The proximal peripheral nervous system is a major site of demyelination in experimental autoimmune encephalomyelitis induced in the Lewis rat by a myelin basic protein-specific T cell clone

Experimental autoimmune encephalomyelitis (EAE) was induced in the Lewis rat by the passive transfer of a cytotoxic CD4⁺ T cell clone specific for the 72–89 peptide of guinea-pig myelin basic protein (MBP). Histological studies on rats with neurological signs showed that inflammation was present in the proximal peripheral nervous system (PNS), namely the spinal roots, as well as in the central nervous system (CNS). The main sites of demyelination were the spinal roots in the PNS, and the spinal cord root entry and exit zones in the CNS. The major involvement of the proximal PNS in autoimmune disease directed at MBP is in marked contrast to EAE induced by immunisation with myelin proteolipid protein, where the inflammation and demyelination are restricted to the CNS. These findings may have implications for the human inflammatory demyelinating diseases including multiple sclerosis, in which MBP is a putative target antigen.     

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.     

Restoration of conduction in the spinal roots correlates with clinical recovery from experimental autoimmune encephalomyelitis

In the Lewis rat, acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants is characterized by tail and hindlimb weakness that resolves spontaneously after several days. In rats with neurological signs of this form of EAE (MBP-EAE) we have previously demonstrated demyelination and nerve conduction block in the proximal peripheral nervous system (PNS) and in the central nervous system (CNS). The present study was performed to assess conduction in the PNS and CNS, after recovery from acute MBP-EAE, using direct recordings from surgically exposed spinal roots and spinal cord dorsal columns. The study revealed that 1–2 weeks after clinical recovery from tail paralysis there was almost complete restoration of conduction in the sacral spinal roots but persistent severe conduction abnormalities in the dorsal columns. Significant restoration of conduction through the dorsal columns occurred over the following 2 weeks. These findings indicate that PNS conduction block due to a demyelinating polyradiculitis is a major cause of the neurological signs of acute MBP-EAE in the Lewis rat.© 1995 John Wiley & Sons, Inc.     

Failure of 5-hydroxytryptophan to increase lumbar MSR amplitude in rats paralyzed with experimental allergic encephalomyelitis

Doses of the 5-hydroxytryptamine precursor, 5-hydroxytryptophan, which markedly increased lumbar monosynaptic response (MSR) amplitude in control rats failed to do so in rats paralyzed with experimental allergic encephalomyelitis (EAE). However, lumbar MSR amplitude could be increased in EAE rats just as in control rats by tetanically stimulating the dorsal root. Post-tetanic potentiation of MSR amplitude occurred in the EAE paralyzed rats both prior to and following 5-hydroxytryptophan injection. It was concluded that, as has been reported for the peripheral system in EAE guinea pigs, central nervous system 5-hydroxytryptamine neurotransmission is impaired, at least in the lumbar spinal cord, in EAE rats with hindlimb paralysis.     

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.     

An analysis of mast cell frequency in the rodent nervous system: numbers vary between different strains and can be reconstituted in mast cell-deficient mice

There is evidence that nervous system mast cells may play a role in the pathogenesis of the experimental autoimmune demyelinating diseases, experimental allergic neuritis (EAN), and experimental allergic encephalomyelitis (EAE). We compared mast cell numbers in the peripheral nervous system (PNS) and central nervous system (CNS) of rodent strains that differed in their susceptibility to experimental demyelination. Mast cells were counted by toluidine blue staining of formalin-fixed tissue. Normal Lewis rats (susceptible to both EAN and EAE) had significantly greater numbers of mast cells in the dura mater (about 6x) of the meninges and the sciatic nerve (3x) than Brown Norway rats (resistant to EAE and EAN induction under normal circumstances). Similarly SJL/J mice (susceptible to EAE and EAN) had significantly greater numbers of CNS (3x) and PNS (8x) mast cells than C3H mice (more resistant to disease induction). Other mouse strains were also examined, and PNS mutant Trembler mice had high numbers of PNS mast cells, while the mast cell deficient W/Wv mice contained no detectable mast cells in either the CNS or PNS. Reconstitution of W/Wv mast cells was accomplished by intravenous injection of bone marrow cells from congenic littermates. After seven months, mast cells could be seen in both the CNS and PNS of reconstituted animals. The possibility that mast cells and mast cell precursors can migrate into the nervous system of animals, in the absence of inflammatory disease, may have implications for their role in the pathogenesis of experimental demyelinating diseases.     

Demyelination and neurological signs in experimental allergic encephalomyelitis

Because of the reported absence of demyelination in some animals with neurological signs of experimental allergic encephalomyelitis (EAE), it has been suggested that these signs are not due to demyelination. The present study demonstrates that there is ample demyelination in the central nervous system (CNS) and peripheral nervous system (PNS) to account for the neurological signs in rats with myelin basic protein (MBP)-induced acute EAE as well as in rats and rabbits with whole-spinal-cord-induced acute EAE. The main reasons for failure to detect demyelination in animals with neurological signs of EAE appear to be inadequate histological techniques and incomplete examination of the nervous system, particularly the PNS and the lumbar, sacral and coccygeal segments of the spinal cord.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Transthyretin is not expressed by dorsal root ganglia cells

Several mutations in transthyretin (TTR) are related to familial amyloidotic polyneuropathy (FAP), a neurodegenerative disorder caused by extracellular deposition of TTR fibrils, particularly in the peripheral nervous system (PNS). TTR is mainly synthesized by the liver and choroid plexus of the brain that contribute to the plasma and cerebrospinal fluid (CSF) pools of the protein, respectively. It has recently been reported that TTR is additionally expressed in the PNS, namely by peripheral glial cells of dorsal root ganglia (DRG). This lead to the hypothesis that TTR synthesis in the DRG might contribute to the PNS involvement in FAP. In this report we clarify this issue by showing that TTR synthesis is absent in both human and mouse DRG. Moreover, by using TTR KO mouse DRG as controls, we demonstrate that TTR-like immunoreactivity in the perineurium is an artifact. As such, and similarly to what has been previously shown in the central nervous system (CNS), TTR amplification by RT-PCR in the DRG most probably results from contamination by the meninges. In conclusion, TTR deposited in the PNS of FAP patients should still be regarded as having blood and/or CSF origin.     

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 hindlimb weakness and paralysis in Lewis rats with EAE.     

The expansion of 300 CTG repeats in myotonic dystrophy transgenic mice does not induce sensory or motor neuropathy

Summary Although many studies have been carried out to verify the involvement of the peripheral nervous system (PNS) in dystrophia myotonica (DM1) patients, the results remain controversial. The generation of DM1 transgenic mice displaying the human DM1 phenotype provides a useful tool to investigate the type and incidence of structural abnormalities in the PNS. In the present study, the morphological and morphometric analysis of semi-thin sections of sciatic and sural nerves, lumbar dorsal root ganglia (DRG) and lumbar spinal cords revealed that in DM1 transgenic mice carrying 300 CTG repeats, there is no change in the number and diameter of myelinated axons compared to wild type. Only a non-significant reduction in the percentage of thin myelinated axons was detected in electron micrographs of ultra-thin sciatic nerve sections. Analysis of the number of neurons did not reveal a loss in number of either sensory neurons in the lumbar DRG or motor neurons in the lumbar spinal cord in these DM1 mice. Furthermore, in hind limb muscle sections, stained with a neurofilament antibody and α-bungarotoxin, the intramuscular axon arborization appeared normal in DM1 mice and undistinguishable from that in wild-type mice. Moreover, in DM1 mice, there was no irregularity in the structure or an increase in the endplate area. Also statistical analysis did not show an increase in endplate density or in the concentration of acetylcholine receptors. Altogether, these results suggest that 300 CTG repeats are not sufficient to induce axonopathy, demyelination or neuronopathies in this transgenic mouse model.     

The neuropathology of chronic relapsing experimental allergic encephalomyelitis induced in the Lewis rat by inoculation with whole spinal cord and treatment with cyclosporin A

Summary Chronic relapsing experimental allergic encephalomyelitis was induced in Lewis rats by inoculation with guinea-pig spinal cord and complete Freund's adjuvant followed by treatment with low-dose cyclosporin A. In most animals, tail and limb weakness developed in a relapsing remitting pattern but in some these signs were persistent or progressive from onset. Histological studies during the early stages of clinically active disease (< 25 days after inoculation) revealed inflammation and primary demyelination in the central nervous system (CNS), particularly the spinal cord, and in the peripheral nervous system (PNS), specifically the ventral and dorsal roots and dorsal root ganglia. Animals studied in the later stages of clinically active disease (> 28 days after inoculation) had extensive spinal cord demyelination but minimal PNS demyelination. In these animals, large plaques of demyelination with gliosis and prominent plasma cells occurred particularly in the thoracic spinal cord, and lesions of different ages were present within the spinal cord. CNS and PNS remyelination by oligodendrocytes and Schwann cells, respectively, was present in all animals studied later than 18 days after inoculation (the time of the first remission, if it occurred). In both early and late clinically active disease electron microscopy revealed macrophages invading and destroying CNS myelin sheaths. Active demyelination was sometimes found in regions of CNS remyelination, suggesting that remyelinated fibres were being attacked. Axonal degeneration occurred in the spinal cord. During clinical remission there was CNS and PNS remyelination and much less inflammation; however, active demyelination still occurred to a limited degree.Supported by the National Health and Medical Research Council of Australia, University of Queensland Foundation and Clive and Vera Ramaciotti Foundations is gratefully acknowledged. During this work Glenn Stanley was a recipient of a Research Training Fellowship of the National Multiple Sclerosis Society of Australia     

The pathophysiology of acute experimental allergic encephalomyelitis induced by whole spinal cord 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 spinal cord and Freund's adjuvants, in order to determine the cause of the neurological signs. These studies demonstrated demyelination-induced nerve conduction block in the large and also the smaller diameter fibres at the ventral root exit zone (VREZ) of the lumbar spinal cord. The demyelination at the VREZ affected both centrally and peripherally myelinated internodes, but predominantly the former. Studies on the H reflex recorded from a hindfoot muscle indicated normal peripheral nerve motor conduction but interruption of the monosynaptic reflex arc, as would be anticipated from this efferent conduction block and previously reported afferent conduction abnormalities. It is concluded that conduction block in alpha, beta and gamma motor fibres at the VREZ is an important cause of hindlimb weakness in whole spinal cord-induced acute EAE.     

Studies with antisera against peripheral nervous system myelin and myelin basic proteins. II. Immunohistochemical studies in cultures of rat dorsal root ganglion neurons and Schwann cells

Antiserum against rat peripheral nervous system (PNS) myelin contained immunoglobulins which bound preferentially to the extracellular surfaces of myelin-related Schwann cells in intact cultures of dorsal root ganglion (DRG) neurons and Schwann cells, while antiserum against basic protein (BP) from central nervous system myelin or the PNS basic protein P₂ did not. We demonstrate the presence of PNS myelin proteins P₁ (identical to BP) and P₂ by immunoperoxidase techniques in DRG cultures that had been treated to disrupt cellular membranes. These observations suggest that P₁ and P₂ are not exposed on the extracellular surfaces of myelin-related Schwann cells in culture. The results also supported the hypothesis concerning the possible mechanisms by which anti-PNS myelin serum demyelinates DRG cultures, while anti-BP serum and anti-P₂ serum do not.     

Experimental allergic neuritis. I. Rat strain differences in the response to bovine myelin antigens

Strain differences among rats to the induction and severity of experimental allergic neuritis (EAN) in response to whole PNS myelin were observed. Lewis rats were highly susceptible and developed severe EAN without central nervous system lesions (EAE), while Brown Norway rats were most resistant. Wistar, Sprague-Dawley, and Buffalo rats were susceptible but developed less severe disease than Lewis rats. Only Lewis rats consistantly developed EAN in response to isolated P₂ with mercaptoehtanol prior to injection. None of the strains developed EAN in response to galactocerebroside and none developed the lesions of EAE in response to any of the bovine myelin antigens tested. Myelin protein profiles from these rat strains were similar which suggests that factors other than target tissue differences, such as genetically determined immune responses to bovine myelin antigens, must be involved in these differing responses.     

Experimental allergic neuritis in the Lewis rat.

Purified myelin from the peripheral nervous system of guinea pig, frog (Rana catesbeiana), rat, rabbit, beef, and human in Freund's adjuvant were injected into the Lewis rat. Groups of rats receiving injections of myelin from different species were examined for signs of dysfunction and lesions in the PNS and CNS. Injection of frog PNS myelin into the Lewis rat did not produce any clinical signs or lesions typical of experimental allergic neuritis (EAN) or experimental allergic encephalomyelitis (EAE). Injection of myelin from the PNS of rat, rabbit, beef, and human elicited clinical signs and lesions characteristic of EAN, while guinea pig myelin injection caused superimposed conditions of EAE and EAN. The myelin proteins from the various species were separated by polyacrylamide gel electrophoresis, the gels were scanned and the individual proteins measured. There did not appear to be a correlation between the amount of P2 protein contained in the different myelin species and the severity of the EAN symptoms and lesions produced. Although the Lewis rat is far more susceptible to EAE caused by guinea pig CNS myelin than by any other species, EAN can be easily induced in this animal by injection of PNS myelin from a number of species.     

P2 protein-induced experimental allergic neuritis. An ultrastructural study

Experimental allergic neuritis (EAN) was induced in 2 groups of inbred Lewis rats by sensitization with P2 protein and peripheral nervous system (PNS) myelin, both purified from bovine intradural roots. Light- and electronmicroscopic study of P2-induced EAN revealed demyelinative lesions in spinal ganglia and root nerves and less frequently in peripheral nerves and root entry zones. Both small and large myelinated fibers were demyelinated, contradictory to the reported selective binding of anti-P2 antibodies to myelin of large fibers. The early lesions were characterised by perivenular lymphocytic infiltration, and subsequent "dissolution" of myelin sheath was associated with invasive of phagocytic cells. The distribution of demyelinative lesions and patterns of demyelination resembled those of PNS myelin-induced EAN except that the disease was milder and dissolution of myelin and intramyelinic edema were more frequently found in P2-induced EAN. The abundance of demyelination in P2-induced EAN strikes contrast to the scarcity of myelin change in experimental allergic encephalomyelitis (EAE) induced by myelin basic protein immunization.     

Phylogenetic preservation of alpha3 Na+,K+-ATPase distribution in vertebrate peripheral nervous systems

The alpha(3) isoform of Na(+),K(+)-ATPase is uniquely expressed in afferent and efferent neurons innervating muscle spindles in the peripheral nervous system (PNS) of adult rats, but the distribution pattern of this isoform in other species has not been investigated. We compared expression of alpha(3) Na(+),K(+)-ATPase in lumbar dorsal root ganglia (DRG), spinal roots, and skeletal muscle samples of amphibian (frog), reptilian (turtle), avian (pigeon and chicken), and mammalian (mouse and human) species. In all species studied, the alpha(3) Na(+),K(+)-ATPase isoform was nonuniformly expressed in peripheral ganglia and nerves. In spinal ganglia, only 5-20% of neurons expressed this isoform, and, in avian and mammalian species, these alpha(3) Na(+),K(+)-ATPase-expressing neurons belonged to a subpopulation of large DRG neurons. In ventral root fibers of pigeons, mice, and humans, the alpha(3) Na(+),K(+)-ATPase was abundantly expressed predominantly in small myelinated axons. In skeletal muscle samples from turtles, pigeons, mice, and humans, alpha(3) Na(+),K(+)-ATPase was detected in intramuscular myelinated axons and in profiles of nerve terminals associated with the equatorial and polar regions of muscle spindle intrafusal fibers. These results show that the expression profiles for alpha(3) Na(+),K(+)-ATPase in the peripheral nervous system of a wide variety of vertebrate species are similar to the profile of rats and suggest that stretch receptor-associated expression of alpha(3) Na(+),K(+)-ATPase is preserved through vertebrate evolution.