Patterns of X and Y optic nerve fibre terminations in the dorsal lateral geniculate nucleus of the cat.

The distributions of X and Y optic nerve fibre terminals in the A and A1 laminae of the dorsal lateral geniculate nucleus (LGNd) of the cat have been determined by a method that eliminates the Y fibres. A pressure-blocking technique was used in a sterile operation to produce anterograde degeneration in the Y fibres with minimal effect on the X fibres. Subsequently the Fink/Heimer technique was used to stain for degenerating fibres. This showed a strong peak of degeneration in the ventral regions of the laminae. Tritiated leucine was injected into one eye either of a normal cat or of one in which the optic nerve had been pressure-blocked at least one week previously. Subsequent examination of the LGNd by autoradiography showed a more uniform distribution of label in the laminae deprived of Y input (i.e. the pattern of distribution of X fibres). Subtraction of this distribution from that produced in a normal cat (i.e. X + Y input) gave the Y distribution. As in the degeneration studies, this revealed a peak of label in the most ventral part of each lamina but also showed a smaller peak in the most dorsal regions.

Activated non-neural specific T cells open the blood-brain barrier to circulating antibodies.

Previous studies have shown that activated T cells can successfully cross endothelial barriers and will accumulate in tissue which contains their specific antigen. Myelin specific T cells (e.g. myelin basic protein specific) are recognized to play an important role in the induction of experimental autoimmune demyelinating disease of the CNS and have been shown to induce blood-brain barrier breakdown effectively. In this study we injected T cells reactive to a non-neural antigen (ovalbumin) systemically into Lewis rats and caused them to accumulate in the thoracic dorsal column by a prior injection of ovalbumin. Selected rats were given systemic demyelinating antibody, antimyelin oligodendrocyte antibody (anti-MOG antibody), to provide evidence of permeability changes to the blood-brain barrier. These animals were compared with control rats given systemic anti-P0 monoclonal antibody and to other rats given a direct micro-injection (3 microliters) of anti-MOG antibody into the thoracic dorsal column. All animals were monitored by serial neurophysiological studies and by histological examination. Direct anti-MOG antibody injection produced a focal block in conduction at the injection site and a large circumscribed area of primary demyelination with axonal preservation within the dorsal column. An even more profound conduction block and more extensive plaque-like region of demyelination were seen in animals given antigen, activated T cells and systemic antibody. However, animals given antigen and T cells without relevant antibody did not show conduction impairment or demyelination, except when very large numbers of T cells were given; such rats developed severe irreversible axonal damage. This study demonstrates the blood-brain barrier is disrupted by activated T cells of non-neural specificity and allows large plaque-like regions of demyelination to form in the presence of circulating antimyelin antibody. The relevance of this finding to multiple sclerosis is discussed.

Bacterial lipopolysaccharide induces a conduction block in the sciatic nerves of rats.

A single injection of Escherichia coli lipopolysaccharide (LPS; intraperitoneally [i.p.] and intravenously [i.v.]) reliably induces peripheral nerve disturbances in the hindlimbs of inbred Australian albino Wistar (AaW) rats. In the series of experiments presented here, we aimed to characterize this syndrome by examining electrophysiologic, immunologic, and immunochemical features. The LPS-induced neurologic sequelae in AaW rats were transient, at least partly reversible by drug treatment, and were not associated with any detectable neuropathologic findings by light microscopy. Neurologic sequelae were prevented by administration of dexamethasone and by pretreatment with the macrophage inhibitor gadolinium chloride, suggesting that they were caused by LPS-induced activation of peripheral macrophages. Sequelae were associated with early decreases in compound muscle-action potential amplitudes, indicating impaired functioning of either proximal sciatic nerve axons and/or neuromuscular synapses. Spinal somatosensory-evoked potential latencies also were increased, indicating impaired somatosensory function at the sciatic nerve, dorsal roots, spinal cord, and/or postsynaptic interneurons, although the precise location of impairment could not be delineated. Similarities between this syndrome and immune-mediated polyneuropathies in humans are discussed.

Intraneural activated T cells cause focal breakdown of the blood-nerve barrier.

Experiments were conducted to investigate the effect of activated T cells on the blood-nerve barrier (BNB) in experimental allergic neuritis (EAN). T cells reactive to the P2 component of myelin (P2 T cells) and known to cause EAN were injected into the sciatic nerve of Lewis rats. Animals were then given daily intraperitoneal (i.p.) injections of serum with known demyelinating activity (rabbit EAN serum) or control serum. Serial nerve conduction studies across the injected segment were performed and nerves were removed at various stages for histology. Focal conduction block and perivascular demyelination were evidence in T cell injected nerves of animals treated with EAN serum. In animals treated with control serum no conduction block was seen and only perivascular infiltrates without demyelination were present. Similar results were obtained with T cells reactive to non-neural antigens, although the effect was less marked. Systemically administered rabbit immunoglobulin (Ig) was demonstrated within the endoneurium of P2 T cell injected nerves by immunofluorescence and the endoneurial blood vessels showed increased permeability to circulating horseradish peroxidase (HRP). These findings demonstrate that activated T cells cause focal breakdown of the BNB, allowing circulating antimyelin antibody to enter the endoneurium with consequent focal demyelination. P2 reactive EAN producing T cells do not cause significant demyelination when injected intraneurally (i.n.) in the absence of circulating antimyelin antibody. Intraneural injection of tumour necrosis factor alpha (TNF-alpha) yielded similar results, causing conduction block and perivascular demyelination in the presence of circulating antimyelin antibody but not in control serum treated animals.

Activated T cells of nonneural specificity open the blood-nerve barrier to circulating antibody.

Recent studies from our laboratory and by other investigators have shown that autoreactive CD4+ cells specific for peripheral nerve P2 protein have a powerful effect on blood-nerve barrier permeability. In this study we injected CD4+ T cells reactive to a nonneural antigen (ovalbumin) systemically and achieved their accumulation in the tibial nerve of Lewis rats by previous intraneural injection of ovalbumin. Selected rats were given systemic demyelinating antibody (antigalactocerebroside) to provide an indicator of changes in the permeability of the blood-nerve barrier, and the animals were monitored by sequential neurophysiological studies and histology. Circulating ovalbumin-specific T cells accumulated at sites of intraneural ovalbumin injection without inducing demyelination in control animals. In rats with circulating galactocerebroside antibodies, local conduction block and demyelination were seen in the region of T-cell accumulation. Electron microscopy demonstrated dissolution of some tight junctions between endothelial cells in areas of T-cell accumulation, and T cells traversing the endothelium between endothelial cells and through their cytoplasm. Endothelial cell damage was evident in these areas. This study demonstrates breakdown of the blood-nerve barrier by activated T cells, even of nonneural specificity, allowing the development of focal conduction block and demyelination in the presence of circulating antimyelin antibodies.

Synergy between antibody and P2-reactive T cells in experimental allergic neuritis.

Studies were conducted in experimental allergic neuritis (EAN) to evaluate the possible interaction of cellular and humoral immune mechanisms in the demyelinating process. EAN was induced in Lewis rats by passive transfer of T cells reactive to P2 myelin protein or by active immunisation with whole myelin. Animals were then given systemic antimyelin antibody or control serum and assessed clinically, electrophysiologically and with semiquantitative histological studies. Animals given intraperitoneal (i.p.) P2-reactive T cells and systemic antimyelin antibody developed much more severe disease than those given i.p. T cells alone (P < 0.001). In actively immunised animals, the addition of systemic antimyelin antibody did not significantly alter disease severity. We believe the more severe disease in animals receiving T cells and antimyelin antibody reflects synergy between cellular and humoral immune mechanisms whereby neural antigen-specific T cells breach the blood-nerve barrier, allowing demyelinating antibody access to the endoneurium. In EAN induced by active immunisation with whole myelin it is likely that both B and T cell activation occurs and that the more severe demyelination characteristic of this disease reflects the involvement of both humoral and cellular immunity.

Transfer of experimental allergic neuritis by intra neural injection of sensitized lymphocytes.

The final mediators of immune injury in EAN were investigated by intraneural injection of sensitized lymphocytes. Unfractionated specifically sensitized cells caused conduction block which was evident within 24 h after injection, reached significance within 3 days and remained depressed for over 12 days. Pathological changes at the site of injection showed infiltrating lymphoid and mononuclear cells and significant demyelination. The latter was only evident several days after the electrophysiological changes. These effects were shown to be specific, as injection of LNC from normal rats or those immunized with CFA alone did not induce the changes. Fractionation of sensitized LNC into the CD4+ and CD8+ subsets of T-cells showed only the former caused a drop in the amplitude ratio of nerve conduction. These changes in conduction were comparable to those observed in rats immunized with myelin/CFA to induce active EAN. Cyclosporin A (CSA) was given to host animals to block production of cytokines by the injected cells. This inhibited macrophage accumulation at the site of injection, but did not stop the electrophysiological changes. This result suggested that there was direct T-cell damage rather than damage consequent upon macrophage activation. These studies developed a model in which the cellular and molecular mechanisms of conduction block and demyelination in EAN can be studied by direct injection of specifically sensitized LNC.