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.

Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbits.

Regeneration of fish optic nerve (representing regenerative central nervous system) was accompanied by increased activity of regeneration-triggering factors produced by nonneuronal cells. A graft of regenerating fish optic nerve, or a "wrap-around" implant containing medium conditioned by it, induced a response associated with regeneration in injured optic nerves of adult rabbits (representing a nonregenerative central nervous system). This response was manifested by an increase of general protein synthesis and of selective polypeptides in the retinas and by the ability of the retina to sprout in culture.

Family of human alpha-interferon-like sequences.

An interferon-alpha-like sequence was isolated from a human genomic library by hybridization with a 15-base oligonucleotide. The sequence also showed homology to alpha-interferon and was most closely related to the leukocyte interferon-M gene fragment. The original isolate cross-hybridized to a family of sequences, 10 of which were isolated as clones. Some of these sequences were located within a few kilobases of alpha-interferon genes, consistent with our assignment of several members of the family to human chromosome 9 which also has the beta 1- and alpha-interferon genes.

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.

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)

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).

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.

A new transorbital surgical approach to the rabbit's optic nerve.

This work describes a surgical approach which establishes the rabbit's visual system as an experimental model for studying CNS regeneration. Using this model, the optic nerve, its cell bodies, and the axons, are easily accessible through an orbital approach, without the need for craniotomy and brain retraction. This surgical approach allows transplantation and 'wrap around' implantations of nerve segments from xenogeneic and syngeneic systems and diffusible substances derived from them, respectively. Furthermore, it enables studies aimed at determining deficiencies in mammalian CNS and investigating methods of augmentating mammalian CNS regeneration.

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.

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.

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)

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.

Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.

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.

Cloning and characterization of DNA complementary to the measles virus mRNA encoding hemagglutinin and matrix protein.

Since cloning and characterization of DNA complementary to measles virus mRNA encoding for the nucleocapsid protein (M. Gorecki and S. Rozenblatt, Proc, Natl. Acad, Sci, U.S.A. 77:3686--3690, 1980), two additional measles-specific clones containing different classes of sequences have been characterized. The cloned plasmids contain inserts of 480 and 530 base pairs as shown by agarose gel electrophoresis and electron microscopy. The sizes of the mRNA species complementary to these inserts are 1,700 and 1,550 nucleotides, respectively as determined by the Northern technique. The cloned DNA fragments were further identified as reverse transcripts of the mRNA coding for the glycoprotein and matrix protein of measles virus. The major cell-free translation products of mRNA selected by hybridization to the individual cloned DNAs comigrated with the 70K in vitro products and matrix proteins. One of the cell-free translation products (70K) was also immunoprecipitated specifically with monoclonal antibodies against measles virus glycoprotein.

Human cytoplasmic superoxide dismutase cDNA clone: a probe for studying the molecular biology of Down syndrome.

The gene locus for human cytoplasmic superoxide dismutase (SOD-1; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is located in or near a region of chromosome 21 known to be involved in Down syndrome. To approach the molecular biology of this genetic disease we have constructed a SOD-1 cDNA clone. Poly(A)-containing RNA enriched for human SOD-1 mRNA was isolated, used to synthesize double-stranded cDNA, and inserted into the endonuclease Pst I site of the plasmid pBR322. The chimeric molecules were used to transform Escherichia coli. Two clones containing SOD-1 cDNA inserts were identified by their ability to hybridize specifically with mRNA coding for SOD-1. Each of these clones carries a 650-base-pair insert, as was determined by restriction enzyme digestion and electron microscopic heteroduplex analysis. Hybridization of labeled cloned cDNA to RNA blots revealed two distinct SOD-1 mRNA classes of 500 and 700 nucleotides. The data suggest that both are polyadenylylated and are coded by chromosome 21.

Substances originating from the optic nerve of neonatal rabbit induce regeneration-associated response in the injured optic nerve of adult rabbit.

We have recently shown that cell bodies of an injured optic nerve of adult rabbit can be induced to express regeneration-associated response by external signals derived from nonneuronal cells of regenerating nerves of lower vertebrates. In this study it is shown that even substances derived from a nonregenerating mammalian system also can trigger such a regenerative response. Thus, substances derived from intact nerves of neonatal rabbits and of adult rabbits, to a lesser extent, were active in triggering a regeneration-associated response, whereas substances derived from injured nerves of adult rabbit were not. However, if subsequent to the injury the nerve was implanted with silicone tube containing medium conditioned by neonatal optic nerves, the substances derived from the implanted injured nerve were active. Thus, it appears that the ability of a periaxonal environment to provide triggering substances correlates with axonal growth. Therefore, we named these substances "growth-associated triggering factors" (GATFs). It is suggested that mammalian cells are unable to express a regenerative response after an injury due to the failure of their nonneuronal cells to produce regeneration-triggering substances. This disability may be circumvented by an appropriate implantation procedure.

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.

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.