Huw G. Jenkins

Tumour necrosis factor causes an increase in axonal transport of protein and demyelination in the mouse optic nerve

Abstract An increase in fast axonal transport of protein by the optic nerve was found in mice following a single combined injection of human recombinant tumour necrosis factor alpha (rTNF) and [3H]proline into the vitreous chamber. Demyelination was observed in optic nerve fibres arising from the eyes of mice which received a single rTNF injection. No such changes were detected when heat-inactivated rTNF was injected with the label. The effects of intravitreal injection of rTNF on the pathophysiology of mouse optic nerve resembled those found in mice infected with Semliki Forest virus (SFV), an animal model of multiple sclerosis. We suggest that TNF could mediate at lease some of the pathophysiological changes found in SFV-infected mice and may provide a clue concerning the disease mechanism in multiple sclerosis.

Morphological changes in oligodendrocytes in the intact mouse optic nerve following intravitreal injection of tumour necrosis factor

Intracellular dye-injection was used to determine the whole-cell morphology of oligodendrocytes in intact optic nerves of mice following intravitreal injection of tumour necrosis factor-alpha (TNF alpha) or heat-inactivated TNF alpha to act as controls. Oligodendrocytes in control nerves had a stereotypic morphology, and provided an average of 20 axons with single internodal myelin segments of around 200 microns internodal length. Oligodendrocytes with abnormal morphological features were identified 8-14 days following intravitreal injection of TNF alpha. Internodal myelin segments developed swellings along their lengths, became attenuated, and in extreme cases receded completely. This study provides a new insight into the process of demyelination, especially of the early stages which are not amenable with other techniques. Furthermore, it confirms that injection of TNF alpha into the vitreous, a fluid compartment of the CNS, instigates a sequence of events which results in oligodendrocyte disruption and demyelination. The mechanism by which intravitreally injected TNF alpha mediates these changes in optic nerve oligodendrocytes are yet unknown.

Optic neuritis in chronic relapsing experimental allergic encephalomyelitis in Biozzi ABH mice: Demyelination and fast axonal transport changes in disease

The encephalitogenicity of optic nerve tissue was demonstrated in Biozzi ABH (H-2(dq1)) mice. Acute experimental allergic encephalomyelitis (EAE) occurred in 11/14 animals and 4/5 exhibited relapse. The involvement of the optic nerve in spinal cord homogenate induced chronic relapsing EAE (CREAE) was demonstrated by mononuclear cell infiltration and myelin degradation in the optic nerve prior to and during clinical disease. During the relapse phase gross pathological assessment revealed swollen and translucent plaques on the optic nerves. Advanced lesions showed widespread demyelination, astrocytic gliosis and fibrotic changes of the blood vessels. Physiologically, the fast axonal transport of proteins from the retina to the optic nerve and superior colliculus was significantly decreased during relapse. The association of inflammation and demyelination with physiological deficit in the optic nerve highlights the usefulness of this model in the study of multiple sclerosis in which acute monosymptomatic unilateral optic neuritis is a common manifestation. Furthermore, the novel induction of CREAE with optic nerve homogenate suggests that optic neuritis is a common significant role in the pathophysiology and progression of neurological disease in CREAE which may be relevant to studies of optic neuritis in multiple sclerosis.

Diurnal variations in the electroretinographic c-wave and retinal melatonin content in rats with inherited retinal dystrophy

The inability of retinal pigment epithelium to phagocytose shed photoreceptor disks is a cause of retinal degeneration in the Royal College of Surgeons rat; retinal pigment epithelial phagocytosis and disk shedding are regulated by the diurnal rhythm of retinal melatonin level. The diurnal rhythms of the electroretinogram (particularly that of the retinal pigment epithelial potential, the electroretinographic c-wave) and retinal melatonin content were thus investigated in Royal College of Surgeons rats from postnatal day 17 to 24, the period preceding retinal degeneration. The amplitudes of both the b- and c-waves of the electroretinogram fell significantly during the peak time of rod disk shedding and rose after the time of expected light off in the control and dystrophic rats. While the b-wave rhythms did not differ between the two strains, diurnal changes in the c-wave were significantly less distinct in the dystrophic rats than in controls. This difference may reflect lack of phagocytosis in dystrophic rats. Furthermore, the ERG c-wave was significantly larger and prolonged, and the retinal melatonin content higher, in dystrophic rats of this age group than in controls. It appears that retinal melatonin metabolism may play an important role in the maintenance of retinal pigment epithelial and photoreceptor function.

Morphology of oligodendrocytes during demyelination in optic nerves of mice infected with Semliki Forest virus

Multiple sclerosis (MS) is a demyelinating disease which affects oligodendrocytes, the myelinating cells of the CNS. Demyelination is known to occur in the optic nerves of Balb/c mice infected with the avirulent A7(74) strain of Semliki Forest virus (SFV), and many of the changes are similar to those of patients with MS. The aim of the present study was to determine how demyelination proceeds in individual oligodendrocytes in SFV infection, to help in understanding the pathology of demyelination and remyelination in MS. The whole‐cell morphology of individual oligodendrocyte units (defined as the oligo‐dendrocyte, its processes and the internodal myelin segments of the axons it ensheaths) was characterized using intracellular dye injection in isolated intact optic nerves. In untreated control mice, oligodendrocytes had a relatively uniform morphology and each cell on average provided 20 or so nearby axons with single myelin sheaths with internodal lengths of ∼ 150 μm. In SFV infected mice, during the peak of demyelination at post inoculation days 14–21, 55% of oligodendrocytes displayed a range of morphological abnormalities, which most likely represented sequential changes in oligodendrocytes during demyelination. Thus, at the earliest stage of demyelination oligodendrocytes developed swellings or vacuolations along their internodal myelin sheaths, which became gradually attenuated and were completely lost in extreme cases. The results show that whole oligodendrocyte units were affected during SFV‐induced demyelination and this is the basis of the focal nature of lesions in this viral model of MS. Individual oligodendrocyte units which had lost their full complement of myelin sheaths had the appearance of immature oligodendrocytes, suggesting they had undergone de‐differentiation. We concluded that these cells may not be destroyed during demyelination and it is possible they are capable of remyelination which is a feature of SFV infection in mice and MS in humans.

Receptor-mediated uptake of β-glucuronidase into primary astrocytes and C6 glioma cells from rat brain

The ability of both primary astrocytes from rat cerebrum and a rat C6 glioma cell line to take up lysosomal enzymes by receptor-mediated endocytosis was compared. The beta-glucuronidase secreted by 3T3 fibroblasts was purified to homogeneity by antibody affinity chromatography, iodinated and used as a typical enzyme to determine the nature of receptors involved in its uptake into glial cells. Both primary astrocytes and C6 glioma cells took up 125I-labelled enzyme in a rapid and saturable manner indicative of specific receptors, while immunostaining with an anti-mouse beta-glucuronidase antibody showed that the enzyme was distributed in a mainly punctate pattern after uptake, characteristic of that of lysosomes. Subcellular fractionation of C6 glioma cells following endocytosis revealed that the enzyme became localised in lysosomes, after first passing through an endosomal compartment. Uptake of enzyme was reduced markedly after its sugar side chains had been removed with N-glycanase, indicating that endocytosis was mediated via a carbohydrate-recognising receptor. A range of carbohydrates and glycoproteins were tested for their ability to inhibit receptor-mediated endocytosis but of these only sialic acid had a notable effect. Further evidence that endocytosis of beta-glucuronidase into primary astrocytes and C6 gliomas may be mediated via sialic acid receptors was provided by the large reduction in rate of uptake observed following removal of this sugar from the enzyme with sialidase.

EVIDENCE FOR A T-CELL RELATED FACTOR AS THE CAUSE OF DEMYELINATION IN MICE FOLLOWING SEMLIKI FOREST VIRUS-INFECTION

Patchy demyelination throughout the central nervous system, including the optic nerve, is known to occur following infection with Semliki Forest virus (SFV) in Swiss/A2G mice. An increase in the fast axonal transport of protein in optic nerves occurred before they showed signs of demyelination in Swiss/A2G mice, heterozygous nude mice and nude mice reconstituted with T-cells. SFV infection, however, caused neither an increase in the fast axonal transport of protein, nor optic nerve demyelination in T-cell-deficient nude mice. Thus, the increase in both axonal transport and demyelination following SFV infection appear to be T-cell-mediated events, rather than direct effects of the virus.

TNF mediates oligodendrocyte damage in vivo

The effect of intravitreally injected cytokines (TNFa, TNF/3, IL-1, IL-6, IFN--/) on glia in mouse optic nerves was determined, by using intracellular dye-injections to characterise their whole-cell morphology. Following intravitreal injection of TNFa or TNFI3, oligodendrocytes initially developed swellings along the length of their myelin segments, and internodal myelin segments became increasingly attenuated, until, in extreme examples, individual oligodendrocytes lost their full complement of myelin segments; oligodendrocyte changes were not observed with IL-1, IL-6 or IFN-7. Widespread astrocyte reactivity, characterised by astrogliosis and an upregulation of GFAP, was observed following intravitreal injection of TNFa, TNF/3, IL-1 or IL-6; IFN-7 alone had no effect. Similar glial changes have been seen during demyelination following inoculation with Semliki Forest virus. This study supports a role for TNF in T-cell mediated demyelination and for TNF, IL-1 and IL-6 in mediating astrogliosis and reactivity.

Tumor necrosis factor causes an increase in axonal transport of protein and demyelination in mouse optic nerve

An increase in fast axonal transport of protein by the optic nerve was found in mice following a single combined injection of human recombinant tumour necrosis factor alpha (rTNF) and [3H]proline into the vitreous chamber. Demyelination was observed in optic nerve fibres arising from the eyes of mice which received a single rTNF injection. No such changes were detected when heat-inactivated rTNF was injected with the label. The effects of intravitreal injection of rTNF on the pathophysiology of mouse optic nerve resembled those found in mice infected with Semliki Forest virus (SFV), an animal model of multiple sclerosis. We suggest that TNF could mediate at least some of the pathophysiological changes found in SFV-infected mice and may provide a clue concerning the disease mechanism in multiple sclerosis.