Heterotopic regeneration of peripheral nerve fibres into the subarachnoid space

Summary Within the spinal cord meninges of SJL/J mice afflicted with chronic relapsing experimental allergic encephalomyelitis for up to seven months postinoculation, networks of aberrant regenerated nerve fibres myelinated by Schwann cells have been observed. These P.N.S. elements are believed to have been derived from incoming sensory fibres from the spinal nerve roots which had been interrupted during acute stages of the disease. This phenomenon might also have been related to the occurrence of marked nerve fibre loss and gliosis within superficial tracts in the spinal cord. P.N.S. elements within the subarachnoid space were first apparent six weeks postinoculation, and were distributed around the entire spinal cord and tended to be more concentrated around meningeal blood vessels. Schwann cells associated with these fibres frequently contained centrioles and cilia and daughter Schwann cells apparently arose from parent cells already committed to axons.     

On the occurrence of Schwann cells within the normal central nervous system

Summary The presence of Schwann cells and P.N.S. myelin are reported in subpial areas of apparently normal spinal cord from one control rabbit, two experimental rabbits and one experimental guinea pig. These P.N.S. elements exerted no adverse effects upon local C.N.S. components. The occurrence of ectopic Schwann cells in the normal C.N.S. has also been reported elsewhere in studies on normal human spinal cord tissue. The propensity for Schwann cells to reside in the normal C.N.S. of several species makes it necessary for experiments and hypotheses on the aetiology of Schwann cell invasion into the abnormal C.N.S. to take the present phenomenon into consideration.     

Schwann cell myelin ensheathing C.N.S. axons in the nerve fibre layer of the cat retina

Summary In the retina of the cat the axons of the nerve fibre layer are unmyelinated and are provided with a C.N.S. myelin sheath only in the extraocular part of the optic nerve. The present study demonstrates that in the apparently normal cat retina close to the optic disc, some axons of the nerve fibre layer run for a short distance in the perivascular space of the retinal arteries. While coursing in the perivascular space, these C.N.S. axons become transiently myelinated by Schwann cells, which form a typical P.N.S. myelin sheath. These P.N.S. myelin sheaths terminate at a heminode in the transitional zone in which the C.N.S. axons penetrate the perivascular glial sheath in order to leave or to re-enter the nerve fibre layer. It is suggested that the Schwann cells, which elaborate the P.N.S. myelin around C.N.S. axons, are descendants of the Schwann cells of the perivascular autonomie nerves. The present study shows that Schwann cells are able to provide previously unmyelinated C.N.S. axons with a P.N.S. myelin sheath.     

Observations on the interactions of Schwann cells and astrocytes following X-irradiation of neonatal rat spinal cord.

Myelination was inhibited in the spinal cord of three day-old rats with 2000 rads of X-irradiation. Myelination subsequently occurred as a result of caudal migration of oligodendrocytes and extensive invasion of the cord by Schwann cells. Although oligodendrocytes were present in areas containing Schwann cells, astrocytes were absent. The presence of Schwann cells in the neuropil of the spinal cord did not stimulate production of basement membrane by astrocytes, so no new glial limiting membrane was formed. Evidence is presented which suggests that if astrocytes do not form a glial limiting membrane when opposed by large numbers of Schwann cells they are destroyed by the invading cells. It is suggested that the glial limiting membrane normally inhibits entry of Schwann cells into the central nervous system; if this is destroyed and not reconstituted, Schwann cells can migrate freely into the neuropil.     

Transplantation of Schwann cells to subarachnoid space induces repair in contused rat spinal cord

Schwann cell transplantation is well known to induce repair in the injured spinal cord which disables millions of injured patients throughout the world. An ideal route of delivering the grafted Schwann cells to the spinal cord should neither cause more injury nor reinitiate inflammatory events and also provide a favorable milieu to the grafted cells. In this study, we have utilized subarachnoid route to transplant Schwann cells and evaluated their effects in a contusive model of spinal cord injury. Adult rats weighing 100-140 g were experimentally injured by crushing the spinal cord with a titanium clip and then divided into four groups (Tracing, Control, Medium-treated and Schwann cell-treated). Cultured Schwann cells (5x10(4) cells in 5 microl) or medium were injected to the animals of corresponding groups via subarachnoid space at the injured site 7 days after injury. In tracing group, Schwann cells (labeled with Hoechst) demonstrated their presence within spinal cord 7 days after transplantation. Evaluation of locomotor performance of animals for 60 days after injury showed that animals treated with Schwann cells had significant improvement (P<0.01). Similarly, the axon density at the site of injury was significantly higher. The results indicate the efficacy of subarachnoid route for the transplantation of Schwann cells in inducing repair of the contused spinal cord. We conclude that this route can be useful for the transplantation of Schwann cells and offers a hope for the patients suffering from spinal cord injury.     

On the association between perinodal astrocytic processes and the node of Ranvier in the C.N.S.

Summary Associations between the axolemma of nodes of Ranvier in the C.N.S. and perinodal astrocytic processes have been confirmed in the guinea pig spinal cord. Since perinodal astrocytic processes were found to be a consistent feature at such nodes, it is proposed that they represent the C.N.S. analogue of the Schwann cell fingers at P.N.S. nodes. Although they assumed a variety of configurations, perinodal astrocytic processes rarely approached the complexity and regularity of Schwann cell profiles at PNS nodes. The findings are discussed in terms of the relevance between perinodal astrocytes and the maintenance of an electrogenic pump at the node.     

Remyelination during remission in Theiler's virus infection.

Inoculation of the cell-adapted WW strain of Theiler's virus into mice produces a chronic demyelinating infection of the central nervous system (CNS) characterized by a remitting relapsing course. During remission, extensive remyelination of spinal cord white matter is observed. Remyelination is carried out by both Schwann cells and oligodendrocytes. This paper examines the possible mechanisms of entry of Schwann cells into the CNS, their possible source in different regions of the white matter, their relations with various CNS elements, and the relative activity of these cells versus that of oligodendrocytes. Observations suggest that Schwann cells, originating from peripheral roots and from perivascular areas, migrate into white matter through gaps in the glial limiting membrane ( GLM ), probably caused by active mononuclear inflammatory cells. Schwann cell invasion and axonal contact appear to be facilitated by the presence of collagen matrix along their pathway of migration. No alterations of astrocytes in the immediate vicinity of Schwann cells were observed, and free contact between Schwann cells and different neuroglial elements was present in the initial stages of Schwann cell migration. While Schwann cells were the predominant myelinating cells in the outer white matter, oligodendrocytes were numerous and very active in the inner portions of the spinal cord column. Although oligodendrocytes produced thinner myelin than normal, in most areas essentially complete remyelination by these cells was observed. These results contrast with those of previous studies of DA infected mice in which remyelination is sporadic in the presence of unabated inflammation which continues without remission for many months after infection. It is suggested that oligodendroglial cells are quite capable of extensive remyelinating activity in this infection, provided the noxa responsible for myelin injury subsides. The host inflammatory response appears to be the most likely noxa impeding remyelination in this model.     

Pattern of Schwann cell remyelination in a spinal cord lesion

Intraspinal injection of mitomycin C into the rat dorsal columns produced extensive demyelination, axonal degeneration and glial cell death. Five weeks post-injection Schwann cell remyelinated fibers were present along the surface of the dorsal columns and around blood vessels within the lesions. Axons near these sites either were enclosed within a Schwann cell but not myelinated or were completely devoid of any cellular ensheathment. Schwann cells were associated only with those blood vessels which no longer retained astroglial end-feet. It is concluded that Schwann cells migrate into spinal cord lesions along such vessels. The marked sub-pial and perivascular distribution of Schwann cell remyelinated fibers may reflect a failure of Schwann cells to disperse quickly elsewhere within the lesion.     

Non-neuronal cells in the spinal cord of nude and heterozygous mice. II. Agranular leukocytes in the subarachnoid and perivascular space

Summary The lumbar spinal cord of the athymic nude mouse and its heterozygous control were examined at the light and electron microscopic levels for differences in the cellular constituents of the sub-arachnoid and perivascular spaces. Both macrophages and lymphocytes were found in these spaces associated with the adventitial and leptomeningeal cells. Occasionally, fixed-cells were associated with extensions of the basal lamina and subjacent astrocytic processes. The appearance of the basal lamina of the externalglia limitans and perivascular space was enhanced by tannic acid treatment. There were twice as many macrophages as lymphocytes, but no significant strain differences. Comparison was made between cells in the lumina of incompletely perfused vessels and neuroglial cells in the spinal cord. The cellular morphology is distinct in each of these compartments and no cell migrations were observed between the blood, cerebrospinal fluid and C.N.S. interstitial space. The normal presence of both macrophages and lymphocytes in the subarachnoid and perivascular space suggests that these cells could penetrate the basal lamina and gain access to the C.N.S.     

Intramedullary schwann cell development following x-irradiation of mid-thoracic and lumbosacral spinal cord levels in immature rats

Intramedullary Schwann cells have been observed following x-irradiation of lumbosacral spinal cords in immature rats. The present investigation was designed to determine whether or not the development of intramedullary Schwann cells within the spinal cord could be influenced by the portion of the spinal cord exposed to radiation. Three groups of rats were irradiated when three days of age. In one group the irradiated zone was limited to a 5 mm length of mid-thoracic spinal cord (T only), in another group the irradiation was limited to a 5 mm length of lumbosacral spinal cord (L only), and in a third group 5 mm lengths of both mid-thoracic and lumbosacral spinal cord (T/L) were irradiated. All of these animals received a single exposure to 4000 R of soft x-rays (HVL 0.16 mm Al). Sham-irradiated littermates served as control animals. Groups of rats were killed at intervals from 9 through 60 days following irradiation, and the spinal cords were prepared for light microscopic examination. Schwann cells appeared in the lateral portion of the lumbosacral dorsal funiculi of L only and T/L irradiated spinal cords as early as 9 days post-irradiation. By 19 days post-irradiation Schwann cells occupied the lateral, medial, and deep medial portions of the dorsal funiculi in the lumbosacral areas. By 25 days post-irradiation Schwann cells were also observed in the dorsal gray horns. In contrast, Schwann cells were not observed in the midthoracic regions of T only and T/L irradiated rats until 11 days post-irradiation. The accumulation of these cells was not extensive, and, in general, the Schwann cells were confined to the lateral portion of the dorsal funiculi in all animals. These findings indicate that intramedullary Schwann cell development is influenced by the region of spinal cord irradiated in immature rats.     

Uncoupled relationship between demyelination and primary infection of myelinating cells in Theiler''s virus encephalomyelitis.

Theiler's virus infection in mice produces a chronic demyelinating disease which appears to be based on an immune pathogenesis rather than on direct viral destruction of myelin-supporting cells. The purpose of the present study is to ascertain whether viral antigen is present in the cytoplasm of such cells in areas of demyelination. Because of the difficulty of identifying oligodendrocytes in tissues rich in infiltrating mononuclear cells and fixed for immunohistochemistry, I turned to a recently described form of Theiler's virus encephalomyelitis which follows inoculation with the attenuated ww strain and is characterized by extensive spinal cord remyelination by invading Schwann cells and by recurrent demyelination of Schwann cell-remyelinated axons. The unlabeled antibody peroxidase-antiperoxidase technique was employed to study whether such spinal cord Schwann cells were primarily infected by virus at the time when recurrent demyelination was occurring. Whereas other types of cells, including neurons, astrocytes, and macrophages, contained abundant viral antigen, no positive immune reaction was observed in Schwann cells. These results correlate with our previous studies which had suggested that demyelination in this viral model is not dependent on primary viral attack on myelinating cells but is probably dependent on the host immune response.     

Immunocytochemical method to identify basic protein in myelin-forming oligodendrocytes of newborn rat C.N.S.

Summary An immunocytochemical method for detecting myelin basic protein in oligodendrocytes and myelin of newborn rat C.N.S. is described. C.N.S. tissue is perfused and fixed in HgCl₂-formaldehyde and 20 m Vibratome sections are treated with antibodies to myelin basic protein using the peroxidase-antiperoxidase method. Oligodendrocytes in the newborn rat are intensely stained by antiserum to basic protein and multiple stained processes extend from the perikaryon to myelin sheaths. With this procedure it is possible to demonstrate the geometric relationships between a single oligodendrocyte and multiple myelin sheaths. Stained oligodendrocytes and myelin are present in newborn cervical spinal cord, medulla oblongata, pons and midbrain. By 25 days of age, staining in oligodendrocytes is less intense than in newborn rats and differences in amount of staining can be detected in areas that are myelinating at different rates. With anticerebroside serum, cerebroside, of newborn and developing rat C.N.S. tissue is localized only in myelin. In the developing P.N.S., myelin basic protein is localized in Schwann cell cytoplasm and myelin sheaths of the trigeminal ganglion. Cerebroside is found only in myelin.     

Glial–glial and glial–neuronal interfaces in radiation-induced, glia-depleted spinal cord

This review summarises some of the major findings derived from studies using the model of a glia-depleted environment developed and characterised in this laboratory. Glial depletion is achieved by exposure of the immature rodent spinal cord to x-radiation which markedly reduces both astrocyte and oligodendrocyte populations and severely impairs myelination. This glia-depleted, hypomyelinated state presents a unique opportunity to examine aspects of spinal cord maturation in the absence of a normal glial population. An associated sequela within 2–3 wk following irradiation is the appearance of Schwann cells in the dorsal portion of the spinal cord. Characteristics of these intraspinal Schwann cells, their patterns of myelination or ensheathment, and their interrelations with the few remaining central glia have been examined. A later sequela is the development of Schwann cells in the ventral aspect of the spinal cord where they occur predominantly in the grey matter. Characteristics of these ventrally situated intraspinal Schwann cells are compared with those of Schwann cells located dorsally. Recently, injury responses have been defined in the glia-depleted spinal cord subsequent to the lesioning of dorsal spinal nerve roots. In otherwise normal animals, dorsal nerve root injury induces an astrocytic reaction within the spinal segments with which the root(s) is/are associated. Lesioning of the 4th lumbar dorsal root on the right side in irradiated or nonirradiated animals results in markedly different glial responses with little astrocytic scarring in the irradiated animals. Tracing studies reveal that these lesioned dorsal root axons regrow rather robustly into the spinal cord in irradiated but not in nonirradiated animals. To examine role(s) of glial cells in preventing this axonal regrowth, glial cells are now being added back to this glia-depleted environment through transplantation of cultured glia into the irradiated area. Transplanted astrocytes establish barrier-like arrangements within the irradiated cords and prevent axonal regrowth into the cord. Studies using other types of glial cultures (oligodendrocyte or mixed) are ongoing.     

Dorsal root ganglion neurons induce transdifferentiation of mesenchymal stem cells along a Schwann cell lineage

It has been reported that mesenchymal stem cells (MSCs) can transdifferentiate into Schwann cell-like cells by a series of treatments with a reducing agent, retinoic acid and a combination of trophic factors in vitro, and can transdifferentiate into myelin-forming cells to repair the demyelinated rat spinal cord in vivo. We now report that when co-cultured with dorsal root ganglion (DRG) neurons, MSCs were induced to transdifferentiate into Schwann cell-like cells that had ensheathed DRG axons. Following differentiation, MSCs underwent morphological changes similar to those of cultured Schwann cells and express GFAP and S100, the marker of Schwann cells. Moreover, 6 weeks later, MSCs wrapped their membrane around DRG axons. Further, initiation of myelination was observed in the co-cultured DRG neurons, which was determined by signals to MBP and this initiation of axon myelination by MSCs is similar to that of Schwann cells. However, electron micrographs show that no compact myelin was present in the MSCs co-cultures, whereas the Schwann cells co-cultures had formed a multilammelar myelin sheath around the axon. These indicate that the release of cytokine by DRG neurons may promote the transdifferentiation of MSCs, but is not sufficient to elicit compact myelination by transdifferentiated MSCs. These results improve our understanding in the mechanism of MSC transdifferentiation, and the mechanism underlying ensheathment and myelination by transdifferentiated MSCs.     

Molecular identity, distribution and heterogeneity of glial fibrillary acidic protein: an immunoblotting and immunohistochemical study of Schwann cells, satellite cells, enteric glia and astrocytes

Summary Glial fibrillary acidic protein has been firmly established as the predominant component of astrocyte intermediate filaments. It has also been detected immunohistochemically in the glial cells of the enteric nervous system and some Schwann cells in the P.N.S. The molecular identity of this GFAP immunoreactivity in the P.N.S. has so far not been investigated. This study compares GFAP in the C.N.S. and P.N.S. of adult rats both immunochemically and immunohistochemically.Using SDS polyacrylamide gel electrophoresis combined with immunoblotting, and a polyclonal antiserum to brain GFAP, we show that the peripheral GFAP immunoreactivity resides in a polypeptide with a molecular weight of 49 kd, which is identical to that of rat brain GFAP. Furthermore, we find that this GFAP reactivity can be detected immunohistochemically in Schwann cells in a wide variety of nerves in the P.N.S. and in some satellite cells in both sensory and sympathetic ganglia, in addition to enteric glia. The pattern of distribution of GFAP filaments in Schwann cells suggests that, in the nerves surveyed, they may be expressed by most or all non-myelin forming Schwann cells but not by myelin-forming Schwann cells. We also show, using a monoclonal antibody to GFAP (anti-GFAP-3) in both immunohistochemical and immunoblotting studies, that the GFAP found in most peripheral glia is not identical to that of astrocytes since it lacks an antigenic determinant, defined by this monoclonal antibody, which is present in astrocytes. An exception to this finding is seen in the myenteric plexuses where immunohistochemically detectable GFAP is found in some, but not all, of the enteric glia, using the monoclonal antibody.Thus, the results suggest that GFA polypeptides may be a heterogeneous group, that share some common determinants and a common molecular weight, and show a widespread and complex distribution in the glia of both the C.N.S. and P.N.S.     

Identification of CD4- CD8- alpha beta T cells in the subarachnoid space of rats with experimental autoimmune encephalomyelitis. A possible route by which effector cells invade the lesions.

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats to elucidate the origin of effector T cells and the route by which they invade lesions. Since mouse studies have suggested that some autoimmune diseases are induced by extrathymic T cells in the liver, we focused our attention on the properties of mononuclear cells (MNC) isolated from the liver and other organs in rats with EAE. A small but significant proportion of LFA-1+ alpha beta T cells was identified in the liver as early as day 7 after immunization with myelin basic protein (MBP). Such LFA-1+ alpha beta T cells were also abundant among MNC attached to the spinal cord (i.e. subarachnoid space), and MNC infiltrated the spinal cord in rats with EAE (day 12). In electron microscopy, MNC attached to the spinal cord were found to be quite unique in terms of their large cell size with well-developed microvilli. More importantly, they were comprised of a considerably large proportion of double-negative CD4- CD8- T cells as well as single-positive CD4+ T cells. However, the cells which infiltrated the spinal cord were mainly CD4+. The present results raise the possibility that the subarachnoid space might be a major site for the expansion of extrathymic T cells in rats with EAE, and that only a limited population of CD4+ T cells invade the spinal cord directly through the outer layer and elicit EAE.     

Transplantation of sciatic nerve segments into normal and glia-depleted spinal cords

 Although peripheral nerves are used as guides in attempts to enhance regeneration in the central nervous system (CNS), surprisingly little is known about the interface that develops between the host tissue and the transplanted or implanted peripheral nerve. This study examines host-nerve interfaces following transplantation of segments of sciatic nerve into the spinal cord under two differing conditions, one in which the spinal cord contains normal numbers of glia and one in which the glial population is reduced. The depletion of the glial population is achieved by exposing the lumbosacral region of the spinal cord in 3-day-old rats to X-rays, a model developed in this laboratory. Twenty days later, segments of fresh or frozen sciatic nerves harvested from other 3-day-old rats were transplanted into the lumbar region of spinal cord in irradiated animals and in their non-irradiated littermate controls. Following a 20-day postoperative period, the interfaces between host spinal cord and sciatic nerves were examined ultrastructurally, and pronounced differences were noted. A distinct scar composed of multiple layers of astrocyte processes completely enveloped the transplant in non-irradiated host spinal cord and confined Schwann cells and fibroblasts to the area enclosed by the scar. Terminals from axons that appeared to have traversed the transplant during this 20-day period ended blindly in the astrocytic scar. In contrast, a complete astrocytic scar failed to form around the transplant in the irradiated, glia-depleted hosts, and Schwann cells intermingled with host tissue. Some Schwann cells migrated away from the transplant, which was placed in the dorsal funiculus, along a perivascular route and extended into the gray matter. In some instances Schwann cells were observed in the ventral gray surrounding blood vessels and motoneurons. From these observations, it is clear that the formation of a distinct astrocytic barrier at the host-graft interface is greatly reduced irradiated host. The effects of astrocyte reduction on enhanced regeneration within the spinal cord are discussed. Received: 15 April 1998 / Accepted: 9 September 1998     

Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function

To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding for green fluorescent protein or saline. AAV-BDNF- and AAV-NT-3-transduced 293 human kidney cells produced and secreted BDNF or NT-3, respectively, in vitro. The secreted neurotrophins were biologically active; they both promoted outgrowth of sensory neurites in vitro. In vivo, transgene expression was observed predominantly in neurons for at least 16 weeks after injection. Compared with controls, a modest though significant improvement in hind-limb function was found in rats that received AAV-BDNF and AAV-NT-3. Retrograde tracing demonstrated that twice as many neurons with processes extending toward the Schwann cell graft were present in the second lumbar cord segment of AAV-BDNF- and AAV-NT-3-injected animals compared with controls. We found no evidence, however, for growth of regenerated axons from the Schwann cell implant into the caudal cord.Our results suggest that AAV vector-mediated overexpression of BDNF and NT-3 in the cord caudal to a Schwann cell bridge modified the local lumbar axonal circuitry, which was beneficial for locomotor function.     

Sustained delivery of dibutyryl cyclic adenosine monophosphate to the transected spinal cord via oligo [(polyethylene glycol) fumarate] hydrogels

This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.