H. deF. Webster

An experimental analysis of interlamellar tight junctions in amphibian and mammalian C.N.S. myelin

SummaryThe distribution of interlamellar tight junctions was examined in myelin sheaths ofXenopus tadpole optic nerve and rabbit epiretinal tissue fixed with aldehydes, postfixed with osmium ferrocyanide and embedded in a water-soluble medium, Durcupan. Intramyelinic zonulae occludentes were clearly formed by fusion of adjacent intraperipd lines which corresponded to the external leaflets of oligodendrocytes. These occurred in register with other tight junctions present within successive lamellae and appeared as a series of radial lines extending either partially or totally across the thickness of the myelin sheath. This distribution of zonulae occludentes corresponded with that of tight junctional particle strands observed in freeze-fracture replicas.Analysis of intramyelinic vacuolation induced by hexachlorophene (HCP) intoxication indicated that lamellar splitting was frequently limited by the tight junctions. The intramyelinic zonulae occludentes also restricted the diffusion of colloidal lanthanum which had penetrated the myelin intraperiod gap followingin vivo perineural injection. The results of this study provide evidence favouring a correspondence between interlamellar tight junctions and the ‘radial component’ of myelin described earlier by other investigators. Furthermore, observations of swollen myelin sheaths, resulting from HCP intoxication, suggest that these junctions may play a major role in maintaining myelin sheath integrity and limiting the extent of breakdown during certain pathological conditions.

ISOLATION AND CHARACTERIZATION OF MYELIN-RELATED MEMBRANES

Abstract— Myelin related membrane fractions from rat brain and spinal cord were isolated from material normally discarded during standard myelin isolation procedures. A fraction which floated on 0.32 M‐sucrose (F) and the material released after subjecting the myelin fraction to osmotic shock at two stages in the purification (W1 and W2) were characterized. These fractions were subjected to subfractionation on three step discontinuous sucrose gradients. Morphologically, the heavier subfrac‐tions of W1 and W2 were shown to consist mainly of single membranes and vesicles. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that, relative to myelin, proteolipid and basic protein were reduced in all subfractions, while the high molecular weight proteins were increased. The specific activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) was up to 2‐fold higher than that of myelin in the heavier subfractions of W1 and W2. The major myelin‐associated glycoprotein was also increased in these subfractions as determined by periodic acid‐Schiff staining. Differential centrifugation of the initial tissue homogenate to remove microsomes prior to myelin isolation gave rise to W1 and W2 subfractions with a CNP specific activity 3–4 times that of myelin. The high molecular weight proteins and glycoproteins were enriched in these microsome‐depleted subfractions, but were qualitatively similar to those of myelin. Some of the membranes in these fractions may be derived from the continuum between the plasma membrane of the oligodendrocyte and compact myelin. Fraction F consisted of small membrane fragments and many vesicles, and was particularly deficient in proteolipid. The specific activity of CNP in fraction F was about the same as myelin, while the major myelin associated glycoprotein could not be detected. Fraction F from normal CNS tissue appears to be similar to the floating fractions previously isolated in larger amounts from pathological brain undergoing edematous demyelination.

Hexachlorophene-induced myelin lesions in the amphibian central nervous system. A freeze-fracture study.

Abstract Myelin membranes in the optic nerves and spinal cord of Xenopus laevis tadpoles were studied with the freeze-fracture technique during the pathogenesis of edematous lesions induced by hexachlorophene (HCP) intoxication. Freeze-fracture replicas of myelin exposed to HCP for as long as 12 days revealed that even chronic intoxication did not significantly alter particle strands associated with zonulae occludentes between tongue processes and their adjacent layers of myelin. Furthermore, experiments using electron-dense tracers demonstrated that the permeability characteristics of these junctions were unchanged. Following subcutaneous perineural injection of lanthanum or horseradish peroxidase (HRP), the tracers easily penetrated the optic nerve and were uniformly distributed throughout its parenchyma. While only limited amounts of lanthanum could penetrate myelin and enter the vacuoles, no HRP was found within the large myelin vacuoles after either brief or long-term exposure to HCP. In contrast to the tight junctions, some modifications were observed in the distribution of other intramembranous particles associated with myelin lamellae adjacent to swollen regions of the sheath. These alterations primarily involved formation of multifocal, particle-free elevations which corresponded to areas in which the fused inner glial leaflets, forming the major dense line, had separated. Such particle-deficient areas were absent in myelin of control as well as normal sheaths of HCP-intoxicated animals. Thus, these findings demonstrated that the structural and physiological characteristics of a tight junctional component of myelin were not affected by HCP. Sparing of these junctions may account for the relatively low incidence of demyelination and reversible nature of HCP lesions. The development of particle-deficient blisters on glial leaflets suggests, however, that HCP may be capable of modifying other aspects of the chemical organization of myelin, thereby producing local alterations in the compact arrangement of myelin lamellae.

Herpes-simplex-related antigen in human demyelinative disease and encephalitis

SummaryUsing immunohistochemical methods optimized to detect herpes simplex virus type 2 (HSV-2) antigen, paraffin sections from human central nervous system tissues from 31 cases pathologically diagnosed as multiple sclerosis (MS), 34 cases of other neurological diseases, 4 adult cases of HSV encephalitis, and mouse brains infected with various HSV strains were examined. Two distinct patterns of immunoreactivity with HSV antisera were seen. In typical acute human and experimental encephalitis, antigen was readily detected using high dilutions of antisera to both HSV types −1 and −2, and was found nonselectiviely in both neurons and glia. Lesions were destructive, with necrosis of all neural cell types, and inflammation was a mixture of polymorphonuclear and mononuclear cells. By contrast, immunoreactivity in lesions in each of three MS cases and in one case of brain stem encephalitis was found only with HSV-2 antisera, and relatively high antiserum concentrations were required to detect it. Reactivity appeared to be largely restricted to glial cell nuclei within and near lesions that were selectively demyelinated. Only mononuclear inflammation was present. These experiments suggest that HSV-related antigen may be found in a broader spectrum of human CNS lesions than has previously been recognized, and that HSV or a related agent may be associated with a selective infection of glial cells and with CNS demyelination.

The effects of low temperature on myelin formation in optic nerves of Xenopus tadpoles

To test the effect of cold on CNS myelin formation, optic nerves of stages 52-55 Xenopus tadpoles were examined electron microscopically after maintenance at 15, 10, 7 and 4 degrees C for 1-7 days. Nerves from tadpoles maintained at 15 degrees C resembled 22 degrees C (room temperature) controls. After 3 days at 10, 7, or 4 degrees C, tongue processes and perikarya of many myelin forming oligodendrocytes were swollen and filled with vesicular membrane profiles. The number of axonal microtubules was decreased in affected fibers but the lamellar structure of their myelin sheaths remained normal. Astrocytes were hypertrophic and contained large aggregates of filaments. Longer exposure to 10 or 7 degrees C increased the number of affected fibers but the changes were not more severe or associated with degeneration. The delayed onset, lack of progression and reversibility of the changes indicated that cold has a direct metabolic effect on myelin forming oligodendrocytes. Alterations produced by nerve transection or exposure to mitotic inhibitors differed, suggesting that cold induced changes were not due primarily to either axonal degeneration or reduced axonal transport.

Po Glycoprotein mRNA distribution in myelin-forming Schwann cells of the developing rat trigeminal ganglion

SummaryA biotinylated Po cDNA was hybridizedin situ to aldehyde-fixed vibratome sections of trigeminal ganglia from day 2, day 7, day 15, day 30 and adult rats. Nickel-enhanced horseradish peroxidase (HRP) was used in an antibody sandwich method to detect hybridization.After postfixation in osmium tetroxide, the sections were dehydrated in ethanol and embedded in epon. At each age, some vibratome sections were used to count the HRP-positive and HRP-negative myelin-forming Schwann cells. The percentage of HRP-positive myelin-forming Schwann cells in ganglia from day 2, 7, 15, 30, and adult rats were 31%, 56%, 47%, 12% and 3%.In sections of ganglia from 2-day-old rats, studied by light and electron microscopy, peroxidase reaction product localizing hybridized Po mRNA was found on profiles of granular (rough) endoplasmic reticulum (RER) in perinuclear regions of Schwann cells which had formed two to three compact myelin lamellae. Peroxidase deposits were larger and more numerous in the cytoplasm of cells with thicker myelin sheaths.At day 7, some Schwann cells had long external mesaxons; the cytoplasm between these mesaxons and the cell surface often contained abundant HRP-stained profiles of RER. In sections from day 7 and day 15 ganglia, substantially more reaction product was found. In each myelin-forming Schwann cell, the amount was generally proportional to the size of the newly formed myelin sheath. HRP deposits were observed all along the outer surfaces of myelin segments at these ages, and their distribution corresponded to that of the RER.Later on, at day 30 and in the adult, fewer Schwann cells contained reaction product, and there was less in each one, indicating that lower levels of Po mRNA are present in Schwann cells during later stages of myelination. The lowest levels were seen in adults and may reflect those needed for Po turnover during myelin sheath maintenance.

Early Viral Proteins As Autoantigens

In multiple sclerosis (MS) the target antigen in the CNS could be a normal component of glia or myelin. Alternatively, partial reactivation of a latent viral infection might allow the immune response to target an early viral antigen without virus replication. If an early viral protein induces immunopathology in the absence of virus particle production, it would mimic an autoantigen. Although the human polyomavirus JC virus (JCV) is not known to latently infect the brain, it occasionally reactivates in the kidney, and in progressive multifocal leukoencephalopathy (PML) it infects oligodendrocytes and astrocytes of immunocompromised adults. Polyomavirus T antigens are early DNA-binding nuclear proteins that can also become targets of cytotoxic T lymphocytes at the cell surface. To study the relation of early and late viral gene expression, we developed a double-label method for simultaneous detection of JCV T antigen and capsid proteins in cryostat sections of PML brain. PML was diagnosed in a 46-year-old man by computerized tomography and magnetic resonance imaging and confirmed pathologically at autopsy. For immunocytochemical methods see figure legends.

Persistence of neurotropic JC virus DNA in hamster tissues six months after intracerebral inoculation

Immunostaining and polymerase chain reaction (PCR) methods were used to examine tissues from 18 6-month-old hamsters intracerebrally inoculated with JC virus (JCV) as newborns. JCV DNA was detected in all hamster brains and urinary bladders, as well as in most kidney, adrenal gland and pancreas samples. While results from reverse transcription PCR (RNA PCR) and immunostaining suggest that T antigen transcription and protein expression were restricted to the brain, the DNA suggests that intracerebrally inoculated JCV enters the systemic circulation and latently infects organs in a tissue specific manner.

Central Nervous System Demyelination in Herpes Simplex Virus Type 2 Infection

The pathogenesis of primary and reactivated herpes simplex virus type 2 (HSV-2) infection is examined in mice to determine its nervous system effects and its potential for causing demyelination. In intracerebrally and vaginally infected mice, there is a broad spectrum of possible disease outcomes. A mainly demyelinative CNS pathology is the consequence of non-fatal infections, while animals with fatal infectious have non-selective, severe infection and disease of both the gray and white matter. The mechanism of demyelination involves the infection and loss of oligodendrocytes, but whether this is a purely cytolytic infectious process is unclear. Elongated spinal cord demyelinative lesions which appear to be tract associated, and presence of virus in axons in acute demyelinative lesions suggest a model of virus spread from neurons to the cells that myelinate their axons, with amplification of infection in the white matter. In the genital infection model, antibody responses may appear late or be undetected, thymic cortical necrosis tends to segregate with severe infection, and the capacity to isolate virus from lymphoid tissues is restricted to animals with fatal infections, raising the possibility that at virus-induced immunosuppression may be a determinant of severe infections. In a model of viral latency established by a genital route in which 50% or more of mice develop small demyelinative cord lesions during primary infections, HSV-2 reactivations were successfully induced in many mice using immunosuppressive treatment. With reactivation, virus could be isolated from the vagina and CNS. Antigen was found in T9-L2 and L6-S2 ganglia, with lesser amounts in distal roots and in the spinal cord; recurrent CNS demyelinative lesions were not found. The potential importance of these findings in understanding human HSV-2 infection and demyelinative disease is discussed.