Da-Lin Yao

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 Freunds 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.

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.

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.

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 counterstained with Luxol fast blue

Transcriptional Controls in the Oligodendrocyte Lineage

Transcriptional controls are operative at all stages in the life cycle of an oligodendrocyte: in the commitment of progenitor cells to the oligodendrocyte lineage, in the coordinated regulation displayed by mature oligodendrocytes of the array of genes that encode myelin proteins and enzymes for myelin lipid synthesis, and in the activation of the myelin program in cells subjected to a demyelinating insult. An appreciation of the molecular events that underly the pinnacle of oligodendrocyte achievement, the compact myelin sheath, requires an understanding of how signalling molecules produced by neuronal and non-neuronal cells transmit information to the transcription factors that ultimately determine the fate and/or regulate myelin production of oligodendrocytes. The specific transcription factors that are involved in each of these events are just beginning to be identified. One of the best characterized factors in precursors of myelinating cells is a POU-homeodomain protein named SCIP for suppressed-cyclicAMPinducible protein (Monuki et al. , 1989; also referred to as Tst-1 or Oct-6). The expression of SCIP in proliferating oligodendrocyte progenitors and subsequent down-regulation of SCIP expression upon differentiation originally suggested that this transcription factor may be tied to the proliferative status of developing oligodendrocytes or the early decision-making steps in this lineage (Collarini et al. , 1992). This suggestion was supported by a number of studies that have documented the role that SCIP plays in myelinating cells of the peripheral nervous system. The most relevant ones are transgenic experiments with mice expressing a dominant-negative antagonist of SCIP in Schwann cells, in which the developmental program of Schwann cell differentiation is disrupted, resulting in severe perturbations of the myelin program in the peripheral nervous system (Weinstein et al. , 1995).

Astrocytes Upregulate Glial Fibrillary Acidic Protein (GFAP), but not Insulin‐like Growth Factor‐I (IGF‐I) during Experimental Autoimmune Neuritis (EAN)

T cell‐mediated autoimmune neuritis produces rapid activation of spinal cord microglia. To determine whether this microglial response upregulates astrocytic expression of IGF‐related proteins, we induced EAN and used in situ hybridization and immunocytochemistry to examine the mRNAs and peptides for glial fibrillary acidic protein (GFAP), insulin‐like growth factor‐I (IGF‐I), IGF‐I receptor (IGFR‐I) and IGF binding protein‐2 (IGFBP‐2). Relative levels of GFAP mRNA and peptide were highest in the lumbar spinal cord 4–10 d following T cell transfer and significant GFAP elevations were still present after three weeks. The astrocytes expressing GFAP mRNA and peptide were localized around motoneurons which were related topographically to axons in peripheral nerve inflammatory lesions. In the nucleus gracilis, where terminals of dorsal root ganglion neurons are located, astrocytic levels of GFAP mRNA and peptide rose later and did not reach their highest levels until 21 d after T cell transfer. Even though microglia were activated in both locations 2–4 d after transfer, astrocytic levels of IGF‐I, IGFR‐I and IGFBP‐2 mRNA and peptide did not differ significantly from those observed in controls. The dissociation of GFAP and IGF‐I expression in EAN suggests that these astrocytic responses may be independently regulated. We also suggest that the type and severity of remote neuronal injury are probably more important inducers and regulators of these astrocytic responses than microglial cell activation.