The roles of reactive gliosis and mitosis on tropic factor production in traumatized nervous system tissue

Proximal stumps of rat sciatic nerves were attached to inlet ends of Y-shaped silastic implants and offered a 'choice' of growing toward an Elvax pellet containing homogenate from previously crushed optic nerve which had been exposed to saline or cytosine arabinofuranoside (AraC). Previous studies indicate that AraC administration inhibits reactive gliosis in crushed optic nerve. Preferential or exclusive growth of axons occurred in implant forks attached to pellets containing saline- (vs drug-) exposed optic nerve homogenates. In contrast, inhibition of Schwann cell mitosis had no discernible effect on tropic factor production in distal stumps of transected sciatic nerves. Tropic activity of homogenates from cultures containing reactive-like astrocytes was nearly 4 times higher than homogenates not containing these cells. Results suggest a possible link between formation of hypertrophic reactive astrocytes and production of neurotropic factor.     

Tropic factors in reactive mammalian central nervous system tissue

Previous studies suggest that distal stumps of transected peripheral nerves contain diffusible factors which can attract/support axonal regeneration over distances of several mm in vivo. The present experiments were undertaken to determine if this is so for distal regions of traumatized central (i.e., optic) nerves. Proximal stumps of transected rat sciatic nerves were inserted into the single inlet ends of 6 mm long Y-shaped Silastic implants. Alternative ‘lures’ were attached to the paired outlets, the ability of these lures to attract/support regeneration of nerve fibers in their associated forks assessed 3.5 weeks postoperatively. Exclusive or preferential growth of nerve fibers occurred in implant forks associated with optic nerve grafts, of Elvax pellets containing homogenate obtained from previously crushed (reactive) optic nerves. Grafts of tendon, as well as homogenate from unoperated optic nerve had no effect.Results suggest that, with repect to the assay used, degenerating optic nerve tissue contains factor(s) which can attract/support regenerating nerve fibers.     

Effect of cytosine arabinofuranoside (AraC) on reactive gliosis in vivo. An immunohistochemical and morphometric study

Glial reactivity is believed to contribute to the lack of functional recovery after injury to the mammalian central nervous system. The role of glial mitosis in the progression of events associated with reactive gliosis has received little attention. In the present study, the expression of reactive gliosis distal to the site of crush in rat optic nerves was assessed in the presence and absence of a chronically administered mitotic inhibitor, cytosine arabinofuranoside (AraC). Right optic nerves were crushed and animals sacrificed 1, 2 or 3 weeks later. Parameters assessed were (1) glial hypertrophy, (2) degradation of myelin sheaths and (3) ability of tissue to stain with antisera raised against glial filament protein (GFA), actin and vimentin. In saline treated animals, greater than 90% of the myelin sheaths distal to the site of axonal injury had degraded within 7 days postoperatively. Glial hypertrophy was evident by the second week after after crush and increased progressively. The number of GFA-positive profiles (i.e., cells) increased between 1 and 3 weeks postoperatively. Vimentin staining increased 4-fold between 1 and 2 weeks after injury and subsequently showed no change. Actin staining rose 3-fold between 1 and 2 weeks after injury, but decreased by the third postoperative week. In AraC treated animals, almost 50% of the myelin sheaths distal to the injury site were preserved a week after surgery. A delay in myelin degradation continued until the second postoperative week. Glial hypertrophy was evident at the 2 and 3 week time points. However, the extent of hypertrophy was substantially lower in drug (vs saline) treated animals. Vimentin staining never rose above minimal levels in AraC treated animals. Actin staining in AraC rats at 2 weeks postoperatively was equivalent to that in saline injected animals, but in contrast to the results in the latter group, increased (3-fold) between 2 and 3 weeks after crush. Results indicate a delay in the expression of reactive gliosis with chronic administration of AraC. It is proposed that this might be due to a delay in the appearance of 'signals' (e.g., myelin debris) which initiate the process of reactive gliosis.     

The role of post-traumatic mitosis in elevation of anaerobic metabolism enzyme (lactic acid dehydrogenase) activity in degenerating central and peripheral nerve

Glial and Schwann cells undergo marked biochemical and morphological alterations following axonal injury. In the present experiments, the extent of enzyme activity associated with anaerobic (LDH, lactic dehydrogenase) vs aerobic (SDH, succinic dehydrogenase) respiration was assessed distal to the site of nerve fiber injury. Studies were performed in rat central (optic) and peripheral (sciatic) nerves at 2, 7 and 14 days postoperatively (d.p.o.). In sciatic nerves, LDH activity rose 3-fold in traumatized (vs unoperated control) nerve tissue between 2 and 7 d.p.o. and remained elevated at 14 d.p.o. SDH activity in traumatized nerve was equal to that in unoperated nerve at 7 d.p.o., but decreased at 14 d.p.o. LDH activity in optic nerve at 2 d.p.o. was equivalent to that in control nerve, but rose approximately two-fold by 7 d.p.o. However, unlike peripheral nerve, activity in traumatized optic nerve decreased to control levels at 14 d.p.o. SDH activity in traumatized optic nerve remained unchanged at any timepoint examined. Taken in concert, these data are consistent with the hypothesis that there is an overall shift in CNS glial and Schwann cell metabolism from aerobic to anaerobic respiration following nerve injury. Additional studies were performed to determine if this shift requires prior Schwann or glial cell mitosis. Administration of mitotic inhibitor (AraC, cytosine arabinofuranoside) inhibited post-traumatic elevations in LDH activity in optic, but not peripheral nerve. No significant effect of the drug on axonal degeneration (as assessed by saxitoxin binding) was observed.     

Reactive glial cells in CNS demyelination contain both GC and GFAP

The glial cell response to anti-galactocerebroside (GC) induced demyelination of the cat optic nerve was studied using electron microscopy and immunocytochemistry. Oligodendrocytes, which are a primary target for anti-GC, were depleted in the early lesions but astrocytes survived and showed reactive changes. Astrocytic processes exhibited dual staining for both GC and glialfibrillary acidic protein, a feature not seen in astrocytes outside the lesion or in normal optic nerve. These reactive astrocytes did not stain for anti-myelin basic protein, nor did they contain myelin debris, making it unlikely that the GC immunoreactivity was due to phagocytosis of myelin. Rather, it is postulated that the presence of GC in these cells represents a process of dedifferentiation to a more primitive state in which both astrocytic and oligodendrocytic determinants are synthesised, and that these reactive glial cells may be precursors of a new population of remyelinating oligodendrocytes.     

Optic Disc and Optic Nerve of the Blind Cape Mole-Rat (Georychus capensis): A Proposed Model for Naturally Occurring Reactive Gliosis

Recent studies of the visual system of animal species that live in a subterranean environment show not only regressive but also progressive morphological features. In this regard the aim of the present investigation is to describe the structural organisation of the eye and optic nerve of the adult Cape mole-rat, with special emphasis on both glial cell population and myelination. The main results are: (a) astrocytes show identical features to those occurring in reactive gliosis; (b) optic fibers vary greatly in diameter; (c) very small axons are myelinated and are often surrounded by a thicker sheath than larger optic fibers; (d) a large onion bulb-like structure composed of optic fibers, glia, and ganglion cells is found within the choriocapillary layer. These results suggest that the Cape mole-rat and probably other subterranean rodents may serve as a model to study spontaneous gliosis as well as mechanisms involved in myelination and degenerative processes.     

Post-traumatic alterations in glutamine synthetase activity in peripheral and central nerves

Glutamine synthetase (GS) was assessed distal to the site of axonal injury in traumatized rat central (optic) or peripheral (tibial) nerve prior to and during periods of reactive PNS and CNS glial cell hypertrophy. GS activity in crushed optic nerve remained equivalent to that in unoperated control tissue at 2 days postoperatively, but rose by 30% at 7 and 12 days after surgery. In contrast, GS activity in traumatized peripheral nerve was significantly lower than in unoperated controls at 7 and 12 days postoperatively. Administration of mitotic inhibitor (AraC, cytosine arabinofuranoside) prevented significant trauma-induced alterations in GS activity in optic nerves at 7, and in peripheral nerves at 7 and 12 days postoperatively. Results suggest significant alterations in glial/Schwann GS activity after nerve fiber injury and that these alterations can be delayed/prevented by administration of mitotic inhibitors.     

Ultrastruktur der Glia- und Axonschädigung durch 6-Aminonicotinamid (6 An) am Sehnerv der Ratte

Summary Light and electron microscopic studies were performed in optic nerves and tracts of 25 adult rats until 21 days following intraperitoneal application of 10 mg/kg 6 AN, an antimetabolite of nicotinamide. The results were: Progressive astrocytic swelling (ER and perinuclear cisternae) in the orbital portion of the optic nerve. The resulting spongy state disappeared within 10–12 days by scar forming and phagocytic fibrous astrocytes. Within 2–6 days mild swelling of the ER in oligodendrocytes occurred together with extreme myelin swelling, probably by splitting of the first inner intraperiod line, leaving the axon unchanged. The resulting spongy state was observed only in the optic tract and the cerebral portion of the nerve. This was followed by myelin break down and axon degeneration in the whole optic nerve; probably the result of both myelin swelling and secondary (Wallerian) degeneration. In the perilaminar portion some special alterations occurred in the third week p.i.: Axonal swelling, remyelination and inner glial processes of myelin sheaths containing glial filaments. The simultaneous application of 10 mg 6 AN and 10 mg nicotinamide per kg produces no detectable pathologic changes. The morphologic changes after the inhibition of the pentose phosphate shunt by 6 AN are considered to represent a glial syndrome in the optic nerve of the rat, i.e. the axonal changes are secondary to glial alterations. The pathomorphogenesis is characterized by a different vulnerability in the orbital and cerebral portion probably due to different glial populations.

     

Immunohistochemical evidence of inducible nitric oxide synthase and nitrotyrosine in a case of clinically isolated optic neuritis.

BACKGROUND: Optic neuritis (ON) is a demyelinating inflammation of the optic nerve that may occur as an isolated disease or related to multiple sclerosis (MS). There is little evidence of whether the immunohistochemistry of ON resembles that of typical cerebral MS lesions. METHODS: Pathologic optic nerves were obtained from a patient who died of causes unrelated to ON after clinical recovery from clinically isolated ON. Normal control optic nerves were obtained from an eye bank. Normal and pathologic tissues were probed with antibodies to pathologic proteins including myelin basic protein (MBP) fragment, the inducible form of nitric oxide synthase (iNOS), macrophage markers CD14 and CD64, nitrotyrosine, and cyclooxygenase (COX-2). We also examined MBP, the oligodendrocyte marker cyclic nucleotide phosphodiesterase (CNPase), and glial fibrillary acidic protein. RESULTS: In the affected pathologic nerve, iNOS-positive macrophages/microglia, iNOS-positive astrocytes, COX-2, and nitrotyrosine were observed. iNOS and COX-2 were occasionally observed in the unaffected nerve.Decreased expression of MBP and CNPase was seen in the pathologic optic nerves, along with evidence of gliosis and ongoing myelin degradation indicated by the presence of MBP fragment. CONCLUSIONS: The immunohistochemistry of clinically isolated optic neuritis, as judged by this single case, resembles that of cerebral lesions of MS in showing abnormally high levels of iNOS and nitrotyrosine as well as other mediators of immune damage.     

Axolemma is a mitogen for human Schwann cells

The mechanisms responsible for the induction of Schwann cell proliferation in peripheral nerves undergoing wallerian degeneration and segmental demyelination are not understood. To determine whether contact with axolemma stimulates mitosis of human Schwann cells, cultured Schwann cells from spinal roots obtained postmortem and from sural nerve biopsy specimens were incubated with axolemmal fractions prepared from human spinal cord or from adult rat central nervous system. Schwann cell proliferation was estimated by autoradiographic assay of tritiated thymidine incorporation. Schwann cell labeling indices after exposure to human or rat axolemmal fractions ranged from 26.7 to 59.9%; labeling indices of Schwann cells cultured without axolemmal fraction were 9.8 to 22.4%. The stimulation index, or ratio of Schwann cell labeling index with axolemmal fraction to that without axolemmal fraction, ranged from 1.97 to 3.40. This study demonstrates that both human and rat axolemma are capable of stimulating human Schwann cell replication in vitro.     

Neurite outgrowth over resting and reactive astrocytes

A classic problem in CNS fiber regeneration is that the glial scar, generated after a lesion, is not crossed by regenerating axons. We know that reactive astrocytes are important in the formation of this barrier and that the barrier is not mechanical. However, its precise nature remains unclear. To study interactions of normal and reactive astrocytes with central neurites, we have attempted to create an in vitro model of the glial scar. We found the following: (1) Cultured astrocytes, independently of their lineage, morphology, immunological type and treatment with differentiating agents, induced profuse neurite outgrowth from various kinds of embryonic CNS neurons. The outgrowth was comparable to that elicited by laminin. (2) Membranes from isomorphic gliotic tissue (induced by deafferentation or excitotoxic injury and containing a large number of reactive astrocytes), inhibited central neurite outgrowth as powerfully as myelin. Reactive astrocyte membranes from areas of anisomorphic gliosis (following penetrating trauma) were permissive for neurite outgrowth, but growth was more limited than on cultured astrocyte membranes. (3) When given a choice, growing neurites actively avoided membranes from isomorphic gliosis (similar to myelin), while they seemed to follow anisomorphic membrane boundaries and crossed unhindered into membranes of cultured astrocytes. In conclusion, reactive glia seem to contain both inhibitory and neurite promoting molecules, the proportion of which depends on the way gliosis has been generated. For isomorphic reactive astrocytes the balance is inhibitory for central neurite outgrowth, while anisomorphic reactive astrocytes probably express inhibitory components at lower levels and the growth promoting factors predominate. Overall, our observations suggest that reactive astrocytes are still the major problem for axonal regeneration in the CNS.     

Retina-derived growth-promoting extract supports axonal regeneration in vivo

The ability of a growth-promoting extract derived from bovine retina was assessed for its ability to support nerve fiber regeneration from proximal stumps of transected sciatic nerves. Proximal stumps of transected rat peripheral nerves were inserted into the single inlet end of a 6 mm long Y-shaped Silastic implant. One of the paired outlets was attached to an Elvax pellet containing the retina-derived growth extract, and the other outlet to a pellet containing an equivalent amount of tissue extract-free Hank's balanced salt solution. At 3 weeks postoperatively, the number of axons and blood vessels in the midportion of implant forks was assessed. The extent of axonal regeneration and blood vessel formation induced were similar to each other and dose-dependent. Results indicate preferential and dose-dependent growth of axons toward pellets containing the retina-derived growth-promoting extract.     

Experimental inhibition of reactive gliotic-like changes in astrocytic cultures

Dibutylcyclic AMP (DiBucAMP) can induce astroblast-containing cultures to form cells which resemble reactive astrocytes observed in vivo. In the present study, myelin- or axolemmal-enriched fractions were assessed for the ability to inhibit (DiBucAMP)-stimulated reactive-like changes in astrocytic cultures. Addition of exogenous myelin- or axolemmal-enriched fractions to DiBcAMP-exposed cultures prevented drug-induced elevation of lactic dehydrogenase (LDH) 2 days after initial addition of the drug and moderated DiBucAMP-induced decreases in the enzyme's activity normally observed 5 days later. The proportion of reactive colonies (i.e., those in which more than 50% of cells are stellate-shape) was significantly lower in cultures exposed to the drug plus myelin or axolemma vs those exposed to DiBucAMP alone. The inhibitory factors in axolemmal fractions were heat sensitive (at 100 degrees C), whereas those in myelin were not. Both fractions were inactivated by trypsin. Whole brain homogenates had no effect on diBucAMP-stimulated changes in culture.     

Tissue culture studies of Schwann cell proliferation and differentiation

Neonatal rat sciatic nerve Schwann cells in monolayer culture are stimulated to proliferate and to express a lipid and a protein characteristic of myelin by agents which raise intracellular cyclic adenosine 3',5'-monophosphate. Both glial growth factor and axolemmal fragments increase the rate of mitosis of cultured rat and human Schwann cells. Rat Schwann cell mitosis is enhanced by a soluble factor produced by concanavalin A-stimulated blood mononuclear cells and inhibited by lead salts. Schwann-like cells cultured from human dermal and plexiform neurofibromas resemble normal human Schwann cells in phenotype and response to mitogens.     

Caprine beta-mannosidosis: development of glial and myelin abnormalities in optic nerve and corpus callosum

In caprine beta-mannosidosis, an inherited dysmyelinating disorder, the myelin deficit shows substantial variation throughout the nervous system. In this study morphometric analysis of optic nerve and corpus callosum sections at selected developmental stages was conducted in order to investigate development and persistence of myelin sheaths, the population of axons ensheathed, and the extent of myelin deficits and glial cell abnormalities. The results show that the myelin deficit is severe at very early stages of development and persists to about the same extent into postnatal life. The corpus callosum, much more severely involved than the optic nerve, contains a substantially smaller percentage of myelinated axons when compared to control. In both regions, larger axons are preferentially myelinated. In the corpus callosum before myelination begins, many glial cells appear abnormal, suggesting an early cellular defect. In the postnatal, myelin-deficient corpus callosum, there is a substantial decrease in glial cell density as compared to control, with abnormal appearance of many of the remaining cell profiles. These results define developmental characteristics of the dysmyelination in caprine beta-mannosidosis and document both the early appearance and the persistence of glial cell body and myelin abnormalities.     

Isolation and partial characterization of rat CNS axolemma enriched fractions

Axolemma-enriched fractions were prepared from rat brain by osmotic shock of a purified preparation of myelinated axons and subsequent separation of myelin, two axolemma-enriched fractions and myelin-free axons by density gradient centrifugation. Compared with the starting whole homogenate, the fractions were enriched in specific activity of Na+K+ ATPase, acetylcholinesterase, 5'nucleotidase as well as 2',3'-cyclic nucleotide 3'phosphohydrolase. Compared with myelin, the axolemmal fractions are greatly enriched in high molecular weight proteins. The 1.0/1.2 fraction has a predominant peak of fucose-labeled glycoprotein with a molecular weight between that of the myelin associated glycoprotein and the Wolfgram protein which is absent from the myelin glycoprotein profile. Polyacrylamide gel electrophoresis showed that the protein profile of myelin isolated by this procedure was similar to that of myelin isolated by other procedures and that the myelin specific basic and proteolipid proteins were virtually absent in the axolemma-enriched fractions. Both axolemma fractions were enriched in higher MW proteins, some of which resembled proteins in the myelin protein profile. Both axolemma-enriched fractions specifically bind between 2 and 3 pmoles of [3H]tetrodotoxin per mg protein. The axolemma-enriched fractions incorporated [3H]leucine and [14C]fucose exclusively into high molecular weight proteins and glycoproteins. In contrast myelin concomitantly isolated with the axolemma-enriched fractions had a significant amount of [3H]leucine labeled protein in myelin proteolipid and basic proteins. In addition to the myelin associated g-ycoprotein the [14C]fucose labeled a glycoprotein of slightly larger apparent molecular weight than proteolipid protein was found in the myelin fraction while the comparable labeled glycoprotein was absent in the axolemma-enriched fractions. The possible extent of contamination of these fractions by myelin or myelin subfractions and relationship of these axolemma-enriched fractions to other axolemma preparations are discussed.     

Gliosis during optic fiber regeneration in the goldfish: an immunohistochemical study.

Antisera directed against the 48 kDa and 50 kDa cytoskeletal antigens were used to examine changes in the astroglial fabric of the goldfish visual pathways following optic nerve crush. Several major observations are described. First, an optic nerve crush lesion in these animals appears to be devoid of glial cells for at least the first month after surgery. As a corollary, regenerating axons that grow across the lesion may do so over an aglial substrate. Once the axons cross the lesion, their growth is confined to the astroglial domains of the proximal nerve stump. In the optic nerve, gliosis comprises hypertrophy of astrocytic processes such that the open framework characterizing the normal nerve is obscured. In addition, during regeneration, optic nerve glia express large amounts of the 50 kDa cytoskeletal protein, which they ordinarily express at only minimal levels. In the optic tract, gliosis is reflected in a markedly increased expression of the 50 kDa protein as well as an apparent increase in the number and complexity of glial processes. In addition, optic tract glia begin to express the 48 kDa antigen during regeneration. This protein is ordinarily confined for the most part to the optic nerve and is not seen in the tract glia. Finally, no obvious changes were seen in the glia of the optic tectum. These results demonstrate many points of similarity between gliosis in the goldfish and in mammals. However, in some particulars the two responses differ, and it is possible that these differences are related to the differing ability of central axons to regenerate in the two groups of organisms.     

Development of macroglial cells in the embryonic chick optic nerve

Macroglia development in the embryonic chick optic nerve was immunohistochemically examined. The astrocytes with glial fibrillary acidic protein immunoreactivity were initially restricted to the retinal end of the optic nerve at stage 40, but had widely dispersed within the optic nerve in an out-side-in manner by stage 44. Oligodendrocytes with myelin basic protein immunoreactivity appeared at stage 38, and were widely distributed at stage 40. Electron microscopic observation confirmed the presence of intermediate filaments in the glial fibers and developing myelin at stages 40-41. The present results suggest that oligodendrocyte precursors undergo terminal differentiation slightly earlier than astrocyte precursors with respect to the expression of marker proteins.     

Oligodendroglial cell development in jimpy mice and controls. An electron-microscopic study in the optic nerve.

Glial development was studied in the optic nerve of 1- to 28-day-old Jimpy mice and controls. Abnormalities were found in oligodelopment and axons were not affected. These consisted in (a) Increased numbers of glioblastic cells containing lipids and increased occurrence of glial cell death in the premyelination stage; (b) Decreased numbers of maturing oligodendrocytes (i.e. young and active oligodendrocytes) in the period of early myelination; (c) An occurrence of abnormal oligodendroglial cells containing lipids and multimembranous tubes in the period of advanced myelination. The decreased number of maturing oligodendrocytes in the premyelination stage indicates that the lack of myelin in Jimpy mice may be secondary to a disturbance in the differentiation of the oligodendroglial cell line. The occurrence of abnormal, lipid-containing glioblasts and oligodendrocytes may be an expression of a defect in metabolism leading to an abnormality in the association of myelin proteins and myelin lipids.     

Migration and myelination by adult glial cells: reconstructive analysis of tissue culture experiments

Adult glia are capable of at least limited myelination of CNS axons. However, it is difficult to quantitate their myelination or migratory capacities and to examine contributions of the CNS environment or exogenous factors that might promote or inhibit this process in situ. We have therefore developed a mouse tissue culture system in which optic nerve glia (in the form of appropriately handled optic nerve) are added to chemically demyelinated cerebellar axons. Optic nerve up to postnatal day 411 (P411) contains cells that can migrate out of the nerve into the cerebellar explant and form myelin around its axons. The success rate for myelin formation in these cultures is 57% for immature (P7-11) glia and 55% for adult (P50-411) glia. Computer-generated reconstructions of cultures containing immature (P8) and adult (P89 and P139) nerves demonstrate that in all 3 cases the glia may migrate more than 0.6 mm before myelinating axons, assuming the shortest possible track. Both the age limit for myelination and distance limit for migration, if any, remain to be determined for these adult glia. In successful cultures, myelin always directly abuts the optic nerve surface, whether or not it also extends further, suggesting that migrating glia may depend upon contact guidance by myelin-receptive axons. We conclude that this culture system is a useful model of adult CNS myelin regeneration, in which one can examine the influence of potential trophic or toxic factors on specific aspects of myelinating glial cell behavior.