H. de F. Webster

CHARACTERIZATION AND PROTEIN ANALYSIS OF MYELIN SUBFRACTIONS IN RAT BRAIN: DEVELOPMENTAL AND REGIONAL COMPARISONS

—Myelin preparations from the whole brains of 16‐day‐old rats and from cortical regions and brainstem, respectively, of 40‐day‐old rats were separated into light, medium and heavy subfractions on a discontinuous sucrose gradient by a procedure previously used for whole adult rat brain (Matthieu, et al., 1973). The total dry weight of myelin recovered from the 16‐day‐old rats was only 2·4mg/g fresh brain in comparison to 20 mg from adult brains. In 16‐day‐old rat brains, the percentage of the total myelin protein in the light fraction was higher than that found in adult brains; the percentage in the medium fraction was only one‐third that in adults; while the percentage in the heavy fraction was about the same at both ages. The heavy fraction from the 16‐day‐old rats contained less basic protein and proteolipid than the light fraction, and the levels of the 2′3′‐cyclic nucleotide 3′‐phosphohydrolase (CNP) and glycoprotein were less than half those in the light and medium fractions. Double labelling experiments with radioactive fucose indicated that the major labelled glycoprotein in the heavy and medium fractions had a slightly higher apparent mol. wt than that in the light fraction. Electron microscopy showed much readily identifiable, compact myelin in the light and medium fractions from the 16‐day‐old rats, whereas the heavy fraction contained more single membranous structures and much less multilamellar myelin. The yield of myelin/g fresh wt from brainstem of 40‐day‐old rats was 4‐fold higher than from cortical regions, and the percentage recovered in the light fraction was greater in the brainstem. In both regions basic proteins decreased from the light to the heavy fraction, whereas high mol. wt proteins, the glycoprotein and CNP increased. The biochemical and morphological results suggest that in both 16‐day‐old and young adult rats the light fraction is enriched multilamellar, compact myelin. In contrast, the heavy fraction at both ages is enriched in loose, uncompacted myelin and myelin‐related membranes, although the heavy fraction from 16‐day‐old rats also may be substantially contaminated with membranes which are unrelated to myelin.

Ia antigens in the normal rat nervous system and in lesions of experimental allergic encephalomyelitis

SummaryThe distribution of the class II major histocompatability (Ia) antigens has been studied in the normal nervous system and in acute lesions of experimental allergic encephalomyelitis (EAE). EAE was induced in Lewis rats with guinea pig spinal cord in Freunds complete adjuvant. Frozen sections from cord, including the roots and ganglia, were stained for Ia antigens, and some sections were also stained for the hydrolytic enzyme acid phosphatase. In the normal CNS and PNS, there were a few vessel-associated cells or small leukocyte-like cells which expressed Ia antigens. No cells were found which expressed both Ia and acid phosphatase [the phenotype used to describe the activated macrophage group of antigen presenting cells (APCs)]. In EAE, Ia positive cells increased in number prior to the detection of clinical signs. Some of these Ia-positive cells were thought to be astrocytes rather than inflammatory cells. At the height of the disease process large numbers of cells in the EAE lesions were Ia-positive. Among these infiltrating cells were some large acid phosphatasepositive cells which also expressed Ia antigens. These double-positive cells appeared to be APCs in the form of activated macrophages, cells known to be involved in the demyelinating processes of EAE. Our results show that some vascular and vessel-associated cells in the normal nervous system express Ia antigens. We suggest that these and other Ia-positive cells in acute EAE lesions may have a role in antigen presentation.

CHARACTERIZATION OF THE FRACTION OBTAINED FROM THE CNS OF JIMPY MICE BY A PROCEDURE FOR MYELIN ISOLATION

Abstract— A subcellular fraction (called the 0·85‐fraction) was isolated from the brains of Jimpy mice by a procedure for obtaining myelin of high purity from immature normal brains. The yield of this fraction obtained from 17‐day‐old Jimpy mice was only 5 per cent of that from age matched controls. In the electron microscope, the O·85‐fractions obtained from 9‐ and 17‐day‐old control mice showed many multilayered whorls of myelin, whereas the corresponding fraction from the Jimpy mice was free of multilayered structures which could be recognized as myelin. Basic proteins, proteolipid protein and galactocerebrosides could not be detected in the 0·85‐fraction from Jimpy mice although they were major components of the 0·85‐fractions from both 9‐ and 17‐day‐old control mice. The specific activity of 2′,3′‐cyclic nucleotide 3′‐ phosphohydrolase in the Jimpy 0·85‐fraction was only 15 per cent of the value for controls. These results can be explained either by the 0·85‐fraction from Jimpy brain being a very abnormal ‘myelin’ or by its being primarily non‐myelin contaminants. Little or none of the major glycoprotein found in normal myelin fractions was found in the 0·85‐fraction from Jimpy brains. This finding is strong evidence indicating that the glycoprotein is closely associated with normal myelin in situ.

Insulin-like growth factor-I treatment reduces immune cell responses in acute non-demyelinative experimental autoimmune encephalomyelitis

To test the effects of insulin‐like growth factor‐I (IGF‐I) on clinical deficits, lesion severity, and immune cell responses in acute, non‐demyelinative experimental autoimmune encephalomyelitis (EAE), we induced EAE in Lewis rats by passive transfer of an MBP‐reactive T lymphocyte line. Four days after receiving 5 × 105 MBPL‐1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 μg IGF‐I i.v. twice daily for 6 days, and the other 10 received the same volume of 0.89% NaCl. Pairs of rats were sacrificed after 4 days and 6 days of IGF‐I and placebo treatment and spinal cord sections were processed for immunostaining, in situ hybridization, and morphological examination. IGF‐I treatment decreased clinical deficits, lesion numbers, and lesion areas significantly. Numbers of CD4‐positive T cells, α/β TCR‐positive cells, and ED‐1‐positive macrophages were also significantly reduced by IGF‐I treatment. Similar reductions were found in our second trial, when 11 days of placebo and IGF‐I injections began the day after transfer. No demyelination was observed in either toluidine blue‐stained semithin sections or in sections immunostained with an antibody raised against myelin basic protein (MBP). We conclude that IGF‐I‐induced reductions in immune cell responses can occur in the absence of demyelination and are of major importance in decreasing clinical deficits and lesion severity in EAE. If IGF‐I has similar effects in multiple sclerosis, we think that it will be useful therapeutically.

Chronic relapsing experimental autoimmune encephalomyelitis: effects of insulin-like growth factor-I treatment on clinical deficits, lesion severity, glial responses, and blood brain barrier defects.

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE), a model for multiple sclerosis, was used to test 2 regimens of insulin-like growth factor-I (IGF-I) treatment. We induced crEAE by injecting 3×07 myelin basic protein—(MBP) sensitized lymph node cells into adult female SJL/J mice. Fifty-one mice, divided randomly into 4 groups, were used in the first trial. Two groups received IGF-I (a gift of Cephalon, Inc.) 0.6 mg/kg/d subcutaneously from day 7 to day 16 and the other two groups received placebo injections. IGF-I treatment reduced clinical deficits during the first attack and during 2 subsequent relapses. Image analysis of immunostained and histological sections showed that IGF-I treatment reduced BBB defects and both the numbers and sizes of inflammatory, demyelinating, and demyelinated lesions. Twelve mice that had recovered from their first attack were used in our second trial to evaluate possible adverse effects of prolonged treatment with a higher dose of IGF-I. Six received 1.2 mg/kg/d for 6 weeks (days 19–63). No adverse effects of IGF-I treatment were identified. The eyes, hearts, livers, and kidneys of IGF-I-treated mice were normal histologically and their spleens also appeared normal except for mild to moderate microscopic increases in lymphopoesis. Our results suggest that prolonged IGF-I treatment is well tolerated and that the anti-inflammatory effects of IGF-I have a major role in reducing clinical deficits and lesion severity in crEAE. These effects, if present in multiple sclerosis, may benefit patients with this disease.

Prominent white matter lesions develop in Mongolian gerbils treated with 100% normobaric oxygen after global brain ischemia

SummaryCarotid arteries were occluded bilaterally for 15 min in two groups of Mongolian gerbils. The first group received 100% oxygen during the first 3 h of reperfusion. During that period, room air was given to the second group. After 3 h, both groups received room air. Brains of gerbils that died within 14 days after occlusion were removed, fixed in formalin and embedded in paraffin. Gerbils that survived 15–28 days were perfused with formalin before their brains were removed and embedded in paraffin. Adjacent, serially cut sections were stained with luxol fast blue (LFB)-H&E, cresyl violet, according to the Bodian method, or immunocytochemically with antisera raised against myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). In brain sections of gerbils receiving 3 h of 100% oxygen, there were circumscribed white matter lesions in the corpus striatum, lateral thalamus, mesencephalon and posterior limb of the internal capsule. Myelin sheaths were swollen, fragmented and were less intensely stained by MBP antiserum. MBP and LFB-stained myelin fragments were present extracellularly and in macrophages. Many axons in these areas appeared undamaged. Previously described ischemic changes were found in gray matter and some areas of white matter in both groups. However, neurons in the deeper laminae of the cerebral cortex appeared to be better preserved in gerbils given oxygen. The results suggest that hyperoxia, if present immediately after transient brain ischemia, may damage myelin more severely than other cellular elements.

Neural and non-neural origin of calcitonin gene-related peptide (CGRP) in the gastric mucosa

We have used immunohistochemistry and in situ hybridization histochemistry to visualize CGRP and the mRNA encoding the CGRP precursor in the stomach. CGRP is present in nerve fibers in the mucosa. CGRP mRNA and CGRP itself are also found in non-neural cells in the lamina propria. These cells are likely to be macrophages or B-lymphocytes.

Immunocytochemical search for JC papovavirus large T-antigen in multiple sclerosis brain tissue.

SummaryThe large T-antigens of papovaviruses JC (JCV) and BK share a C-terminal subsequence with myelin basic protein (MBP). Since this sequence functions as a phosphate acceptor site in MBP, expression of a competing T-antigen sequence in oligodendroglia might adversely affect their ability to post-translationally process MBP and thus to maintain myelin. We have used techniques which demonstrate JCV T-antigen in small oligodendroglial cells from progressive multifocal leukoencephalopathy tissue to search for a possible latent JCV infection expressing T-antigen in nine cases of multiple sclerosis (MS) and three normal brains. No cells expressing T-antigen were detected in plaque or periplaque regions of the MS brains or in control CNS tissue.

Immunocytochemical study of myelin-associated glycoprotein (MAG), basic protein(BP), and glial fibrillary acidic protein (GFAP) in chronic relapsing experimental allergic encephalomyelitis (EAE)

SummaryChronic relapsing experimental allergic encephalomyelitis (EAE) lesions that resemble those seen in multiple sclerosis (MS) were produced in young Hartley and strain 13 guinea pigs (Lassmann and Wisniewski 1979). To study distributions of myelin-associated glycoprotein (MAG), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) in these lesions, paraffin and semithin epon sections of CNS from eight of these guinea pigs were immunostained with antisera to these proteins according to the peroxidase-antiperoxidase (PAP) method. In lesions with active myelin sheath breakdown, changes in anti-MAG and anti-BP immunoreactivity corresponded closely. Abnormal and/or decreased anti-MAG staining did not extend beyond margins of lesions into surrounding areas containing myelin sheaths stained normally by anti-BP and by histological stains for myelin. GFAP-stained astrocyte processes were more numerous and much larger in more chronic lesions. Anti-MAG and anti-BP both stained regenerating myelin sheaths which were very numerous in both paraflin and epon sections. In the latter, anti-MAG also stained some myelin-forming oligodendroglia. The results are additional evidence suggesting that in chronic relapsing EAE, myelin sheaths are the primary target. Oligodendroglia appear to be relatively unaffected and remyelinate most of the demyelinated axons.

Virus spread and initial pathological changes in the nervous system in genital herpes simplex virus type 2 infection in mice

SummaryMice were infected by the vaginal route with the MS strain of herpes simplex virus type 2 (HSV-2). Serial vaginal cultures were used to confirm infection and to select mice for this study. Two mice were killed by perfusion on days 2–6 post infection (p.i.) and lumbar and sacral cord with cauda were fixed and embedded for electron microscopy. Semithin Epon-sections were stained for viral antigen using a rabbit anti-HSV-2 antiserum and the Avidin-Biotin (ABC) method. Thin sections from antigen-positive blocks were examined by electron microscopy, and the number and types of infected cells detected by these two methods were compared. A good correlation was found between detection of infected cells by these methods. Infected cells included neurons of dorsal root ganglia and spinal cord, satellite cells of dorsal root ganglia, non-myelinating Schwann cells, astrocytes, oligodendrocytes and arachnoidal cells. Infected cells were first detected in the cauda on day 3 p.i. and in the spinal cord on day 5 p.i. The temporal and spatial distribution of infected cells was consistent with neural spread to and within the CNS. The pathological lesions showed a good correlation with the distribution and number of infected cells and are probably due to a direct virus effect. The similar sensitivity of the Epon-ABC method to electron microscopy in detecting infected cells indicates that this method may have useful applications in both experimental and diagnostic work.