Efficacy of keratinocyte growth factor-2 in dextran sulfate sodium-induced murine colitis.

The purpose of this study was to determine the efficacy of a novel human protein, keratinocyte growth factor-2 (KGF-2), in a model of murine colitis induced by ad libitum exposure to a 4% solution of dextran sulfate sodium (DSS) in the drinking water. Initial evaluation of KGF-2 was based on its ability to reduce weight loss, stool score, and histological score in mice exposed to DSS for 7 days. When KGF-2 (0.1-10.0 mg/kg i.p. or s.c.) was injected daily into DSS-treated mice from day 0 to 7, it significantly reduced all three parameters in a dose-response fashion, with a minimum effective dose of between 1 and 3 mg/kg. When KGF-2 was given therapeutically, starting 4 days after initiation of the 7-day DSS treatment, the 3- but not the 0.5-mg/kg dose significantly enhanced weight recovery after discontinuation of DSS treatment. When DSS treatment was prolonged beyond the normal 7 days, therapeutic intervention on day 2 or 4 also significantly reduced mortality, weight loss, and stool score at the 1- and 3-mg/kg dose. Therapeutic treatment also resulted in reduction of colon myloperoxidase levels by more than 50%. These experiments suggest that KGF-2 may be clinically useful in the treatment of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

Model development and behavioral assessment of focal cerebral ischemia in rats.

Stroke in humans is usually focal and occurs in the Middle Cerebral Artery (MCA) distribution. There are several rat models that mimic strokes clinically seen in human. Severity of ischemia can be determined by occlusion time, arteries occluded i.e. MCA alone or combined with one or both Common Carotid Arteries (CCA), and/or location of the occlusion. In this study three focal cerebral infarctions (stroke) were induced for 90 and 120 minutes due to the occlusion of: the MCA alone (MCAo); MCA plus unilateral CCA (MCAo+1CCAo); and MCA plus bilateral CCA (MCAo+2CCAo). Histological parameters included infarct lesion size and hemispheric swelling. Since functional recovery of clinical deficits in stroke often correlates with the efficacy of anti-ischemic therapy, we focused on the behavioral recovery. We combined many sources to obtain comprehensive guidelines for clinical behavior evaluation. Tests included limb flexion, torso twisting, circling, lateral push resistance, beam balancing and walking, hindlimb placing, and inverted angle-board gripping. Occlusion lasting 90 minutes was found to have consistent and repeatable deficits. Results from our study demonstrate 120 minutes of occlusion produced a 60% morality rate and was therefore dropped. Body weight changes between groups showed that increased occlusion time produced more weight loss. Behavior changes indicated that MCAo+2CCAo for 90 minutes demonstrated assessable and consistent clinical deficits for the screening of potential therapeutics.

Pretreatment with intravenous FGF-13 reduces infarct volume and ameliorates neurological deficits following focal cerebral ischemia in rats.

Fibroblast growth factor-13 (FGF-13), novel member of FGF family has recently been molecularly cloned as a result of high throughput sequencing of a ovarian cancer cell, hippocampal, and kidney cDNA libraries. The human gene encodes for a protein with a molecular weight of 22 kDa that is most homologous to FGF-8 (70% similarity). In the current study, we tested the effects of intravenously administered FGF-13 in a model of permanent focal cerebral ischemia in Sprague-Dawley rats. FGF-13 or the vehicle was administered systematically via the tail vein 30 min prior, and 30 min and 24 h after the occlusion of the left middle cerebral artery (MCAo). Animals were weighed and evaluated behaviorally prior to and at 24 and 48 h after MCAo. The volume of cerebral infarct and swelling were determined using an image analysis system (BioQuant) and cresyl violet stained sequential sections from the forebrain region. Histopathology was evaluated to compare the therapeutic effects. We found a 63% reduction in infarct volume in FGF-13- vs. vehicle-treated animals (infarct volume was 21.9+/-3.8% in vehicle- and 8.1+/-1.6% in FGF-13-treated rats, p=0.0016) and a moderate inhibition of brain swelling by FGF-13. The reduction in infarct volume and brain swelling were associated with improvement of clinical deficits in FGF-13 treated animals (p<0.001). Histopathological examination determined that nervous tissue was better preserved in FGF-13 treated rats than those of controls. These data show that pretreatment with intravenous FGF-13 reduces infarct size and ameliorates neurological deficits following permanent focal cerebral ischemia in rats.

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.

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.

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.

Insulin-like growth factor I treatment reduces clinical deficits and lesion severity in acute demyelinating experimental autoimmune encephalomyelitis.

Our goal was to test the effects of insulin-like growth factor I (IGF-I) treatment on clinical deficits, lesion number and lesion size in acute demyelinating experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats with an emulsion containing guinea pig spinal cord. In this EAE model, there is severe immune-mediated demyelination, which resembles that seen in actively demyelinating MS lesions. On day 12-13 after EAE induction, a total of 23 pairs of rats with the same mild degree of tail and hind limb weakness were given either intravenous IGF-I or placebo twice daily for 8 days. The daily IGF-I dose used in the first trial was 200 micrograms (about 0.6 mg kg-1) and in the second and third trials was 1 mg (about 3.0 mg kg-1). IGF-I treatment reduced permeability of the blood-spinal cord barrier to Evans blue-albumin. Maximum clinical deficit scores of IGF-I-treated rats were significantly lower and treated rats recovered faster than controls. IGF-I treatment produced significant reductions in weight loss and hind limb weakness. Treatment also improved treadmill walking, stride length and climbing performance. Morphometric analysis showed that spinal cord inflammatory lesions were significantly smaller and fewer in IGF-I-treated rats. The higher IGF-I dose produced a greater reduction in clinical and pathological deficits. We conclude that IGF-I treatment promotes clinical recovery by reducing EAE-induced blood-spinal cord barrier changes and the associated immune-mediated inflammatory lesions. Our results suggest that IGF-I may be useful in treating patients with multiple sclerosis and other demyelinating diseases.

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.

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.

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.

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.