In vivo three-dimensional MR microscopy of mice with chronic relapsing experimental autoimmune encephalomyelitis after treatment with insulin-like growth factor-I.

PURPOSE: The purpose of this study was to determine the ability of three-dimensional in vivo MR microscopy to depict the treatment effects of insulin-like growth factor-I (IGF-I) in SJL mice with chronic relapsing experimental autoimmune encephalomyelitis (crEAE). METHODS: The experiments were performed at 4.7-T on 10 crEAE mice and on one set of control animals. Five crEAE mice were treated with IGF-I and five were treated with a placebo. RESULTS: In the crEAE mice treated with the placebo, in vivo MR microscopy showed areas of abnormal signal throughout the cerebrum, brain stem, and cerebellum. These findings were not present in either the IGF-I-treated mice or the normal control animals. The diffuse alterations in signal intensity in the placebo-treated crEAE mice were not identified on histologic sections of the same areas. CONCLUSION: Differences between the IGF-I- and placebo-treated groups may reflect changes in stabilization or permeability of cell membranes and/or of the blood-brain barrier, although other alternative contrast mechanisms could be playing a role. In vivo MR microscopy depicted changes resulting from treatment of crEAE with IGF-I.

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 3x10(7) 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.

Growth factors and myelin regeneration in multiple sclerosis.

Insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) are multifunctional growth factors which are found in the CNS. Oligodendroglia are the cells that form and maintain myelin sheaths and many in vitro experiments have shown that these growth factors promote the proliferation, differentiation and survival of cells in the oligodendroglial lineage. Since myelin breakdown is often severe in multiple sclerosis (MS), the possibility of growth factor use in the treatment of MS has been considered and recently, IGF-I treatment has been shown to reduce lesion severity and promote myelin regeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This review briefly summarizes the structural characteristics of these growth factors and the actions which might help reduce oligodendrocyte-myelin sheath injury in MS and promote myelin regeneration.

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 response 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 x 10(5) MBPL-1 T cells intravenously, ten pairs of rats had the same mild degree of tail and hind limb weakness. Ten were given 300 micrograms 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, alpha/beta 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 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.

Vasoactive intestinal peptide: mediator of laminin synthesis in cultured Schwann cells.

To learn more about neuropeptide-induced glial responses which accompany axon regeneration, we studied effects of VIP on laminin production by cultured Schwann cells. Schwann cells were isolated from sciatic nerves of neonatal mice, purified, and incubated for 5 days in either control medium (DMEM + 15% FCS) or control medium containing 10-7 -10-11 M VIP. At 10-7 and 10-8 M VIP, laminin levels measured by enzyme-linked immunosorbent assay were significantly higher (55% and 35%) than those in control cultures. Lower VIP concentrations (10-9 -10-11 M) produced smaller increases which were not significant. Low-affinity VIP receptors which mediated this effect were demonstrated on Schwann cells by radioligand binding studies. The increased Schwann cell synthesis of laminin induced by VIP was blocked when either a VIP antagonist or a VIP receptor antagonist was added to the VIP-containing incubation medium. In contrast to astrocytes, when Schwann cells were loaded with fura-2, VIP did not increase cytosolic Ca2+. This indicates that Schwann cells and astrocytes may have different intracellular transduction pathways; their receptor subtypes also may differ. We suggest that the VIP-induced increase in laminin synthesis which we have observed in cultured Schwann cells may also occur in vivo and might be an important component of axon-Schwann cell interactions during nerve regeneration.

Insulin-like growth factor-I given subcutaneously reduces clinical deficits, decreases lesion severity and upregulates synthesis of myelin proteins in experimental autoimmune encephalomyelitis.

To extend our evaluation of insulin-like growth factor-1 (IGF-1) treatment for human demyelinating diseases, we compared effects of s.c. and i.v. IGF-1 in an in vivo model with lesions resembling those seen in multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats with an emulsion containing guinea pig spinal cord and treatment with placebo or with s.c. or i.v. IGF-1 was started when definite clinical weakness was present. IGF-I given subcutaneously significantly reduced clinical deficits and lesion severity. The clinical improvement, as measured by clinical deficit scores, stride lengths and exercise wheel rotations, was evident in 48 hrs and was comparable to that produced by the same IGF-I dose administered intravenously. Subcutaneously administered IGF-I also increased relative mRNA levels of myelin basic protein (MBP), proteolipid (PLP) and 2',3' cyclic nucleotide 3'-phosphodiesterase (CNP), thereby promoting myelin regeneration. We conclude that s.c. IGF-I produces dramatic improvement in acute, demyelinating EAE. Our results also suggest that this growth factor may be useful in treating multiple sclerosis patients with active demyelination.

Insulin-like growth factor I treatment reduces demyelination and up-regulates gene expression of myelin-related proteins in experimental autoimmune encephalomyelitis.

To compare effects of insulin-like growth factor I (IGF-I) and placebo treatment on lesions that resemble those seen during active demyelination in multiple sclerosis, we induced experimental autoimmune encephalomyelitis in Lewis rats with an emulsion containing guinea pig spinal cord and Freund's adjuvant. On day 12-13, pairs of rats with the same degree of weakness were given either IGF-I or placebo intravenously twice daily for 8 days. After 8 days of placebo or IGF-I (200 micrograms/day or 1 mg/day) treatment, the spinal cord lesions were studied by in situ hybridization and with immunocytochemical and morphological methods. IGF-I produced significant reductions in numbers and areas of demyelinating lesions. These lesions contained axons surrounded by regenerating myelin segments instead of demyelinated axons seen in the placebo-treated rats. Relative mRNA levels for myelin basic protein, proteolipid protein (PLP), and 2',3'-cyclic nucleotide 3'-phosphodiesterase in lesions of IGF-I-treated rats were significantly higher than they were in placebo-treated rats. PLP mRNA-containing oligodendroglia also were more numerous and relative PLP mRNA levels per oligodendrocyte were higher in lesions of IGF-I-treated rats. Finally, a significantly higher proportion of proliferating cells were oligodendroglia-like cells in lesions of IGF-I-treated rats. We think that IGF-I effects on oligodendrocytes, myelin protein synthesis, and myelin regeneration reduced lesion severity and promoted clinical recovery in this experimental autoimmune encephalomyelitis model. These IGF-I actions may also benefit patients with multiple sclerosis.

Cryogenic spinal cord injury induces astrocytic gene expression of insulin-like growth factor I and insulin-like growth factor binding protein 2 during myelin regeneration.

To study injury-induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45: 742-757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin-like growth factor I (IGF-I), IGF-I receptor (IGFR-I), IGF binding protein 2 (IGFBP-2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3-56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin-positive, GFAP-negative astrocyte-like cells in the lesion expressed IGF-I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP-positive, hypertrophic astrocytes, many of which also were vimentin-positive. Levels of IGF-I, IGFBP-2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21-28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7-14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin-positive, oligodendroglia-like cells also were immunostained by anti-IGFR-I. Our findings suggest that early astrocytic production of IGF-I and IGFBP-2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury.

Astrocytes express insulin-like growth factor-I (IGF-I) and its binding protein, IGFBP-2, during demyelination induced by experimental autoimmune encephalomyelitis.

To assess the distribution of insulin-like growth-factor-related proteins during autoimmune CNS demyelination and remyelination, experimental autoimmune encephalomyelitis was produced by injecting Lewis rats with an emulsion containing guinea pig spinal cord and complete Freund's adjuvant. Tail weakness appeared at 10-12 days and was followed by hind and forelimb weakness. Paraplegia and incontinence were observed in some animals. From 8-40 days postinoculation (dpi), spinal cord sections were used to correlate lesion location and severity with mRNA distributions of insulin-like growth factor I (IGF-I), IGF-binding protein 2 (IGFBP-2), IGF-I-receptor (IGFR-I), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP). These were determined semiquantitatively by in situ hybridization. Fourteen dpi, there were inflammatory infiltrates and demyelination in both white matter (WM) and grey matter (GM). IGF-I and GFAP mRNAs were increased in these lesions and transcripts encoding myelin basic protein (MBP) were greatly reduced. Large lesions with extensive demyelination were evident in both WM and GM when mRNA levels of GFAP and IGF-I peaked 26 dpi. MBP mRNA levels began increasing 21 dpi and peaked 26 dpi, when a few thin regenerating myelin sheaths were found morphologically. Astrocytes, identified by their morphology and GFAP immunoreactivity, expressed very low levels of IGFBP-2 mRNA and peptide in normal controls; their levels were significantly higher 14 dpi, peaked 26 dpi, and then gradually decreased. Some neurons, as well as oligodendroglia in areas undergoing remyelination, expressed IGFR-I. Although levels of IGF-I, IGFBP-2, and GFAP mRNAs were highest in lesion areas, levels were also elevated around lesions and in some normal-appearing areas of WM and GM 14-40 dpi. The gene expression of both IGF-I and IGFBP-2 by hypertrophic GFAP-positive astrocytes was demonstrated 14-40 dpi by combined in situ hybridization and immunocytochemistry as well as by double immunostaining. Coexpression of IGF-I and IGFBP-2 in the same astrocyte was a frequent finding. Relative increases in both IGF-I, GFAP, IGFBP-2, IGFR-I, and MBP mRNAs peaked at about the same time. This suggests that during lesion progression and recovery, astrocytic expression of IGF-I-related peptides may reduce immune-mediated myelin injury. We also suggest that astrocytic IGFBP-2 in lesions may help target IGF-I to IGFR-I-expressing oligodendrocytes and promote remyelination of demyelinated axons.

Human immunodeficiency virus (HIV) distribution in HIV encephalitis: study of 19 cases with combined use of in situ hybridization and immunocytochemistry.

Brains of 19 AIDS patients with HIV encephalitis were examined by immunohistochemistry and in situ hybridization using antisense HIV DNA and RNA probes. Double immunohistochemical labeling, using antibodies against viral and cell-type specific antigens, was utilized to study lesions in some brains. Other combined studies included use of in situ hybridization and immunohistochemical labeling of the same section, using antibodies against either viral or cell-type specific antigens. Hybridization signals were abundant and were concentrated mainly in the white matter. Heavy labeling was found in the subcortical white matter, the corpus callosum, the internal capsule, and white matter regions of the brainstem and cerebellum. Deeper cortical layers often contained cells with hybridized probe when the subcortical white matter was intensely labeled. HIV nucleic acid sequences were found almost exclusively in macrophages. Counts showed that 16-25% of macrophages contained viral antigens and exhibited hybridized HIV probe. Almost all of these macrophages contained proviral DNA, viral RNA and viral proteins; i.e. they were actively replicating HIV. We also examined brains from three AIDS cases without clinical or pathological evidence of HIV encephalitis; no HIV sequences or immunoreactive proteins were detected.

Expression of insulin-like growth factor-I and related peptides during motoneuron regeneration.

The regulation of insulin-like growth factor-I (IGF-I) and related peptides during motoneuron regeneration was examined in the facial nerve following facial nerve transection. One to 39 days after axotomy, the mRNAs and peptides of IGF-I, type-I insulin-like growth factor receptor (IGFR), insulin-like growth factor binding proteins 1-5 (IGFBP-1-5), and glial fibrillary acidic protein (GFAP) were assayed in brain stem sections by in situ hybridization and immunohistochemistry. Relative mRNA levels of IGF-I, IGFR, IGFBP-2, and GFAP in the ipsilateral facial nucleus were highest 4-7 days after transection and declined thereafter. Double immunostaining experiments showed that both IGF-I and IGFBP-2 were localized in GFAP-positive astrocytic processes, many of which were perineuronal. Peak staining intensity was found 4-7 days after transection and immunoreactivity still was present after 21-35 days. IGFR mRNA was found in some regenerating neurons; however, IGFR peptide was not detected in these neurons or in any other cells in the facial nucleus. Our findings suggest that astrocytic production of IGF-I and IGFBP-2 may accompany regeneration of neurons undergoing retrograde changes induced by axotomy.

Effects of nerve segment supernatants on cultured Schwann cell proliferation and laminin production.

Mouse sciatic nerves were transected and 3 hr to 16 days later proximal segments were removed and homogenized. Supernatants of these segments or of normal sciatic nerves were added to Schwann cells maintained in Dulbecco's modified Eagle's medium (DMEM) + 15% fetal calf serum (FCS). After 6 days, Schwann cells were solubilized and the protein content was measured using a Bio-Rad (Melville, NY) protein assay. Samples containing the same amounts of protein were then applied to microtiter plates and the laminin content was determined by enzyme-linked immunosorbent assay (ELISA). Lysates of cultures treated with 24 hr proximal segment supernatants contained significantly higher levels of laminin than those prepared from other intervals, from distal segments, or from control nerves. Increased surface and cytoplasmic anti-laminin immunoreactivity also was found in Schwann cells treated with 24 hr supernatants. To identify the source(s) of this effect, proximal segments removed 24 hr after transection were bisected; supernatants were prepared from each half and tested. Significant increases in laminin production were produced by supernatants from both halves. When supernatants from proximal and distal halves were compared, the latter produced significantly higher laminin levels. Electron microscopic examination of both halves showed that distal halves contained sprouting neurites and growth cones ensheathed by Schwann cells which had a basal lamina and resembled those seen during development and regeneration. Proximal halves appeared normal. Schwann cell proliferation also was compared in supernatant-treated cultures by using a bromodeoxy-uridine (BrdU) ELISA. The 24 hr and 2 day supernatants increased Schwann cell proliferation significantly; 12 hr, 4 day, and 8 day supernatants produced smaller increases. Our observations suggest that axons undergoing early regenerative changes are one of several possible sources of substance(s) in our proximal segment supernatants which increased Schwann cell proliferation and laminin production.

Myelinated axons demonstrated in the CNS and PNS by anti-neurofilament immunoreactivity and Luxol fast blue counterstaining.

A method which demonstrates myelinated axons in the central and peripheral nervous systems will be described. Paraffin, frozen or semi-thin epoxy-embedded sections were immunostained first with a monoclonal antibody raised against a 200 kilo-Dalton neurofilament protein and then counter-stained with Luxol fast blue.

Concentric sclerosis (Baló): morphometric and in situ hybridization study of lesions in six patients.

Brain tissues from 6 patients with concentric sclerosis (Baló) were examined by in situ hybridization, immunocytochemistry, morphometry, and histological methods. The patients were 24 to 48 years old and had progressive cerebral symptoms and signs that lasted 15 to 100 days. Large demyelinative lesions, most frequent in the frontal white matter, contained alternating bands of demyelinated and partly myelinated white matter that were arranged in concentric or mosaic patterns. In the areas of demyelination, axons were relatively well preserved and there were perivascular inflammatory infiltrates. In 2 specimens, lesions contained regions with the characteristic appearance of actively demyelinating multiple sclerosis plaques. Oligodendroglial densities were highest in normal-appearing white matter, lower in partially myelinated areas, and lowest in demyelinated areas, which also contained many hypertrophic astrocytes closely associated with oligodendroglia. Messenger RNA levels for myelin-related proteins followed the same pattern; they were lowest in demyelinated areas, higher in partially myelinated areas, and highest in normal-appearing white matter beyond lesion margins. Our findings suggest that concentric sclerosis is a variant of multiple sclerosis, that oligodendroglial loss is important in the pathogenesis of demyelination, and that partially myelinated areas probably represent stages of ongoing myelin breakdown rather than remyelination of previously demyelinated areas.

Effects of psychosine (galactosylsphingosine) on the survival and the fine structure of cultured Schwann cells.

The cytotoxicity of psychosine (galactosylsphingosine) for cultured rat Schwann cells was studied by maintaining them in medium containing 1, 10, 50, 75 and 100 microM psychosine for 24, 48 and 72 hours (h). When incubated in 50-100 microM concentrations of psychosine for 24 h, 52-99% of cultured Schwann cells did not survive. Lower concentrations (1-10 microM) did not significantly reduce Schwann cell numbers for the first 24 h. However, only 43-69% of cultured Schwann cells survived in these low concentrations for 48 h, and substantially fewer remained after 72 h of incubation. During incubations in psychosine, bipolar processes of Schwann cells retracted; the resulting oval and rounded Schwann cells still were S-100 positive. When these Schwann cells were transferred into normal medium, their processes elongated quickly. When examined with the electron microscope, the cytoplasm of Schwann cells incubated in psychosine contained numerous membranous inclusions and fewer mitochondria, some of which were swollen. There also were fewer profiles of granular endoplasmic reticulum and some had widely dilated cisternae. These results suggest that 1) exogenous psychosine in concentrations of 1 microM and greater is cytotoxic for cultured rat Schwann cells; 2) psychosine has reversible toxic effects and its turnover is rapid; and 3) psychosine produces membranous inclusions and abnormalities in the mitochondria and granular endoplasmic reticulum of cultured Schwann cells. Our findings support the hypothesis that the accumulation of psychosine in human and murine globoid cell leukodystrophy is toxic for Schwann cells, produces changes in their capacity to maintain myelin, and leads to Schwann cell dysfunction.

Expression of viral T-antigen in pathological tissues from transgenic mice carrying JC-SV40 chimeric DNAs.

Immunostaining methods were used to detect viral T-antigen and the cellular protein p53 in pathological tissues obtained from transgenic mice carrying JC-SV40 hybrid viral DNAs. A transgenic mouse carrying the SV40 regulatory region and JC virus (JCV) T-antigen-coding sequences exhibited an SV40-characteristic choroid plexus papilloma that expressed JCV T-antigen and p53. JCV-associated pathology was observed in two other mice in which the JCV regulatory signals directed SV40 T-antigen-induced adrenal neuroblastomas and brain neoplastic cells. However, these mice also exhibited an SV40-characteristic osteosarcoma and abdominal lymphoma that contained SV40 T-antigen and p53-positive cells. Contrasting thymic pathology was observed in the two types of mice where the SV40 regulatory region directed a JCV T-antigen-induced thymoma in one mouse, and the JCV regulatory region directed SV40 T-antigen-induced thymic hypoplasia in two other mice.

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.

Myelinated fiber regeneration after sciatic nerve crush: morphometric observations in young adult and aging mice and the effects of macrophage suppression and conditioning lesions.

To study myelinated nerve fiber regeneration during aging, the right sciatic nerves of 6- and 24-month-old mice were crushed at the sciatic notch. Two, 4, and 8 weeks later, both groups of mice were perfused. The sciatic nerves were processed so that the transverse sections of each nerve subsequently studied by light and electron microscopy included the entire posterior tibial fascicle 5 mm distal to the crush site. Two weeks after axotomy, fascicles of aging mice contained significantly fewer regenerated myelinated fibers than those of young adults. After 4 weeks, the difference in the number of myelinated fibers was less. However, measurements of myelinated fibers in fascicles of aging mice showed that areas of Schwann cell cytoplasm and myelin were significantly reduced at all intervals. In contrast, although axon diameters in aging mice were somewhat less 2 weeks after crushing, the difference decreased with time, suggesting that in nerves of aging mice, regenerative responses of Schwann cells were more affected than those of axons. Other experiments in young mice showed that myelinated fiber regeneration could be retarded by suppressing macrophage responses and was not significantly changed by conditioning lesions before crush injury.

Brain vascular endothelial cells express JC virus large tumor antigen in immunocompetent and cyclophosphamide-treated hamsters.

When injected intracerebrally into newborn hamsters, the human polyomavirus JC virus (JCV) establishes a nonproductive infection resulting in brain tumor formation. Using immunostaining methods to detect the JCV regulatory protein, large tumor antigen (T antigen), we have now demonstrated JCV infection of brain vascular endothelial cells (EC) in infected hamsters. JCV T antigen was detected in lectin-labeled EC as well as in von Willebrand factor-expressing EC in both cyclophosphamide-treated and nonimmunosuppressed hamster brains 16, 21, and 31 days after birth. Cyclophosphamide-treated hamsters exhibited a greater number of JCV-infected EC, whereas T-antigen expression in nonvascular cells was not affected. The influence of cyclophosphamide was most pronounced in the cerebellum where increased numbers of JCV-infected EC were located predominantly at the internal granular layer-white matter junction, also a prominent location for T-antigen-expressing neoplastic foci. The hamster model demonstrates in vivo infection of EC by a human polyomavirus and directs interest toward the role of these cells in human JCV infection.