Ascending Impairment of Nociception in Rats with Experimental Allergic Encephalomyelitis

An ascending impairment of tail nociception is a previously undescribed clinical sign of acute experimental allergic encephalomyelitis (EAE) in the rat. It occurs in EAE induced by inoculation with purified central nervous system (CNS) myelin basic protein (MBP) as well as with whole spinal cord. It is invariably present and consists of an absence of the vocalization response to noxious mechanical stimulation of the tail. This impairment of nociception evolves over 1-3 days, simultaneously with the development of tail weakness, and resolves more rapidly than the tail weakness. Light-microscopic, electron-microscopic and electrophysiological studies indicate that it is due to demyelination-induced conduction block in the small diameter myelinated afferent (A delta) fibres in the sacral and coccygeal dorsal root ganglia, dorsal roots and dorsal root entry zones. Unmyelinated fibres appear to be largely spared

Hypothermia Due to an Ascending Impairment of Shivering in Hyperacute Experimental Allergic Encephalomyelitis in the Lewis Rat

Severe hypothermia and an ascending impairment of shivering are previously undescribed clinical signs in hyperacute experimental allergic encephalomyelitis (EAE) in the Lewis rat. These occurred in hyperacute EAE induced by inoculation with guinea pig spinal cord homogenate and heat-killed Bordetella pertussis. Hypothermia was first detected on day 6-7 post-inoculation, within 12-24 h of the onset of neurological signs, and became more severe as the disease progressed. Rectal temperatures less than or equal to 30 degrees C were common at ambient temperatures of 19-22 degrees C. Shivering was assessed by palpation and by cold tremor electromyography. Shivering was absent in the tail by day 6-7 post-inoculation. The impairment then progressed to affect the hindlimbs, thorax and occasionally the forelimbs. Shivering was absent in hindlimbs with only mild or moderate weakness. Histological studies revealed perivascular inflammation with polymorphonuclear and mononuclear cells, oedema, fibrin deposition, haemorrhage, primary demyelination and axonal degeneration in the spinal cord, dorsal root ganglia and spinal roots. The brainstem was also involved but the cerebral hemispheres, including the hypothalamus, were spared. The close relationship between the severity of hypothermia and the extent of shivering impairment indicates that reduced shivering is an important cause of hypothermia in hyperacute EAE. It is concluded that this impairment of shivering is due not to hypothalamic damage but to lesions elsewhere in the central and peripheral nervous systems

The Pathophysiology of Myelin Basic Protein-Induced Acute Experimental Allergic Encephalomyelitis in the Lewis Rat

Histological and electrophysiological studies were performed on Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with guinea-pig myelin basic protein (MBP) and Freund's adjuvant. The histological studies showed demyelination in the lumbar, sacral and coccygeal dorsal and ventral spinal roots and to a lesser extent in the spinal cord, including the dorsal root entry and ventral root exit zones. The electrophysiological studies demonstrated reduced conduction velocities between the lumbar ventral roots and sciatic nerve. Conduction block was demonstrated at the ventral root exit zone of the lumbar spinal cord but was less severe than in rats with whole spinal cord-induced acute EAE. Recordings of the M wave and H reflex elicited in a hindfoot muscle by sciatic nerve stimulation showed a normal M wave, indicating normal peripheral nerve motor conduction, but a markedly reduced H reflex. The reduction in the H reflex is accounted for by demyelination-induced nerve conduction block in the dorsal and ventral spinal roots, intramedullary ventral roots and at the dorsal root entry and ventral root exit zones of the spinal cord. Demyelination and nerve conduction abnormalities were well established in the relevant lumbar segments on the day of onset of hindlimb weakness. It is concluded that demyelination in the lumbar ventral roots and to a lesser extent in the lumbar spinal cord, including the ventral root exit zone, is an important cause of hindlimb weakness in myelin basic protein-induced acute EAE in the Lewis rat

Involvement of the Dorsal Root Ganglion in Acute Experimental Allergic Encephalomyelitis in the Lewis Rat - A Histological and Electrophysiological Study

Histological and electrophysiological studies were performed in Lewis rats with acute experimental allergic encephalomyelitis (EAE) in order to determine the extent of dorsal root ganglion (DRG) involvement. Histological studies showed inflammation and demyelination in both the central nervous system (CNS) and peripheral nervous system (PNS). The DRG was the most affected region of the PNS and its involvement increased caudally. Nerve conduction abnormalities were demonstrated in the regions of the lumbar, sacral or coccygeal DRGs in some of the rats with EAE. However, the overall DRG involvement was much less severe, both histologically and functionally, than what we recently found in rabbits with EAE. Conduction through the lumbar dorsal root entry zone was normal. We conclude that lesions of the afferent pathway to the spinal cord do not contribute significantly to the disturbances of hindlimb motor function in Lewis rats with EAE

Conduction Block Due To Demyelination At The Ventral Root Exit Zone In Experimental Allergic Encephalomyelitis

Histological and electrophysiological studies were performed in Lewis rats with experimental allergic encephalomyelitis (EAE) to determine the cause of the neurological signs. The ventral root exit zone of the spinal cord was shown to be a major site of demyelination and conduction block. It is concluded that demyelination-induced conduction block in this region is an important cause of hind-limb weakness and paralysis in Lewis rats with EAE

Apoptosis in the Nervous System in Experimental Allergic Encephalomyelitis

We report here for the first time the occurrence of apoptosis of cells in the spinal cord in experimental allergic encephalomyelitis (EAE), an autoimmune, T-cell-mediated demyelinating disease. Four different forms of EAE were studied in the Lewis rat: (i) acute EAE induced by inoculation with whole spinal cord and adjuvants; (ii) acute EAE induced by inoculation with myelin basic protein (MBP) and adjuvants; (iii) acute EAE induced by the passive transfer of MBP-sensitized spleen cells; (iv) chronic relapsing EAE induced by inoculation with whole spinal cord and adjuvants followed by treatment with low-dose cyclosporin A. Cells undergoing apoptosis were recognized at light and electron microscopy by the presence of either crescentic masses of condensed chromatin lying against the nuclear envelope or rounded masses of uniformly dense chromatin. They were found in both the white and grey matter of the spinal cord in all 4 forms of this disease. Although it was not possible to identify definitively the types of cells undergoing apoptosis, the size and location of some of the affected cells suggested that they were oligodendrocytes. As there is now a large body of evidence that T-cell-induced target cell death takes the form of apoptosis, it is attractive to hypothesize that oligodendrocyte apoptosis is occurring in EAE as a result of oligodendrocyte-directed T-cell cytotoxicity. However, other apoptotic cells were located within the myelin sheath, meninges and perivascular spaces and were clearly not oligodendrocytes but were most likely blood-derived mononuclear cells. The sparsity of their cytoplasm and the absence of phagocytosed material suggested that they were mainly lymphocytes rather than macrophages. Apoptosis has been shown to be involved in deleting autoreactive T-cells during the normal development of tolerance. Thus apoptotic deletion of myelin/oligodendrocyte-specific lymphocytes in the central nervous system in EAE might explain both the subsidence of inflammation and the acquisition of tolerance in this autoimmune disease

Rapid entry and downregulation of T Cells in the central nervous system During the reinduction of experimental autoimmune encephalomyelitis

We investigated the mechanisms whereby a previous attack of experimental autoimmune encephalomyelitis (EAE) modifies a subsequent attack in the Lewis rat. Active immunization with myelin basic protein (MBP) and complete Freund's adjuvant 28 days after the passive transfer of MBP-sensitized spleen cells induced a second episode of EAE, which occurred earlier than in naive control animals, but was less severe overall. The pattern of neurological signs was also different in rechallenged rats, which had less severe tail and hindlimb weakness but more severe forelimb weakness. In rechallenged rats, inflammation was more severe in the cervical spinal cord, cerebellum, brainstem and cerebrum, but less severe in the lumbar spinal cord, than in controls. The early onset of EAE in rechallenged rats was explained by a memory T cell response to MBP72-89 in the draining lymph node and spleen, and by the enhanced entry of T cells into the central nervous system (CNS). However, the number of alpha beta T cells in the spinal cord of rechallenged rats declined faster than in controls, especially in the lumbosacral cord, where the number of V beta 8.2+ T cells and the frequency of T cells reactive to MBP72-89 rapidly decreased, indicating rapid downregulation of the immune response in the previously inflamed spinal cord. Apoptosis of inflammatory cells in the CNS was increased in the rechallenged rats and is likely to contribute to this downregulation. Furthermore, during the disease course the generation of encephalitogenic T cells in the peripheral lymphoid organs was limited compared with controls. Thus, a previous attack of EAE modifies a subsequent attack through the interaction of the following processes: a memory T cell response to MBP; facilitated T cell entry into the CNS; downregulation of the immune response in the CNS, including increased apoptosis of inflammatory cells; and a limited generation of encephalitogenic T cells in the peripheral lymphoid organs

Corticosteroid Treatment Of Experimental Autoimmune Encephalomyelitis In The Lewis Rat Results in Loss of V Beta 8.2+ and Myelin Basic Protein-Reactive Cells from the Spinal Cord, with Increased Total T-Cell Apoptosis but Reduced Apoptosis of V Beta 8.2+

We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8-12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1-12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5+ and TCR alpha beta + cell populations. At 1-4 h after dexamethasone treatment there was a reduction in the selective apoptosis of V beta 8.2+ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of V beta 8.2+ cells cannot explain the reduction in the numbers of V beta 8.2+ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8-12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE

Expression of CD45RC and Ia Antigen in the Spinal Cord in Acute Experimental Allergic Encephalomyelitis: An Immunocytochemical and Flow Cytometric Study

We performed immunocytochemical studies to analyze the inflammatory infiltrate and major histocompatibility complex class II (Ia) antigen expression in the spinal cord of Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with myelin basic protein and adjuvants. Using antibodies to lymphocyte markers and other monoclonal antibodies we found that during clinical episodes the inflammatory infiltrate was chiefly composed of T lymphocytes and macrophages. The majority of cells in the inflammatory infiltrate were stained by the W3/25 antibody to CD4 and a proportion was stained by OX22 which labels the high molecular weight form of the leucocyte common antigen (CD45RC). CDB+ T cells were sparse and B cells were not detected. There was minimal staining with the OX39 antibody to the interleukin-2 receptor. Presumptive microglia, identified by their dendritic morphology, expressed Ia antigen during the clinical episodes and after recovery. The prominence of Ia antigen expression after recovery could indicate that this la expression was associated with downregulation of the encephalitogenic immune response. We also performed flow cytometry studies on cells extracted from the spinal cord of rats before and during attacks of EAE. With flow cytometry, we found that in established disease a mean of 83(SD, 23)% of CD2+ cells were CD4+, and a mean of 27(SD, 12)% of CD2+ cells were CD45RC+. In rats sampled on the first day of signs, a mean of 43(SD, 22)% of CD2+ cells were CD45RC+. In the cells extracted from the spinal cord of rats with established disease a mean of 47(SD, 32)% of macrophages were CD45RC+. Our study has combined an immunocytochemical assessment of tissue sections with quantitative flow cytometry assessment of cells extracted from the spinal cord of rats with acute EAE. We have shown that the majority of T lymphocytes in the spinal cord are CD45RC-. We have also found prominent Ia expression on dendritic cells in acute EAE and after clinical recovery

A Study Of Human T-Cell Lines Generated From Multiple Sclerosis Patients And Controls By Stimulation With Peptides Of Myelin Basic Protein

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s) by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8+ T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4+. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP