Dexanabinol (HU-211) has a beneficial effect on axonal sprouting and survival after rat optic nerve crush injury.

Dexanabinol (HU-211) is a synthetic non-psychotropic cannabinoid and a non-competitive NMDA-receptor antagonist. The beneficial effect of dexanabinol on prevention of degeneration and promotion of regeneration was studied on the crush-injured rat optic nerve model. Sprague-Dawley rats were subjected to a calibrated crush injury of the optic nerve and treated with a single intraperitoneal injection of dexanabinol (7 mg/kg), its vehicle only or were untreated. Transmission electron microscopic analysis of the excised optic nerves was performed after 30 days. In the dexanabinol treated rats, the site of injury was traversed by unmyelinated and thinly myelinated axons, possibly indicative of regenerative growth. No such growth was detectable in the controls. Viable axons were found 0.5 mm distal to the site of injury in 6 of 8 dexanabinol treated rats, but in only 1 of 10 rats in the control groups. These results have clinical implications for the prevention of secondary degeneration and promotion of regeneration after injuries to the central nervous system.

Dexanabinol (HU-211) effect on experimental autoimmune encephalomyelitis: implications for the treatment of acute relapses of multiple sclerosis.

Dexanabinol (HU-211) is a synthetic non-psychotropic cannabinoid which suppresses TNF-alpha production in the brain and peripheral blood. The effects of dexanabinol in rat experimental autoimmune encephalomyelitis (EAE) were studied using different doses, modes of administration and time regimes. Dexanabinol, 5 mg/kg i.v. given once after disease onset (day 10), significantly reduced maximal EAE score. Increasing the dose or treatment duration resulted in further suppression of EAE. Drug administration at earlier phases during disease induction was not effective. Histological studies supported the clinical findings demonstrating reduction in the inflammatory response in the brain and spinal cord in animals treated with dexanabinol. The results suggest that dexanabinol may provide an alternative mode of treatment for acute exacerbations of multiple sclerosis (MS).

Complete transection of rat optic nerve while sparing the meninges and the vasculature: an experimental model for optic nerve neuropathy and trauma.

In this study we present a method to achieve a complete transection of optic nerve axons in adult rat, while preserving the vasculature and retaining the continuity of the meninges. Under deep anesthesia, the optic nerve of adult rat is exposed. Using specially designed instruments built from disposable glass microsampling pipettes, a small opening is created in the meninges of the optic nerve, 2-3 mm behind the eye globe. A glass dissector is introduced through the opening and is used to cut all the axons through the whole width of the nerve. Complete transection of the optic nerve axons was achieved, while retaining the continuity of the meninges and avoiding damage to the nerve's vascular supply. Transection was confirmed by transillumination showing a complete gap in the continuity of the nerve axons, and by both morphological and electrophysiological criteria. Nerve transection performed by the conventional technique leads to neuroma formation and hampers regeneration. Crush injury may cause nerve ischemia, which is detrimental to axonal recovery. Both of these disadvantages are avoided by the method of transection presented here. The opening created in the 'meningeal tube' can be used to inject substances that may be of benefit in recovery, rescue and/or regeneration of the injured axons. The model is particularly suitable for in vivo studies on nerve regeneration, and especially for screening of putative therapeutic agents.

Transplantation of activated macrophages overcomes central nervous system regrowth failure.

Macrophages have long been known to play a key role in the healing processes of tissues that regenerate after injury; however, the nature of their involvement in healing of the injured central nervous system (CNS) is still a subject of controversy. Here we show that the absence of regrowth in transected rat optic nerve (which, like all other CNS nerves in mammals, cannot regenerate after injury) can be overcome by local transplantation of macrophages preincubated ex vivo with segments of a nerve (e.g., sciatic nerve) that can regenerate after injury. The observed effect of the transplanted macrophages was found to be an outcome of their stimulated activity, as indicated by phagocytosis. Thus, macrophage phagocytic activity was stimulated by their preincubation with sciatic nerve segments but inhibited by their preincubation with optic nerve segments. We conclude that the inability of nerves of the mammalian CNS to regenerate is related to the failure of their macrophages recruited after injury to acquire growth-supportive activity. We attribute this failure to the presence of a CNS resident macrophage inhibitory activity, which may be the biochemical basis underlying the immune privilege of the CNS. The transplantation of suitably activated macrophages into injured nerves may overcome multiple malfunctioning aspects of the CNS response to trauma, and thus may be developed into a novel, practical, and multipotent therapy for CNS injuries.

Vitronectin overrides a negative effect of TNF-alpha on astrocyte migration.

Morphogenesis and tissue repair require appropriate cross-talk between the cells and their surrounding milieu, which includes extracellular components and soluble factors, e.g., cytokines and growth factors. The present work deals with this communication needed for recovery after axotomy in the central nervous system (CNS). The failure of CNS axons to regenerate after axonal injury has been attributed, in part, to astrocyte failure to repopulate the injury site. The goal of this work was to provide an in vitro model to mimic the in vivo response of astrocytes to nerve injury and to find ways to modulate this response and create a milieu that favors astrocyte migration and repopulation of the injury site. In an astrocyte scratch wound model, we blocked astrocyte migration by tumor necrosis factor alpha (TNF-alpha). This effect could not be reversed by astrocyte migration-inducing factors such as transforming growth factor beta 1 (TGF-beta 1) or by any of the tested extracellular matrix (ECM) components (laminin and fibronectin) except for vitronectin (Vn). Vn, added together with TNF-alpha, counteracted the TNF-alpha blockage and allowed a massive migration of astrocytes (not due to cell proliferation) beyond that allowed by Vn only. Heparan sulfate proteoglycans (HSPG) were shown to be involved in the migration. The results may be relevant to regeneration of CNS axons, and may also provide an example that an extracellular component (Vn) can overcome and neutralize a negative effect of a growth factor/cytokine (TNF-alpha) and can act in synergy with other features of this cytokine to promote a necessary function (e.g., cell migration) that is otherwise inhibited.

Recovery of visual response of injured adult rat optic nerves treated with transglutaminase.

Failure of axons of the central nervous system in adult mammals to regenerate spontaneously after injury is attributed in part to inhibitory molecules associated with oligodendrocytes. Regeneration of central nervous system axons in fish is correlated with the presence of a transglutaminase. This enzyme dimerizes interleukin-2, and the product is cytotoxic to oligodendrocytes in vitro. Application of this nerve-derived transglutaminase to rat optic nerves, in which the injury had caused the loss of visual evoked potential response to light, promoted the recovery of that response within 6 weeks after injury. Transmission electron microscopy analysis revealed the concomitant appearance of axons in the distal stump of the optic nerve.

Vimentin immunoreactive glial cells in the fish optic nerve: implications for regeneration.

The poor regenerative ability of neurons of the central nervous system in mammals, as compared with their counterpart in fish or amphibians, is thought to stem from differences in their immediate nonneuronal environment and its response to axonal injury. We describe one aspect of the environmental response to axonal injury in a spontaneously regenerating system--the fish optic nerve. The aspect under investigation was the reaction of glial cells at the injury site. This was examined by the use of antibodies that specifically recognize vimentin in fish glial cells. In the present study, affinity-purified vimentin antibodies were raised against a nonconserved N-terminal 14-amino acid peptide, which was predicted from the nucleotide sequence of vimentin. These antibodies were found to react specifically with glial cells in vitro. Moreover, the antivimentin antibodies stained both the optic nerve and the optic tract, but with different patterns. Specificity of the antibodies was verified by protein immunoblotting, tissue distribution, and labeling patterns. After injury, vimentin immunoreactivity initially disappeared from the site of the lesion due to cell death. Early signs of glial cell migration toward the injury site were evident a few days later. It is suggested that the reappearance of vimentin-positive glial cells at the site of injury is associated with axonal elongation across it, and that they contribute to the regenerative ability of the fish optic nerve.

A new method for expressing axonal size: rat optic nerve analysis.

Analysis of the shape of the cross sections of adult rat optic nerve axons reveals that the majority of axons do not have a true circular shape. Therefore, determination of axonal size has to utilize methods of approximation. The method presented here utilizes three calculated parameters for expression of axonal size: (i) axonal diameter, as calculated from its area, or (ii) axonal diameter, as calculated from its perimeter, both assuming axonal shape to be a perfect circle and (iii) axonal shape factor, which represents the divergence of the axon from a perfect circular shape. The use of the calculated axonal diameter, with a correction for its shape factor, provides a normalized way of expressing axonal size.

Axonal regeneration is associated with glial migration: comparison between the injured optic nerves of fish and rats.

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted at the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Gangliosides attenuate axonal loss after optic nerve injury.

Gangliosides have been shown to be capable of protecting nerve tissue from mechanical and biochemical insults and promoting their repair. The present study provides morphologic evidence that monosialogangliosides attenuate the degenerative process at the distal stump of the rat optic nerve after crush injury. Injured rat optic nerves were treated for 7 days after injury with daily intraperitoneal injections of monosialogangliosides (30 mg/kg/day), and compared with untreated injured controls with respect to the number of viable axons 2 and 4 weeks after injury, as indicated by transmission electron microscopy. After 2 weeks, the mean number of viable axons in the treated optic nerves (n = 5) was slightly higher than in the controls (n = 5). Four weeks after injury, although the absolute number in both the experimental and the control groups had dropped, it was about seven-fold higher in the treated animals (1696 +/- 1149, n = 7) than in the untreated animals (216 +/- 65, n = 6); this difference was statistically significant. These findings, which offer some insight as to how monosialogangliosides affect injured nerves, may have important implications for treatment in cases of optic nerve injury.

GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.

This study demonstrates the earliest reported effects of GM1 treatment on crush-injured axons of the mammalian optic nerve. GM1, administered intraperitoneally immediately after injury, was found to reduce the injury-induced metabolic deficit in nerve activity within 2 hr of injury, as measured by changes in the nicotine-amine adenine dinucleotide redox state. After 4 wk, transmission electron microscopy 1 mm distal to the site of injury revealed a sevenfold increase in axonal survival in GM1-treated compared to untreated injured nerves. These results emphasize the beneficial effect of GM1 on injured optic nerves as well as the correlation between immediate and long-term consequences of the injury. Thus, these results have implications for treating damaged optic nerves.

Disappearance of astrocytes and invasion of macrophages following crush injury of adult rodent optic nerves: implications for regeneration.

Injury to the mammalian central nervous system results in loss of function because of its inability to regenerate. It has been postulated that some axons in the mammalian central nervous system have the ability to regenerate but fail to do so because of the inhospitable nature of surrounding glial cells. For example, mature oligodendrocytes were shown to inhibit axonal growth, and astrocytes were shown to form scar tissue that is nonsupportive for growth. In the present study we report an additional phenomenon which might explain the failure of axons to elongate across the site of the injury, namely, the absence of astrocytes from the crush site between the glial scar and the distal stump. Astrocytes began to disappear from the injury site as early as 2 days after the injury. After 1 week the site was necrotic and contained very few glial cells and numerous macrophages. Disappearance of glial cells was demonstrated in both rabbit and rat optic nerves by light microscopy, using antibodies directed against glial fibrillary acidic protein, and by transmission electron microscopy. Results are discussed with reference to possible implications of the long-lasting absence of astrocytes from the injury site, especially in view of the differences between the present findings in rodents and our recent observations in fish.

Horseradish peroxidase labeling of growth cones and axons beyond the site of injury in injured rabbit optic nerve axons growing in their own environment.

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.

Tumor necrosis factor facilitates regeneration of injured central nervous system axons.

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.

New surgical approach to overcome the inability of injured mammalian axons to grow within their environment.

We present a new method for creating conditions conductive to axonal growth in injured optic nerves of adult rabbits. The surgical approach consists of making a cavity in the adult rabbit optic nerve, into which a piece of nitrocellulose soaked with conditioned medium originating from regenerating fish optic nerves is implanted. In addition, daily irradiation (10 days, 5 min, 35 mW) with low energy He-Ne laser is carried out. Such a combined treatment may open a door to neurobiologists and clinicians, hoping to unravel the enigma of mammalian CNS regeneration.

Growth of injured rabbit optic axons within their degenerating optic nerve.

Spontaneous growth of axons after injury is extremely limited in the mammalian central nervous system (CNS). It is now clear, however, that injured CNS axons can be induced to elongate when provided with a suitable environment. Thus injured CNS axons can elongate, but they do not do so unless their environment is altered. We now show apparent regenerative growth of injured optic axons. This growth is achieved in the adult rabbit optic nerve by the use of a combined treatment consisting of: (1) supplying soluble substances originating from growing axons to be injured rabbit optic nerves (Schwartz et al., Science, 228:600-603, 1985), and (2) application of low energy He-Ne laser irradiation, which appears to delay degenerative changes in the injured axons (Schwartz et al., Lasers Surg. Med., 7:51-55, 1985; Assia et al., Brain Res., 476:205-212, 1988). Two to 8 weeks after this treatment, unmyelinated and thinly myelinated axons are found at the lesion site and distal to it. Morphological and immunocytochemical evidence indicate that these thinly myelinated and unmyelinated axons are growing in close association with glial cells. Only these axons are identified as being growing axons. These newly growing axons transverse the site of injury and extend into the distal stump of the nerve, which contains degenerating axons. Axons of this type could be detected distal to the lesion only in nerves subjected to the combined treatment. No unmyelinated or thinly myelinated axons in association with glial cells were seen at 6 or 8 weeks postoperatively in nerves that were not treated, or in nerves in which the two stumps were completely disconnected. Two millimeters distal to the site of injury, the growing axons are confined to a compartment comprising 5%-30% of the cross section of the nerve. A temporal analysis indicates that axons have grown as far as 6 mm distal to the site of injury, by 8 weeks postoperatively. Anterograde labeling with horseradish peroxidase, injected intraocularly, indicates that some of these newly growing axons arise from retinal ganglion cells.

Optic nerve regeneration--new aspects.

Injury to the optic nerve and its environment provokes a process of degeneration that leads to a degenerative process resulting in an irreversible loss of visual function. We succeeded in stimulating regeneration in injured optic nerves of adult rabbits. The stimulus to regeneration was achieved by implanting in injured optic nerves of adult rabbits substances originating from growing (regenerating) optic nerves of fish or developing optic nerves of neonatal rabbits, and by delaying the degenerative process by irradiating the injured optic nerve with a low energy laser. The effect was manifested by abundant growth of new fibers across the injury site and by other manifestation of regeneration characteristics.

Morphological response of injured adult rabbit optic nerve to implants containing media conditioned by growing optic nerves.

Adult rabbit retina can express regeneration-associated characteristics after optic nerve injury, provided it is supplied with appropriate diffusible substances originating from media conditioned by regenerating fish optic nerves or by optic nerves of a newborn rabbit [Hadani et al., Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7965; Schwartz et al., Science, 228 (1985) 600]. This was shown by applying the active substances to the injured axons in the form of 'wrap-around' implants, consisting of collagen-coated silicone tubes which had been soaked in the conditioned media (CM). The regeneration-associated response was manifested biochemically and by sprouting of nerve fibers in culture. The present work provides morphological evidence that the implantation prolongs survival of ganglion cells and optic nerve fibers and induces new growth. Light microscopic analysis (using horseradish peroxidase (HRP) for labeling the fibers) revealed, 1 week following optic nerve injury, labeled fibers and ganglion cells in both the implanted and control (injured only or injured and implanted with collagen-coated silicone tubes free of CM) nerves. However, from the second week after the injury, distinct differences in the appearance of viable ganglion cells and labeled fibers, were seen between experimental and control preparations. In sections taken through the optic nerve, at the region distal to the site of injury, HRP-labeled fibers were seen in the experimental nerves 1 week, 2 weeks and to a significantly lesser extent 1 month after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and cellular aspects of axon-glia interaction in CNS regeneration.

The relationships of neurons and non-neuronal cells are vital for the maintenance and function of neurons. Trauma alters these relationships causing proliferation of non-neuronal cells and, in adult mammalian CNS, presumably disturbs the environmental support needed for regeneration. A supportive environment can be restored by introducing a regenerating nerve to injured mammalian CNS. This response is probably due, at least in part, to diffusible substances secreted by the non-neuronal cells. We have obtained diffusible substances from either regenerating fish optic nerves or neonatal rabbit optic nerves and applied them around crushed adult rabbit optic nerves. This manipulation caused the adult nerve to show regenerative changes: a general increase of protein synthesis in the retinas; selective increase in synthesis of a few polypeptides in the retinas; sprouting from the retinas in vitro; increased viability of nerve fibers as shown by HRP staining; and the appearance of growth cones adjacent to glial limitans in the injured nerves. We termed these diffusible, active substances "Growth Associated Triggering Factors" (GATFs). In addition to the phenomena described above, the active substances (obtained in the form of media conditioned by regenerating fish optic nerve or neonatal rabbit optic nerve) caused various other changes in the injured nerve itself: acceleration of non-neuronal cell proliferation; changes in the protein pattern, e.g. an increase in a 12 kDa polypeptide which might be a second mediator in the cascade of events leading to regeneration; increased laminin immunoreactive sites in the nerve; and the acquisition of growth supportive activity in media conditioned by the implanted injured nerves.(ABSTRACT TRUNCATED AT 250 WORDS)