Insulin-like growth factor I increases myelination and inhibits demyelination in cultured organotypic nerve tissue.

The implication of insulin-like growth factor I (IGF-I) in the myelination and the repair of myelin that occur after a demyelinating process was evaluated in organotypic cultures of embryonic nerve tissue. The amount of myelin of mouse spinal cord explants exposed to media supplemented with IGF-I beginning on the first day of explantation was recorded by light-microscopic examination and quantitation of the 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity. After 9 days in vitro (DIV), the cultures treated with medium supplemented with 0.1-1 microgram/ml IGF-I showed a greater amount of myelin and an increase over the controls in CNPase activity between 50 and 80% at 16 DIV and 100% at 21 DIV. Total demyelination with a concomitant reduction of about 80% in the CNPase activity resulted when anti-white matter antiserum and complement were added to the nutrient medium of fully myelinated cultures. This effect was partially reverted when IGF-I was included in the demyelinating medium. The higher inhibition, about 50%, was obtained with concentrations of IGF-I between 0.1 and 0.5 micrograms/ml. To study the effect of IGF-I on remyelination, well-myelinated cultures were completely demyelinated, maintained in that state for 2 or 15 DIV and after that allowed to remyelinate for 14 days. Cultures exposed to medium supplemented with 0.01-0.1 microgram/ml IGF-I showed a degree of remyelination similar to that of the normal nutrient medium-fed cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

CSF-1 expression is upregulated in astrocyte cultures by IL-1 and TNF and affects microglial proliferation and morphology in organotypic cultures.

Astrocytes produce factors that control the growth and differentiation of many cell types within the CNS as well as play a role in the generation of the immune response. The extent to which these two functions interact has received less attention. We now report that astrocyte cultures established from rat brain endogenously express mRNA and low levels of secreted biologically active protein for the monocyte growth and differentiation factor colony stimulating factor-1 (CSF-1). Exposure of astrocytes to interleukin-1 (IL-1) and/or tumor necrosis factor (TNF) upregulated the expression of CSF-1 mRNA and protein. Following treatment with 100 U/ml of TNF, IL-1, or TNF+IL-1, maximum CSF-1 mRNA expression was observed at 3 hr. In the presence of IL-1 an increase in biologically active CSF-1 was detected in the astrocyte conditioned medium at 6 hr. These data indicate that the expression of CSF-1 by astrocytes can be modulated by exposure to the cytokines IL-1 and TNF. To determine whether CSF-1 provides a mitogenic signal for microglia during development, mouse spinal cord organotypic cultures were exposed to recombinant mouse CSF-1 (rmCSF-1), resulting in proliferation of microglia by 7 days and an increase in the number of ramified microglia over ameboid microglia by 14 days.

A placebo-controlled, double-blind, randomized, two-center, pilot trial of Cop 1 in chronic progressive multiple sclerosis.

We found Cop 1 to be effective and relatively safe in a previous (exacerbating-remitting) clinical trial. This current trial involves 106 chronic-progressive patients. The major end point, confirmed progression of 1.0 or 1.5 units (depending on baseline disability) on the Kurtzke Expanded Disability Status Scale, was observed in nine (17.6%) treated and 14 (25.5%) control patients. The differences between the overall survival curves were not significant. Progression rates at 12 and 24 months were higher for the placebo group (p = 0.088) with 2-year probabilities of progressing of 20.4% for Cop 1 and 29.5% for placebo. We found a significant difference at 24 months between placebo and Cop 1 at one but not the other center. Two-year progression rates for two secondary end points, unconfirmed progression, and progression of 0.5 EDSS units, (p = 0.03) are significant.

Delta 9-tetrahydrocannabinol: a novel treatment for experimental autoimmune encephalomyelitis.

Since multiple sclerosis (MS) is believed to be an immune-mediated disease, it follows that its therapies should be directed towards modulating the immune system. Current MS treatments, which include the use of exogenous steroids that are immunosuppressive, do not meet therapeutic objectives. delta 9-Tetrahydrocannabinol (THC), an active component of marijuana, has been shown to be immunosuppressive. To test THC's ability to suppress an immune-mediated disease, experimental autoimmune encephalomyelitis (EAE), the laboratory model of MS, was used. Lewis rats and strain 13 guinea pigs were administered THC either before inoculation for EAE or treated with THC after injection. Control animals received placebo. The effect of dose, in addition to the timing of treatment, was also investigated. All animals treated with placebo developed severe clinical EAE 10-12 days post-injection (d.p.i.) and more than 98% died by 15 d.p.i. THC-treated animals had either no clinical signs or mild signs with delayed onset (13-15 d.p.i.) with survival greater than 95%. Examination of central nervous system tissue revealed a marked reduction of inflammation in the THC-treated animals. Therefore, as THC has been shown to inhibit both clinical and histologic EAE, it may prove to be a new and relatively innocuous agent for the treatment of immune-mediated diseases.

Electrophysiological analysis of factors involved in the primary demyelinating diseases: the rabbit eye model system.

This study explores the longitudinal assessment of visual evoked potentials (VEPs) in the rabbit as a method for defining factors underlying functional and structural changes associated with optic neuritis and the inflammatory demyelinating diseases. In rabbits with experimental autoimmune encephalomyelitis (EAE) induced by sensitization with guinea pig spinal cord myelin, injection of lymphokines into the posterior chamber of one eye (monocular challenge) produces an early inflammatory response in the retina and optic nerve, and an alteration in the VEP, all limited to the injected eye and its projections. The earliest changes in the timing and distribution of the cortical VEP occur within hours of ocular challenge and precede histopathological evidence of structural demyelination at the light microscope level. Prechallenge assessment allows the induced monocular prechiasmal effects to be distinguished from the more diffuse electrophysiological findings associated with EAE (i.e. those due to sensitization alone). In sensitized/challenged animals there is a clear correspondence between electrophysiological and morphological measures of dysfunction at the time points sampled. These results suggest that this model system afford an excellent opportunity to examine the precise structural correlates of the early functional changes associated with the onset of inflammatory demyelination within the CNS. Furthermore, the stability of the system provides the capacity to monitor alterations over the complete course of inflammation, demyelination and remyelination, induced by experimental manipulations.

Clinical experience with COP-1 in multiple sclerosis.

COP-1 is one of a series of polypeptide preparations developed to stimulate myelin basic protein (MBP), a natural component of the myelin sheath. MBP in Freund's complete adjuvant induces experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). In saline, MBP suppresses EAE. This is the rationale for the use of COP-1 in MS.

Quantitation of antigen-specific T-cell-induced demyelination in vitro.

To test the hypothesis that T lymphocytes sensitized to central nervous system (CNS) antigens may quantitatively induce more demyelination in neural tissue than T cells sensitized to non-CNS antigens, we established T cell lines specific for myelin basic protein (MBP) or the purified protein derivative (PPD) of M. tuberculosis. The potential of T cells to cause myelin pathology was determined by measuring the activity of the myelin-associated enzyme 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in organotypic cultures of syngeneic spinal cord after incubation with the T cell lines. The activity of CNPase in neural tissue has been shown to correlate positively with the amount and integrity of CNS myelin. Although both MBP- and PPD-specific T cells caused decreases in CNPase activity, the MBP line caused significantly greater and consistent changes. This finding indicates that T cell-mediated CNS demyelination may be comprised of CNS antigen-specific and CNS non-specific components, the former causing more pathology.

A pilot trial of Cop 1 in exacerbating-remitting multiple sclerosis.

Cop 1 is a random polymer (molecular weight, 14,000 to 23,000) simulating myelin basic protein. It is synthesized by polymerizing L-alanine, L-glutamic acid, L-lysine, and L-tyrosine. It suppresses but does not induce experimental allergic encephalomyelitis, an animal model of multiple sclerosis. It is not toxic in animals. In a double-blind, randomized, placebo-controlled pilot trial, we studied 50 patients with the exacerbating-remitting form of multiple sclerosis, who self-injected either 20 mg of Cop 1 dissolved in 1 ml of saline or saline alone daily for two years. Six of 23 patients in the placebo group (26 percent) and 14 of 25 patients in the Cop 1 group (56 percent) had no exacerbations (P = 0.045). There were 62 exacerbations in the placebo group and 16 in the Cop 1 group, yielding two-year averages of 2.7 and 0.6 per patient, respectively. Among patients who were less disabled on entry (Kurtzke disability score, 0 to 2), there were 2.7 exacerbations in the placebo group and 0.3 in the Cop 1 group over two years. Among patients who were more affected (Kurtzke disability score, 3 to 6), there was an average of 2.7 exacerbations in the placebo group and 1.0 in the Cop 1 group. Over two years, less disabled patients taking Cop 1 improved an average of 0.5 Kurtzke units; those taking placebo worsened an average of 1.2 Kurtzke units. More disabled patients worsened by 0.3 (Cop 1 group) and 0.4 (placebo group) unit. Irritation at injection sites and rare, transient vasomotor responses were observed as side effects. These results suggest that Cop 1 may be beneficial in patients with the exacerbating-remitting form of multiple sclerosis, but we emphasize that the study is a preliminary one and our data require confirmation by a more extensive clinical trial.

Antigen-specific T cells can mediate demyelination in organotypic central nervous system cultures.

To investigate a role for T lymphocytes in primary demyelination of central nervous system (CNS) tissue, antigen-specific T cell lines sensitized to myelin-associated and myelin-unrelated antigens were developed from SJL mice and tested on myelinated organotypic cultures of syngeneic spinal cord. Demyelination was assessed morphologically by electron microscopy. Antigen responsiveness and specificity, and the phenotypes of the cell lines, were determined by thymidine uptake (3H-TdR) assays and flow cytometry (FC), respectively. Although all T cell lines caused pathologic changes in myelin, the CNS-antigen-specific line induced the most pronounced effects. 3H-TdR uptake assays and FC showed that after three cycles of incubation in the presence of interleukin-2 (IL-2) or antigen, the T cell lines had increased specificity and responsiveness to the priming antigen and were enriched for the L3T4 (helper/inducer) phenotype. This represents the first direct demonstration of T-cell-mediated demyelination, supports a role for the helper/inducer subset in CNS lesion development, and may prove relevant to the human demyelinating disease multiple sclerosis.

Rapid degradation restricts measles virus matrix protein expression in a subacute sclerosing panencephalitis cell line.

Measles virus matrix protein expression is restricted in the persistently infected brain cells of patients with the chronic neurological disease subacute sclerosing panencephalitis (SSPE). Prior studies of the nature of this restriction have identified polyadenylylated matrix gene-encoded RNA transcripts unable to direct effective translation. The defective nature of these mRNAs readily accounted for the inability to detect matrix protein in these persistently infected cells and suggested that in SSPE the restriction of matrix protein expression is achieved by preventing its synthesis. Recently, however, we reported evidence that matrix protein is synthesized in at least one example of this persistent infection, the SSPE cell line IP-3-Ca. In this case, failure of matrix protein to accumulate normally accounted for its restricted expression [Sheppard, R. D., Raine, C. S., Bornstein, M. B. & Udem, S. A. (1985) Science 228, 1219-1221]. To clarify the nature of the restriction displayed by IP-3-Ca cells, the synthesis and fate of the matrix protein of this SSPE cell line were examined in detail. No evidence of constraints on the efficiency of matrix protein mRNA transcription or translation was found. Instead, the restricted expression proved to be the result of rapid posttranslational degradation of matrix protein. We suggest that matrix protein gene mutations incurred in the course of genome replication are likely to be responsible for the diversity of observed mechanisms restricting matrix protein expression. In that event, the nature and position of the nucleotide substitution(s) would be the determinants of the level at which restricted expression is achieved.

Sustained hyperexcitability elicited by repetitive electric stimulation of organotypic hippocampal explants.

Sustained or complex evoked extracellular slow-wave field potentials were recorded in the CA3/2 areas of organotypic hippocampal explants following stimulation of the dentate area. After repetitive electric stimulation, these discharges became more complex and/or self-sustaining. Self-sustaining discharges continued to occur for the duration of the experiment (15 min-10 h). These slow-wave discharges were evoked (or occurred spontaneously) over a wide range of extracellular K+ concentrations (3-9 mM) without addition of pharmacologic inhibitory antagonists, whereas in some explants raising extracellular K+ from 5.9 to 8-9 mM resulted in spontaneous discharges. The observation that epileptiform discharges in hippocampal explants often occurred spontaneously, were elicited by repetitive electric stimulation, and were recorded at K+ levels which are generally ineffective in acute adult hippocampal slices, indicates that excitability of these CNS explants may be significantly increased following altered neuronal and synaptic development (and/or reorganization) under isolated conditions in culture.

Chemical neurotoxicity: detection and analysis in organotypic cultures of sensory and motor systems.

Screening of chemical substances for human neurotoxic (and therapeutic) properties may be carried out with the aid of organotypic tissue cultures composed of foetal explants of mouse sensory and neuromuscular tissues that develop in vitro their characteristic cytoarchitectural and functional organization. Supporting this statement is a wealth of studies describing a range of specific, chemically-induced responses in organotypic neural cultures that parallel changes induced in the nervous system of humans and animals.

Effect of antiglycolipid antisera on the lipid composition of cultured mouse spinal cords.

To examine the effect of antiglycolipid antibodies on demyelination, myelinated cultures of embryonic mouse spinal cords were treated by antigalactocerebroside (anti-GC), anti-GM1; and anti-GM4 antisera, and the lipid composition of the cultures were studied. The anti-GC antiserum-treated cultures, which exhibited severe morphologic signs of demyelination, revealed a significant reduction of cerebroside. The anti-GM4 or anti-GM1 antiserum-treated cultures, which exhibited mild degrees of demyelination, also had low contents of cerebroside. These results support our previous data showing that antiglycolipid antibodies cause demyelination in cultured mouse spinal cords, and suggest a possible role of myelin-specific glycolipids in the demyelination process.

Transformation of cells of astrocyte lineage into macrophage-like cells in organotypic cultures of mouse spinal cord tissue.

Phagocytic cells on the surface of the explants and their relationships to the surface were examined morphologically and immunocytochemically in organotypic cultures of mouse spinal cord tissue. Phagocytic cells were rounded, had smooth cytoplasmic surfaces and were occasionally closely apposed to underlying cells by junctional complexes. These cells contained dense bodies, vacuoles, smooth and coated vesicles, a few microtubules and bundles of intermediate filaments similar to astroglial filaments. The superficial layer of the explant which usually consisted of astroglial cell bodies and their processes, sometimes contained immature neuroepithelial cells with numerous free ribosomes, centrioles, Golgi apparatus, microtubules and infrequently, intermediate filaments. Overall, the cells resembled poorly differentiated astrocytes. Numerous dense bodies and coated vesicles were observed in some of these immature cells as well as in astrocytes in the surface layer of the explant. Cytoplasmic bridges between immature cells within the explant and phagocytic cells on the surface were observed. Immunocytochemistry revealed the presence of glial fibrillary acidic protein within these surface phagocytic cells. It thus appears that immature neuroepithelial cells of astrocytic lineage are capable of transforming into macrophage-like cells in organotypic culture.

Effect of insulin, proinsulin and pancreatic extract on myelination and remyelination in organotypic nerve tissue in culture.

The effect of insulin, proinsulin and crude pancreatic extract was studied in organotypic nerve tissue cultures, principally in relation to the development of myelin. Cultures were exposed to media supplemented with these substances beginning on the first day of explantation. By 4 days in vitro, there was a good neuritic outgrowth from all the fragments. That from the insulin and pancreatic extract-fed were more profuse and extended further than from the control group. By 8-12 days in vitro it was also possible to observe more myelinated axons in these treated groups. The pattern of changes in the myelin associated enzyme activity, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) paralleled the differential increase in myelination. Insulin-fed cultures showed a more rapid increase in CNPase activity, which, after 21 days in vitro reached a plateau about 30-50% over that of the controls. Cultures treated with pancreatic extract showed a similar pattern of increased activity, while in proinsulin-treated explants the activity was only significantly higher after 21 days in vitro. To study the effect of these substances on remyelination, well myelinated cultures were completely demyelinated by exposure to anti-white matter antiserum and were subsequently exposed to the same normal control or supplemented media. The amount of myelin and concomitantly the CNPase activity increased rapidly and in the same proportion between the various groups as was observed previously during primary myelination. Insulin as well as crude pancreatic extract and, to some extent, proinsulin demonstrated a marked effect on the time of onset and principally on the total amount of myelin developed by treated cultures as compared to those maintained in normal nutrient medium.

Immunogenic potentials of copolymer I in normal human lymphocytes.

Copolymer I (COP I), a nonencephalitogenic polypeptide analogous to myelin basic protein, is currently being tested for possible effectiveness in treating MS. Peripheral blood mononuclear cells from normal human donors respond blastogenically to the L-form of COP I. This response was greater than that obtained with either bovine or guinea pig myelin basic protein (GPMBP), with no specificity for a particular T-cell subset. Analyses of culture supernatants demonstrated the presence of interleukin-2 and gamma interferon. Mononuclear cells from human fetal cord blood also responded blastogenically to COP I, but the magnitude of the response did not differ from that induced by GPMBP.