneration, Brain Research, 537 (1990) 24-32. 7 Christopher, D.B., Dinarello, C.A. and Saper, C.B., Interleukin-1 immunoreactive innervation of the human hypothalamus, Science, 240 (1988) 321-324. 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 Guth, L. and Windle, W.F., Regeneration in the vertebrate central nervous system, Exp. Neurol., 5 (1970) 1-43. 10 Hadani, M., Harel, A., Solomon, A., Belkin, M., Lavie, V. and Schwartz, M., Substances originating from the optic nerve of neonatal rabbit induce regeneration-associated response in the injured optic nerve of adult rabbit, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)7965-7969. 11 Heumann, R., Korching, S., Bandtlow, C. and Thoenen, H., Changes in nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection, J. Cell. Biol., 104 (1987) 1623-1631. 12 Kiernan, J.A., Hypotheses concerned with axonal regeneration in the mammalian nervous system, Biol. Rev., 54 (1979) 155-197. 13 Lavie, V., Harel, A., Doron, A., Solomon, A., Lobel, D., Belkin, M., Ben-Bassat, S., Sharma, S. and Schwartz, M., Morphological response of injured adult rabbit optic nerve to implants containing media conditioned by growing optic nerves, Brain Research, 419 (1987) 166-173. 14 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. Neurol., 298 (1990) 293-317.
15 Misantone, L.J., Gershenbaum, M. and Murray, M., Viability of retinal ganglion cells after nerve crush in adult rats, J. Neurocytol., 13 (1984) 449-465. 16 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. Comp. Neurol., 168 (1976) 175-196. 17 Perry, V.H., Brown, M.C. and Gordon, S., The macrophage response to central and peripheral nerve injury: a possible role for macrophages in regeneration, J. Exp. Med., 165 (1987) 1218-1223. 18 Ram6n y Cajal, S., Degeneration and Regeneration of Nervous System, Vol. 1, R.M. May (Transl.), Hafner Publ. Co., New York, 1959. 19 Reier, P.J., Stensaas, L. and Guth L., The astroeytes' scar as an impediment to regeneration in the central nervous system. In C.C. Kao, R.P. Bunge and P.J. Reier (Eds.), Spinal Cord Reconstruction, Raven, New York, 1983, pp. 163-196. 20 Robbins, D.S., Shirazi, Y., Drysdale, B., Liberman, A., Shin, H.S. and Shin, M.L., Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes, J. lmmunoL, 139 (1987) 2593-2597. 21 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. 22 Schwab, M.E. and Thoenen, H., Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors, J. Neurosci., 5 (1985) 2415-2423. 23 Schwab, M.E. and Caroni, P., Oligodendrocytes and CNS myelin are non-permissive substrates for neurite growth and fibroblast spreading in vitro, J. Neurosci., 8 (1988) 2381-2393. 24 Schwartz, M., Belkin, M., Harel, A., Solomon, A., Lavie, V., Hadani, M., Rachailovich, I. and Stein-Izsak, C., Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbit~, Science, 228 (1985) 600-603. 25 Selmaj, K.W. and Raine, C.S., Tumor necrosis factor mediates myelin and oligodendrocyte damage in vivo, Ann. Neurol., 23 (1988) 339-346. 26 Sivron, T., Cohen, A., Duvdevani, R. and Schwartz, M., Glial response to axonal injury in vitro manifestation and implication for regeneration, Glia, 3 (1990) 267-276. 27 Skoff, R.P. and Hamburger, V., Fine structure of dendritic and axonal growth cones in embryonic chick spinal cord, J. Comp. Neurol., 153 (1974) 107-148. 28 Solomon, A., Belkin, M., Hadani, M., Harel, A., Rachaiiovich, I., Lavie, V. and Schwartz, M., A new transorbital surgical approach to the rabbit's optic nerve, J. Neurosci. Methods, 12 (1985) 259-262. 29 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.
appropriate time. Exogenous TNF, applied to mammalian injured CNS, might overcome the temporal or spatial
Brain Research, 545 (1991) 334-338 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0006-8993/91/$03.50 ADONIS 000689939124603J
BRES 24603
Tumor necrosis factor facilitates regeneration of injured central nervous system axons M. Schwartz 1, A. Solomon 2, V. Lavie ~, S. Ben-Bassat 2, M.
Belkin 2 and A. Cohen 1
1Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100 (Israel) and 2Goldschleger Eye Research Institute, Sheba Medical Center, Sackler School of Medicine, Tel Aviv University, Tel Hashomer (Israel)
(Accepted 8 January 1991) Key words: Central nervous system; Regeneration; Mammal; Tumor necrosis factor
The results of this study attribute to tumor necrosis factor (TNF) a role in regeneration of injured mammalian central nervous system (CNS) axons which grow into their own degenerating environment. This is the first time that a specific factor involved in axonal regeneration has been identified. The axonal environment is occupied mostly by glia cells, i.e., astrocytes and oligodendrocytes. Previous studies have shown that mature oligodendrocytes are inhibitory to axonal growth. Therefore, it seemed likely that application of a factor such as TNF, which has been shown to be cytotoxic to oligodendrocytes, would contribute to the creation of permissive conditions for axonal regeneration. In the present work, injured adult rabbit optic nerves were treated with human recombinant TNF (rhTNF). As a result, abundant newly growing axons (circa 9000, about 4% of the total estimated number of axons in an intact adult rabbit) were observed traversing the site of injury. M a m m a l i a n CNS neurons have a negligible capacity to r e g e n e r a t e after lesions 8'~2'15. In contrast, central neurons of lower vertebrates reliably regenerate after axotomy 9'12'16'18. The prevailing hypothesis that axotomized neurons, m a m m a l i a n as well as n o n - m a m m a l i a n , are capable of regeneration after injury TMis s u p p o r t e d by the fact that axotomized m a m m a l i a n central neurons can regenerate their axons over long distances, if special conditions are provided, such as replacement of a severed central nerve by a segment of a peripheral autologous nerve 1"29. Thus, the success or failure of a regenerative process d e p e n d s upon the response of the non-neuronal cells (astrocytes, oligodendrocytes, microglia, macrophages) in CNS or Schwann in PNS to the axonal injury. As a result of neuronal injury in mammals, astrocytes form glial scar, which is non-supportive to axonal growth 19. M a t u r e oligodendrocytes, which are present in the adult m a m m a l i a n system, have been shown in vitro to inhibit axonal growth 4'22'23. The inhibitory effect of the oligodendrocytes can be circumvented by antibodies specific to the observed inhibitors, as was shown lately 21. We have previously shown that the CNS of lower vertebrates, specifically the regenerating fish optic nerve, is a source of factors which, when applied to injured adult m a m m a l i a n optic nerves, can support regenerative axonal growth 1°'13"j4. For example, in injured adult rabbit optic nerves which were treated with soluble substances derived from regenerating fish optic nerves, along with
low energy H e - N e laser irradiation, which has been shown to delay the d e g e n e r a t i o n process 2, r e m a r k a b l e growth was observed TM. Thus, a b u n d a n t newly growing axons, in a typical CNS environment, were observed traversing the site of injury and extending into the distal stump of nerves, up to a distance of 6.5 mm, 8 weeks postsurgery 14. The observed axons, organized into a special compartment, were indeed newly growing axons, as was evident by the facts that they were either unmyelinated or thinly myelinated and appeared together with abundant growth cones. The total number of unmyelinated axons was greater than in normal unoperated nerves. Anterograde transport of horseradish peroxidase injected intraoculady provided evidence that these newly growing axons were emerging from the retinal ganglion cells. In control injured but untreated nerves, no viable axons or growth cones were observed distal to the site of the injury from 4 - 6 weeks postinjury and beyond. The effect of the applied soluble substances was attributed to the presence of molecules which have been shown to have a cytotoxic effect on oligodendrocytes in vitro 6'26, and to molecules which activate astrocytes to p r o d u c e high levels of laminin 5. This brought to light the intriguing possibility that substitution of the soluble substances with a factor which has b e e n suggested to have a lethal effect on m a t u r e oligodendrocytes, such as T N F 2°'25, might facilitate axonal regeneration across the site of injury and distally.
Correspondence: M. Schwartz, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.
335 Adult rabbits (albinos, from the Weizmann Institute Animal House) were deeply anesthetized with xylazine (5 mg/kg) and ketamine (35 mg/kg). The left optic nerves were exposed, as previously described 2a, and transected almost completely, except for the meningeal membrane, at a distance of 5-6 mm from the eyeball, using a sharpened needle. In all operated nerves, a piece of nitrocellulose, 2-4 mm long and 1 mm wide, was inserted at the site of the injury. In the experimental group, the nitrocellulose was soaked with rhTNF (100 U/nerve; 6 x 107 U/mg); in the control groups it was soaked in medium, free of any active substances. The meningeal membrane was partially spared to ensure continuity of the injured nerve and retention of the applied nitrocellulose. Animals of the experimental group and one control group were also treated daily with low energy He-Ne laser irradiation (10 days, 5 min/day, 35 mW) 14. Six and 8 weeks postsurgery, the experimental and control injured nerves were examined qualitatively and quantitatively by transmission electron microscopy, which allows distinction between newly growing axons and spared ones. Quantitative analyses were carded out as have been described previously 14. Excised experimental and control nerves (6 and 8 weeks postoperatively), which were processed for electron microscopy, were closely analyzed. Characteristics of growth (distal to the site of injury) were observed only in the experimental but not control nerves. Both nerves were systematically examined. Fig. 1 shows some characteristics of the experimental injured nerve after treatment with TNF and laser irradiation. The electron micrographs were taken from cross-sections of experimental nerves, from an area within the second millimeter distal to the site of injury. This area was chosen because in the control nerve it was free of any non-myelinated axons or any other reminiscence of growth. The pictures depict characteristics of newly growing axons, including abundant unmyelinated and thinly myelinated axons embedded in a typical astrocytic environment (Fig. 1A). Among these axons, structures resembling growth c o n e s 14'27, w e r e also observed, one of which is shown in Fig. lB. Some axons were seen in early stages of myelination, as they were surrounded by oligodendrocyte dark cytoplasm (Fig. 1C). In control-injured but untreated nerves, into which nitrocellulose free of active substances was applied, the area corresponding to 2 mm distal to the site of injury was in a state of degeneration (Fig. 1D), containing astrocytic processes and degenerating axons. In a few cases, both in control and in treated operated animal groups, the lesion was less extensive, and there were regions throughout the entire length of the nerve in which viable axons showed the organization, density and thickness of myelin sheath characteristic of
the intact uninjured nerves. Such axons were considered to be spared axons and these animals were not included in our studies. That those spared axons kept their myelin in the presence of TNF, the putative oligodendrocyte cytotoxin, could be due either to the fact that they were not directly exposed to the applied TNF or that they have already undergone a process of remyelination, 6 weeks after the exposure to T N E This issue should be further investigated. In order to evaluate the extent of growth in the experimental treated nerves and to compare it to the situation in the injured non-treated animals, we used the sampling method 14 and we counted the number of unmyelinated and myelinated axons in 4 regions along the nerve: 1 mm proximal to the site of injury, within the second, third and fifth mm distal to the injury, and deep within the distal stump (see Table I). In the treated animals, viable, unmyelinated and myelinated, axons were abundant at areas proximal to the site of injury; their number decreases upon increase in the distance from the globe and the site of injury. In the treated nerves, the maximal number (8466) of unmyelinated axons is seen within the second m m distal to the site of injury. In operated control nerves which were treated with nitrocellulose soaked with medium only, the degeneration process was evident and showed that by 6 and 8 weeks postinjury, no unmyelinated axons were observed at 2 mm to the site of injury. At 8 weeks post-operation only a very small number of myelinated axons were found in the area 2 m m distal to the site of injury (Table I). This small number (127) might be even an overestimation of the concrete number, since it was calculated by the sampling method in which very few axons were actually counted. The fact that no viable axons were observed in the control animals from 2 mm on distal to the site of injury, rules out the possibility that the high number of surviving axons found in the section taken from the area proximal to the site of injury were spared axons, possibly resulting from an incomplete transection but that they were injured ones which would eventually degenerate retrogradely (Table I). The percentage of unmyelinated axons out of the total number of viable axons in the two groups is shown in Table I. The most striking result is the high percentage (93%) of unmyelinated axons in the experimental nerves, 2 mm distal to the site of injury. In the experimental nerve, the area 2 m m distal to the site of injury, in which 93% of the viable axons were unmyelinated, probably represents the growth frontier. The apparent increasing number of myelinated axons in the area located 3 mm distal to the site of injury, relative to the area located 2 mm distal to the site of the lesion, might be due to myelination of some of the growing unmyelinated axons seen in the former (Table I). Such
336
Fig. 1. Characteristics of newly growing axons in TNF-treated nerves. At a distance of 5-6 mm from the eyeball, the nerves were transected except for part of the meningeal membrane26. A piece of nitrocellulose (NC), 3 mm long by 1 mm wide, was inserted at the site of injury; the NC was soaked in rhTNF (100 U, 6 x 107 U/mg) for 1 h prior to the insertion. Beginning within 30 rain after surgery, the rabbits were transocularly irradiated for 5 min each day, for 10 consecutive days, with low energy (6328 nm, 35 roW) He-Ne laser14. Six weeks after the injury, the nerve was removed, fixed by immersion overnight with 1% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer 0.1 M pH 7.4 containing 2.5 mM CaCI2, and then cut serially into 1 mm segments from the retina to the chiasm, which were further processed for electron microscopy (see ref. 14). The electron micrographs shown in this figure were taken from the area of the second millimeter distally to the site of injury. Note in (A) abundant unmyelinated axons (Ax) grouped together surrounded by astrocytic processes (Ap); (B) a typical growth cone (gc) in an astrocytic environment; (C) unmyelinated axons in an early process of myelination by dark cytoplasm of oligodendrocytes (arrows); and (D) control injured untreated rabbit optic nerve, excised 6 weeks postinjury at a distance of 2 mm distal to the site injury, showing only interdigitated astrocytic processes, representing glial sear (gs), and degenerating axonal profiles. Bar represents 1/~m. (A, B, C the same magnification.) myelination is carried out by oligodendrocytes (Fig. 1C), p r e s u m a b l y those which spared the cytotoxic effect of TNF. Alternatively, these myelinated axons at the distal region might represent axons which were injured but in which the degenerative process had not yet reached the e x a m i n e d a r e a (4 m m from the injury). Three-dimensional reconstruction of the treated nerve (Fig. 2) shows that the area occupied by the viable unmyelinated and m y e l i n a t e d axons, as well as their total number, decreases upon distance from the site of injury, emphasizing that the observed unmyelinated axons are, indeed, newly growing axons, rather than spared axons that have lost their myelin sheaths.
Summarizing, we have seen regenerative growth of injured CNS axons into their own injured environment, achieved, for the first time, by a t r e a t m e n t which consisted of the application of a single factor - - T N E T N F was the factor of choice, because it has been shown in vitro by us (M. Lotan, A . C o h e n and M. Schwartz, in p r e p a r a t i o n ) and by others 2°, that rhTNF, in a crossspecies m a n n e r , is active as a cytotoxic factor on oligodendrocytes. The conclusion that the unmyelinated and thinly myelinated axons o b s e r v e d distal to the site of injury are regenerating axons, and not axons spared by the lesion, is based on the following observations: (1) in o p e r a t e d controls, no u n m y e l i n a t e d axons could be
337
mAx IAx ~
GIs+dAx
1mmt__.~,. 1mm Fig. 2. Three-dimensional reconstruction of an injured nerve treated with TNF and laser irradiation, 6 weeks postinjury. The injured rabbit optic nerve was operated and treated as described in Fig. 1. Six weeks later, the animal was anesthetized and the nerve excised. Thin sections were cut from each 1 mm segment and were mounted on grids, photographed at x 140. Compartments identified in each cross-section contained the following: unmyelinated, presumably growing axons (Ax) and myelinated axons (mAx); nitrocellulose (NC); and glial scar and degenerating axons (Gls + dAx). Arrow indicates first section in which nitrocellulose is seen. TABLE I
Quantitative analysis of operated nerves Animals from the experimental and operated control groups were quantitatively analyzed using the sampling method. Further, 6 experimental and 10 operated control animals were qualitatively analyzed. Experimental nerves which contained an area with the organization, and density and thickness of myelin sheath characteristic of intact uninjured nerves, and in which this area ran the entire length of the nerve, were not included in the analysis14,since they were considered to overcome an incomplete injury, In the experimental nerves, the site of injury is between 6 and 7 mm distal to the globe, whereas the last region in which nitrocellulosecould be observed, was 10 mm distal to the globe. In control operated nerves examined 6 weeks post-injury, no viable axons, myelinated or unmyelinated, could be observed at any of the examined regions. The table intentionally gives an example of an operated control nerve, excised 8 weeks post-injury, in which a few unmyelinated axons were observed, but only proximally to the site of the injury.
Nerve region
Number of counted axons (% of total viable axons) Experimental Unmyelinated
1 mm 7530 (19%) proximal to injury
Operated control Myelinated Unmyelinated Myelinated 31357
195 (11%)
1666
2 mm distal to injury
8466 (93%)
591
0
127
3 mm distal to injury
200 (9%)
2272
0
0
5 mm distal to injury Deep distal
0
200
0
0
detected distal to the site of injury; (2) in the TNFtreated nerves, the n u m b e r of viable axons decreases with increasing distance from the site of injury (Table I). If u n m y e l i n a t e d axons were axons spared by the injury, and demyelinated by the TNF, survival of these axons, either as u n m y e l i n a t e d or myelinated, at more distal locations and throughout the entire length of the nerve would be seen; and (3) along with the u n m y e l i n a t e d axons, a b u n d a n t growth cones do appear. These newly growing axons could be sprouts from the cell bodies of the injured axons, or sprouts from the cut tip. Whatever the nature of these sprouts, they were absent from sites distal to the injury in all operated control animals. The newly growing axons observed 6 weeks postsurgery traversed the site of injury. The lack of further growth might be a reflection of (a) the limited time elapsed postinjury before the nerves were examined; (b) the limited a m o u n t of T N F provided by the single application; (c) the limited accessibility to TNF, provided by the nitrocellulose up to a distance of 4 m m distal to the lesion; and (d) the need for additional factor(s), like those which activate the astrocytes and are present among the soluble substances in regenerating fish optic nerves 5. In the absence of such activating factors, these astrocytes eventually develop into non-supportive scar° forming cells, and elongation of the growing axons will be prevented. U n d e r normal physiological conditions, T N F may be a product of macrophages, which invade nerves after injury 17. If that is the case, one would expect T N F to
338 facilitate axonal growth and regeneration, provided that the invasion of macrophages is in temporal coordination with the injury-induced activation of the cell bodies of the injured axons. A temporal shift of the local invasion of macrophages after injury might lead to absence of T N F at the critical period. It is also possible that T N F is not the product of macrophages but of other cells, such as activated astrocytes 2°, which, in the m a m m a l i a n CNS, might not be activated or, at least, not activated at the
thus supporting their regeneration. A t this stage, we cannot rule out the possibility that the applied T N F might have additional effects on additional cells, such as astrocytes, resident microglia, invading macrophages and/or lymphocytes. Such an effect might be primarily manifested by changes in IL-1 and IL-2 levels 7 and, consequently, possibly on neurotrophic factors 11.
shortage of TNF, possibly helping to eliminate inhibitors from the e n v i r o n m e n t surrounding injured axons and
We thank Prof. D. Wallach and Dr. H. Engelman, for the fruitful discussions and for their generosity in giving us rhTNF and anti-TNF antibodies. This work is supported by Farmitalia (Milan) and by the Daniel Heumann Fund for Spinal Cord Injury Research. M. Schwartz is an incumbent of the Maurice and Ilse Katz Professorial Chair in Neuroimmunology.
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15 Misantone, L.J., Gershenbaum, M. and Murray, M., Viability of retinal ganglion cells after nerve crush in adult rats, J. Neurocytol., 13 (1984) 449-465. 16 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. Comp. Neurol., 168 (1976) 175-196. 17 Perry, V.H., Brown, M.C. and Gordon, S., The macrophage response to central and peripheral nerve injury: a possible role for macrophages in regeneration, J. Exp. Med., 165 (1987) 1218-1223. 18 Ram6n y Cajal, S., Degeneration and Regeneration of Nervous System, Vol. 1, R.M. May (Transl.), Hafner Publ. Co., New York, 1959. 19 Reier, P.J., Stensaas, L. and Guth L., The astroeytes' scar as an impediment to regeneration in the central nervous system. In C.C. Kao, R.P. Bunge and P.J. Reier (Eds.), Spinal Cord Reconstruction, Raven, New York, 1983, pp. 163-196. 20 Robbins, D.S., Shirazi, Y., Drysdale, B., Liberman, A., Shin, H.S. and Shin, M.L., Production of cytotoxic factor for oligodendrocytes by stimulated astrocytes, J. lmmunoL, 139 (1987) 2593-2597. 21 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. 22 Schwab, M.E. and Thoenen, H., Dissociated neurons regenerate into sciatic but not optic nerve explants in culture irrespective of neurotrophic factors, J. Neurosci., 5 (1985) 2415-2423. 23 Schwab, M.E. and Caroni, P., Oligodendrocytes and CNS myelin are non-permissive substrates for neurite growth and fibroblast spreading in vitro, J. Neurosci., 8 (1988) 2381-2393. 24 Schwartz, M., Belkin, M., Harel, A., Solomon, A., Lavie, V., Hadani, M., Rachailovich, I. and Stein-Izsak, C., Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbit~, Science, 228 (1985) 600-603. 25 Selmaj, K.W. and Raine, C.S., Tumor necrosis factor mediates myelin and oligodendrocyte damage in vivo, Ann. Neurol., 23 (1988) 339-346. 26 Sivron, T., Cohen, A., Duvdevani, R. and Schwartz, M., Glial response to axonal injury in vitro manifestation and implication for regeneration, Glia, 3 (1990) 267-276. 27 Skoff, R.P. and Hamburger, V., Fine structure of dendritic and axonal growth cones in embryonic chick spinal cord, J. Comp. Neurol., 153 (1974) 107-148. 28 Solomon, A., Belkin, M., Hadani, M., Harel, A., Rachaiiovich, I., Lavie, V. and Schwartz, M., A new transorbital surgical approach to the rabbit's optic nerve, J. Neurosci. Methods, 12 (1985) 259-262. 29 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.
appropriate time. Exogenous TNF, applied to mammalian injured CNS, might overcome the temporal or spatial
