Comparative postnatal development of spinal, trigeminal and vagal sensory root entry zones

Somatic and visceral sensory information enters the central nervous system (CNS) via root entry zones where sensory axons span an environment consisting of Schwann cells in the peripheral nervous system (PNS) and astrocytes and oligodendrocytes in the CNS. While the embryonic extension of these sensory axons into the CNS has been well-characterized, little is known about the subsequent, largely postnatal development of the glial elements of the root entry zones. Here we sought to establish a comparative developmental timecourse of the glial elements in the postnatal (P0, P3, P7, P14) and adult rat of three root entry zones: the spinal nerve dorsal root entry zone, the trigeminal root entry zone, and the vagal dorsal root entry zone. We compared entry zone development based on the expression of antigens known to be expressed in astrocytes, oligodendrocytes, oligodendrocyte precursor cells, Schwann cells, radial glial fibres and the PNS extracellular matrix. These studies revealed an unexpected distribution among glial cells of several antigens. In particular, antibodies used to label mature oligodendrocytes (RIP) transiently labelled immature Schwann cell cytoplasm, and a radial glial antigen (recognized by the 3CB2 antibody) initially decreased, and then increased in postnatal astrocytes. While all three root entry zones had reached morphological and antigenic maturity by P14, the glial elements comprising the PNS–CNS interface of cranial root entry zones (the trigeminal root entry zone and the vagal dorsal root entry zone) matured earlier than those of the spinal nerve dorsal root entry zone.     

Remyelination by cells introduced into a stable demyelinating lesion in the central nervous system

Schwann cells from an autogeneic peripheral nerve source were injected into an established demyelinating lesion produced by the direct micro-injection of diphtheria toxin into the cat spinal cord. In control diphtheria toxin lesions, which were not injected with Schwann cells, demyelination and some oligodendrocyte remyelination was seen but Schwann cell remyelination was not observed. In diphtheria toxin lesions which were wholly confined to the posterior columns, Schwann cell myelin was not seen before 3 weeks after cell injection. The Schwann cell myelinated fibres occurred singly or in small groups within the posterior columns and were considered to have been myelinated by injected Schwann cells. By one month Schwann cell myelinated fibres had thick myelin sheaths but many demyelinated axons remained. By contrast, in more extensive diphtheria toxin lesions there was widespread Schwann cell remyelination of central axons at all stages examined after cell injection. The Schwann cell myelinated fibres were grouped in large numbers around the damaged dorsal root entry zones, the likely source of Schwann cells in these lesions. It is concluded that CNS remyelination may be improved by the injection of peripheral Schwann cells although the extent of remyelination is limited. One facet limiting remyelination may be the concentration of Schwann cells that it is possible to inject with present techniques. Functional recovery remains to be investigated.     

Remyelination of central nervous system lesions in experimental genital herpes simplex virus infection

To study spinal cord remyelination in a model of genital herpes simplex virus type 2 (HSV-2) infection, adult female mice were inoculated by a vaginal route. At intervals up to 6 months after infection, cord tissues were removed and examined by light and electron microscopy and by immunohistochemical methods. As a consequence of acute infection, 60% of mice developed multifocal central nervous system (CNS) demyelinative lesions in the lower thoracic, lumbar, or upper sacral cord. These lesions, already present 10 days after infection, contained naked axons and mononuclear cells, including macrophages. At 2 weeks, while active myelin breakdown was still ongoing, numerous Schwann cells were present in lesions and surrounded denuded axons. At 3 weeks, the earliest remyelination was seen, and was carried out by Schwann cells and to a lesser extent by oligodendrocytes. Remyelination was extensive by 6-10 weeks and was apparently completed after 3 months. Immunocytochemical studies using antisera to myelin proteins showed relatively distinct zones of central and peripheral remyelination in some lesions, whereas remyelination was of mixed type in others. Thus the remyelinative response following experimental HSV-2-induced CNS demyelination begins promptly, proceeds briskly and goes to completion. With a natural route of inoculation and a relatively avirulent strain of this human pathogen, we have produced a model of CNS white matter injury and repair in a high proportion of infected mice that may be useful in understanding mechanisms of human demyelinative disease.     

Progressive demyelination and reparative phenomena in chronic experimental allergic encephalomyelitis.

Chronic experimental allergic encephalomyelitis (EAE), produced in inbred guinea pigs given a single inoculation during the juvenile period with isologous spinal cord in complete Freund's adjuvant, has been studied by light and electron microscopy. Most animals showed a delayed onset of nurologic signs from 12 to 68 weeks post-inoculation (PI), while several were asymptomatic up to 74 weeks PI. Two animals showed a relapsing clinical course. Examination of the spinal cords of all animals revealed chronic demyelination, remyelination, and recent demyelination. Marked perivascular inflammation, including plasma cells, was seen within demyelinated plaques. The usual type of central nervous system (CNS) remyelination was documented but in addition, remyelination of CNS axons by invading Schwann cells was noted. This Schwann cell invasion, not previously seen in EAE, was predominantly in the area of the root entry zone, and occasionally involved extensive areas of the dorsal or ventral horns. The extent of Schwann cell invasion, as well as the usual CNS-type remyelination, demonstrates the reparative capacity of the CNS. The recurrent clinical and morphologic changes in these long-term animals provides further evidence that this model of chronic EAE has many features reminiscent of multiple sclerosis. The underlying immunologic mechanisms responsible for the recurrent disease in these animals are unknown. The presence of plasma cells in the inflammatory exudates might suggest a role for B cells in these chronic animals. The possibility of an intermittent release of loculated adjuvant/antigen accounting for the recurrent disease was considered.     

Virus-induced demyelination in herpes simplex virus-infected mice

Herpes simplex virus (HSV) infection of the mouse trigeminal ganglia and the brain stem is associated with demyelination of axons in the central part of the trigeminal root and inflammatory cell infiltration and perivascular demyelination in the brain stem. Cyclophosphamide (CPA) treatment prior to or soon after HSV inoculation caused increased axonal spread of infective virus from the peripheral site of inoculation, more widespread and severe demyelination and increased mortality, suggesting that by CPA the virus invasion of the CNS was facilitated. A direct cytocidal effect of HSV on myelinating cells seemed one plausible explanation for the demyelination. Influence on demyelination at late stages of infection by cytotoxic immune reactions are not excluded by the results reported but seemed not to dominate the picture. Schwann cells from the peripheral part of the nerve root invaded demyelinated areas in the brain stem and remyelinated the axons.     

Recurrent demyelination in chronic central nervous system infection produced by Theiler's murine encephalomyelitis virus

A morphologic study of demyelination produced by Theiler's encephalomyelitis virus (TMEV) infection in C3H/He mice was performed. Demyelination in this strain of mouse was less intense and had a milder gliomesodermal response than that observed in SJL mice. As early as 80 days after infection numerous remyelinated axons were present in C3H/He mice, and later, extensive remyelination was observed and was mainly by Schwann cells. About one-third of remyelinated plaques showed recurrent demyelinating activity at 200 days. The best evidence of recurrent demyelination was the loss of myelin by abons which had been previously remyelinated by Schwann cells. In addition, acute areas of demyelination were also seen in spinal cords which contained chronic or quiescent plaques. The demonstration of recurrent demyelination in TMEV infection is important for it increases the relevance of this model to multiple sclerosis (MS). In addition TMEV infection of C3H/He mice appears to be an excellent model for further studies of Schwann cell remyelination and recurrent demyelination in the central nervous system (CNS).     

Macrophage response to herpes simplex encephalitis in immune competent and T cell-deficient mice

Corneal inoculation of Nude (athymic) mice and Balb/c mice with herpes simplex virus Type I produces a brainstem encephalitis with demyelination of the trigeminal root entry zone. The extent of CNS demyelination is less in the immune-deficient athymic mice 7 days after infection compared to the immune-competent Balb/c mice. Both groups demonstrate a macrophage response and beginning myelin disruption approximately 3 days after corneal infection when herpes viral particles are first observed within central nervous system cells. Five to seven days after infection when differences in the extent of demyelination between the immune-competent and immune-deficient animals become evident, the Balb/c mice demonstrate T cells and increasing numbers of macrophages at the trigeminal root entry zones. These findings suggest an interaction between macrophages and T cells which leads to an extension of the demyelination in the immune competent Balb/c mice and that lack of T cells is important in limiting demyelination in Nude (athymic) mice.     

Schwann cell remyelination is not replaced by oligodendrocyte remyelination following ethidium bromide induced demyelination

Demyelinated CNS axons can be remyelinated by Schwann cells. A recent study concluded that with time Schwann cell remyelination is replaced by oligodendrocyte remyelination [9]. To examine this, the extent of Schwann cell and oligodendrocyte remyelination at 4, 6.5 and 24 weeks was determined for ethidium bromide lesions made in the spinal cords of rats. Although the extent of oligoden-drocyte remyelination increased with time there was no significant change in the amount of Schwann cell remyelination. This indicates that Schwann cell remyelination is stable and is not replaced by oligodendrocyte remyelination.     

Glial cell transplants that are subsequently rejected can be used to influence regeneration of glial cell environments in the CNS

Transplantation of glial cells into demyelinating lesions in CNS offers an experimental approach which allows investigation of the complex interactions that occur between CNS glia, Schwann cells, and axons during remyelination and repair. Earlier studies have shown that (1) transplanted astrocytes are able to prevent Schwann cells from participating in CNS remyelination, but that they are only able to do so with the cooperation of cells of the oligodendrocyte lineage, and (2) transplanted mouse oligodendrocytes can remyelinate rat axons provided their rejection is controlled by immunosuppression. On the basis of these observations, we have been able to prevent the Schwann cell remyelination that normally follows ethidium bromide demyelination in the rat spinal cord by co-transplanting isogeneic astrocytes with a potentially rejectable population of mouse oligodendrocyte lineage cells. Since male mouse cells were used it was possible to demonstrate their presence in immunosuppressed recipients using a mouse Y-chromosome probe by in situ hydridisation. When myelinating mouse cells were rejected by removal of immunosuppression, the demyelinated axons were remyelinated by host oligodendrocytes rather than Schwann cells, whose entry was prevented by the persistence of the transplanted isogeneic astrocytes. The oligodendrocyte remyelination was extensive and rapid, indicating that the inflammation associated with cell rejection did not impede repair. If this host oligodendrocyte remyelination was prevented by local X-irradiation, the lesion consisted of demyelinated axons surrounded by processes from the transplanted astrocytes. By this approach, it was possible to create an environment which resembled the chronic plaques of multiple sclerosis. Thus, these experiments demonstrate that in appropriate circumstances the temporary presence of a population of glial cells can alter the outcome of damage to the CNS. © 1995 Wiley-Liss, Inc.     

The demyelinating effect of corneal HSV infections in normal and nude (athymic) mice

Nude (athymic) mice and their immune competent Balb/c litter mates were infected with herpes simplex virus (HSV) type I on the scarified cornea. Seven to eight days after infection the trigeminal root entry zone was examined by light and electron microscopy. The immune competent Balb/c litter mates demonstrated marked central nervous system (CNS) demyelination when compared to the T cell-deficient animals who showed mild edematous change and minimal CNS demyelination ultrastructurally 7 days after infection. Viral titers were similar in both groups at this time. These studies further substantiate a role for the cell-mediated immune system in the CNS demyelination 1 week after corneal infection with HSV.     

Schwann cells genetically engineered to express PSA show enhanced migratory potential without impairment of their myelinating ability in vitro

Schwann cells, the myelin-forming cells of the PNS, are attractive candidates for remyelination therapy as they can remyelinate CNS axons. Yet their integration in CNS tissue appears hampered, at least in part, by their limited motility in the CNS environment. As the polysialylated (PSA) form of NCAM regulates migration of neural precursors in the CNS and is not expressed by developing Schwann cells, we investigated whether conferring sustained expression of PSA to Schwann cells derived from postnatal rats enhances their motility. Cells were transduced with a retrovirus encoding polysialyl-transferase STX, an enzyme that synthesizes PSA on NCAM. Migration of wild type and transduced cells expressing STX or the marker gene alkaline phosphatase was examined using a gap bridging assay in dissociated cells and by grafting cells in slice cultures of postnatal brain. Migration of PSA expressing cells was significantly increased in both models, as compared to control cells, and this effect was abolished by endoneuraminidase-N stripping of PSA. PSA-positive Schwann cells retained the ability to differentiate in vitro and expressed the Krox20 and P zero myelination markers. When grafted in neonatal cerebellar slices, STX-transduced cells started to myelinate Purkinje cell axons like control cells and make myelin internodes after 2 to 3 weeks. PSA was redistributed on the cell membrane and downregulated during differentiation in pure Schwann cell cultures and slice co-cultures. Thus, migratory properties of PNS myelin-forming cells within the CNS can be enhanced without altering their differentiation program. This finding may be beneficial for the development of remyelination therapies.     

Central nervous system susceptibility to herpes simplex infection.

Four days after inoculation of herpes simplex virus (HSV) on the rabbit cornea, distinctive and reproducible lesions appear in the trigeminal root entry zone. These viral lesions, situated in the central nervous system (CNS) portion of the root, consist of severe myelin destruction accompanied by mononuclear cell infiltration and partial sparing of axons. Immunofluorescent study demonstrated abundant viral antigen, and by electron microscopy viral nucleocapsids were found to be numerous within astrocytes and were rarely found in other cell types. In contrast, the adjacent peripheral nervous system (PNS) tissue appears unaffected by the presence of virus. The mechanism for this marked difference in response of the central nervous system and the peripheral nervous system may depend upon the susceptibility of astrocytes to viral infection and replication. The selective nature of the lesion provides an easily reproducible model for further investigation of the response of nervous system tissue to HSV.     

Herpes simplex virus type I (HSV I)-induced multifocal central nervous system (CNS) demyelination in mice.

Multifocal central nervous system (CNS) demyelination develops in the brains of SJL/J, PL/J, and A/J mice following lip inoculation with a specific strain of herpes simplex virus I (HSV I). The lesions in all three inbred strains of mice share similar characteristics including demyelination, relative preservation of axons, and a mononuclear cell (MNC) infiltrate. The lesions, developing during the early phase of demyelination, also appear sequentially in the CNS (trigeminal root entry zone of the brainstem greater than cerebellum greater than cerebral hemispheres) of all three strains of mice but differ in the time of their initial appearance following infection as well as their morphology. In SJL/J mice, new areas of demyelination are observed for only 24 days following lip inoculation with virus. Late stage multifocal CNS demyelination persists throughout 28 weeks postinoculation (pi) in PL/J mice while in A/J mice the development of new areas of demyelination are restricted to 8 weeks pi. Although mononuclear inflammatory cells are present in the new areas of demyelination in either PL/J or A/J mice, viral antigens are not detected in the CNS beyond 12 days pi. In contrast, in situ hybridization studies using 35S-cDNA HSV probes and performed beyond day 12 pi identify probe-positive cells central to a number of the multifocal CNS demyelinating lesions in A/J mice. Results from studies with inbred and congenic strains of mice indicate that the major histocompatibility complex (H-2) does not determine the development of multifocal CNS demyelination following lip inoculation with HSV I but does influence the morphological appearance of the lesions that do develop.     

Limited remyelination of CNS axons by Schwann cells transplanted into the sub-arachnoid space

Areas of primary demyelination which did not subsequently remyelinate spontaneously were prepared in the cat spinal cord by injecting small volumes of ethidium bromide into tissue which had previously been exposed to 40 Grays of X-irradiation. Autologous peripheral nerve tissue was placed in the sub-arachnoid space over such lesions, either at the time of injecting ethidium bromide, or at 14 days or 28 days after injecting ethidium bromide. The extent of Schwann cell remyelination was assessed 28 days after transplantation. In no case were all the demyelinated axons remyelinated; rather, remyelination was limited to axons near to blood vessels. It was concluded that Schwann cells migrated from the transplanted tissue into the lesion via the perivascular space and that they failed to remyelinate the bulk of demyelinated axons because of an absence within the CNS of suitable extracellular matrix.     

Semliki Forest virus-induced demyelination and remyelination--involvement of B cells and anti-myelin antibodies

Semliki Forest virus (SFV) infection induces a demyelinating encephalomyelitis in the central nervous system (CNS) of mice and serves as a model for multiple sclerosis (MS). This study investigated CNS immune responses at different stages of infection and during SFV-induced demyelination and remyelination. Following the initial CNS inflammation, pathology and viral clearance on days 6-10 post-infection (pi), primary demyelination was observed in cerebellar, brainstem and corpus collosal white matter by days 15-21 pi, with plasma cells and microglia as main participants, and this was followed by remyelination. By day 35 pi, the tissue appeared almost normal. Fluorescent antibody cell sorter (FACS) analysis showed that brain CD8(+) T cells increased during the initial inflammatory response and gradually decreased thereafter. Brain B cell (B220(+)CD19(+)) numbers did not change significantly during the course of infection; however, from days 14 to 35 pi, they matured and produced antibodies to viral and myelin proteins (and peptides) during the period of demyelination and remyelination. The proportion of CD3(-)B220(-)CD11b(+) cells also progressively increased throughout the periods of de- and remyelination. Our results suggest that CD8(+) T cells are involved in the initial destruction of CNS tissue during the first weeks of SFV infection, while B cells, antibodies and microglia may contribute to the myelin pathology seen after recovery.     

Caspr reveals an aggregation of nodes and flanking node free zones at the rat trigeminal sensory root and dorsal root entry zones

The sensory root entry zone demarcates the transition from the peripheral nervous system (PNS) to the central nervous system (CNS). In this study, we describe the organization of nodes of Ranvier at the trigeminal sensory and dorsal root entry zones of the rat. Caspr immunoreactivity (IR) was used to identify the paranodal region of nodes of Ranvier, while L-MAG-IR was used to identify CNS oligodendrocytes. Immunofluorescence confocal microscopy revealed a dense aggregation of nodes precisely at the PNS to CNS transition with prominent node-depleted zones on either side, while L-MAG-IR was confined to ensheathing fibers on the central side of nodes located in this dense band and identified these as transitional nodes. Morphometric analysis of the PNS and CNS sides of the trigeminal and the PNS side of the dorsal root entry zones confirmed the presence of virtually node-free domains flanking the transitional zone. Further, the reappearance of nodes on the far side of the node-free zones strongly correlated with nodal diameter, with small nodes reappearing first. These findings suggest that the PNS/CNS transition may represent the initial site of myelination of the primary afferent axon within this area.     

Latent herpes simplex virus trigeminal ganglionic infection in mice and demyelination in the central nervous system.

Mice were inoculated with herpes simplex virus (HSV) type 1 by gently scraping the skin of the nose with a fine needle. About 80% of the animals developed latent inapparent HSV infections in trigeminal ganglia. Virus was demonstrable for at least 6 months post inoculation (p.i.) by cocultivation of ganglionic tissue with GMK cells. Histologically, trigeminal ganglia revealed infiltrations of inflammatory cells even 6 months p.i. In addition, lesions occurred in the brainstem corresponding to the entry of trigeminal roots, trigeminal tracts and nuclei. Inflammatory cell infiltration, disruption of myelin sheaths and macrophages laden with myelin degradation products were observed 7 days p.i. Fourteen to 30 days p.i. electron microscopy demonstrated completely naked axons. In the transitional region of the trigeminal root denuded axons occurred in the central part of the region while the peripheral myelin, bordering the demyelinated central segments, was intact. Small areas of demyelination were still detectable 3 and 6 months p.i. but there were then also signs of remyelination. Possible mechanisms causing the demyelinations are discussed.     

HSV (Type 1) infection of the trigeminal complex

Following corneal inoculation with herpes simplex virus (Type 1) (HSV), virus spreads to the CNS by axonal transport in the central branches of trigeminal ganglion cell neurons. Although this mode of viral entry to the CNS is rare for humans, it appears to be the principal route of entry into the CNS in animal models of herpetic corneal disease. In this study, the corneas of BALB/c mice were unilaterally inoculated with HSV, and the distribution of HSV-immunoreactive label was studied to identify the central branches of the axons of infected trigeminal ganglion cells. Virus was first noted in the brainstem trigeminal complex 4 days after corneal inoculation, when HSV-labeled afferents were found throughout the course of the descending tract of V as well as in interstitial neurons in the tract. By 5 days labeled neurons were also found not only in the n. caudalis and portions of the n. interpolaris of the trigeminal complex but also in laminae I–IV of the dorsal horn of the upper cervical levels of the spinal cord. No immunoreactivity was seen in other regions of the complex, including the n. oralis or the main sensory n. of V. By 6 days, however, the infection had spread to the main sensory division of V.     

Type 1 astrocytes fail to inhibit Schwann cell remyelination of CNS axons in the absence of cells of the O-2A lineage.

The extent to which Schwann cells are able to remyelinate demyelinated CNS axons is influenced by the presence of astrocytes. In order to study further the nature of astrocyte control of Schwann cell remyelination in the CNS, cultures containing type 1 astrocytes and a small proportion of Schwann cells, but depleted of O-2A lineage cells by exposure to cytosine arabinoside and complement-mediated immunocytolysis, were transplanted into glial-free lesions in adult rat spinal cord in which the host response to demyelinated axons was suppressed by X-irradiation. Following transplantation of these O-2A lineage-depleted cultures into X-irradiated, demyelinating lesions, there was extensive remyelination of demyelinated axons by Schwann cells, a result which contrasted with those obtained from earlier experiments in which O-2A lineage cells were present within the transplant, and/or recruited from host tissue. This experiment shows that the presence of O-2A lineage cells is required in order for transplanted type 1 astrocytes to organise in a manner which inhibits extensive Schwann cell remyelination of CNS axons.     

Demyelination and early remyelination in experimental allergic encephalomyelitis passively transferred with myelin basic protein-sensitized lymphocytes in the Lewis rat

Histological studies were performed on Lewis rats with experimental allergic encephalomyelitis (EAE) passively transferred by myelin basic protein (MBP)-sensitized syngeneic spleen cells in order to determine the relationship between demyelination and neurological signs. Neither inflammation nor demyelination was present on the day prior to the onset of neurological signs but both were present in the spinal roots and spinal cord on the day of onset of tail weakness (4 days after passive transfer). Demyelination and the neurological signs both increased over the next 48 h. There was evidence that the caudal roots were more severely affected than the rostral roots. The peripheral nerves were spared. Demyelination in the spinal cord was concentrated in the dorsal root entry and ventral root exit zones. The initial stages of repair of demyelinated spinal root fibres by Schwann cells were observed on the earliest day that clinical recovery commenced (day 7). At this time some demyelinated fibres were closely associated with debris-free Schwann cells, and occasional fibres were completely invested by 1-2 layers of Schwann cell cytoplasm. Remyelination (compact myelin lamellae formation) by Schwann cells was first observed in the spinal roots on day 9. By the time of complete clinical recovery (day 11) the majority of affected spinal root cores had thin new myelin sheaths. Repair of central nervous system myelin by oligodendrocytes was slower than peripheral nervous system myelin repair. Investment of demyelinated spinal cord axons by oligodendrocytes was observed on day 9, and remyelination by these cells was seen on day 10. We conclude that the neurological signs of passively induced MBP-EAE can be accounted for by demyelination of the lumbar, sacral and coccygeal spinal roots and spinal cord root entry and exit zones, and that the subsequent clinical recovery can be explained by investment and remyelination of demyelinated peripheral and central nervous system fibres by Schwann cells and oligodendrocytes respectively.