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Biomedical subjects

F H Gage

Publications and source records attributed to F H Gage.

At least 163 records · Page 9Linked to original sources
PubMed1993

Gene therapy for neurologic disease.

Gene therapy is a potentially potent new method of treating a number of neurologic disorders previously considered refractory to current conventional therapeutic treatments. Numerous advances have been made in the construction of expression vectors, cellular and viral transgene carriers, and the characterization of target cells for neuronal gene therapy. Two primary approaches to nervous system gene transfer have emerged as a result of these advances. The in vivo approach concentrates on direct transfer of genetic material to cells in vivo using viral and chemical agents. The ex vivo approach relies on genetic transfer to cultured cells that are subsequently implanted into a host organism. Both of these methods have been used in preliminary experiments designed to test the efficacy of gene transfer strategies in the amelioration of nervous system dysfunction.

Adenoviridae↗
PubMed1993

Changes in protein kinase C isozymes in the rat hippocampal formation following hippocampal lesion.

The cellular and regional distribution of the four protein kinase C (PKC) isoforms in the rat hippocampal formation and the response of PKC to lesions were determined by employing immunohistochemical and immunochemical techniques with antibodies specific to PKC(alpha), -(beta I), -(beta II), and -(gamma). PKC(alpha) intensely stained the periphery of the pyramidal cell in the stratum pyramidale. The granule cells, glial cells, and mossy fibers were anti-PKC(alpha) negative. The cytoplasm, axons, and dendrites of basket cells and interneurons in the hilus were labeled with anti-PKC(alpha). Anti-PKC(beta I) immunoreactivity was localized on the periphery of pyramidal cells and interneurons of the hilus, as well as the oriens, radiatum, and molecular layers of the CA regions. Anti-PKC(beta II) immunoreactivity was mainly cytoplasmic, extending into the dendrites in the hippocampal pyramidal cells and the dentate granule cells, and also in some glial cells. In the stratum radiatum of the CA1, anti-PKC(gamma) immunoreactivity localized to the pyramidal cell cytoplasm, extending into the dendrites. Following fimbria-fornix (FF) lesions, the anti-PKC(alpha) and -(beta I) staining of the pyramidal cell periphery was markedly reduced. The anti-PKC(gamma) staining of the pyramidal and granular cells of the dentate gyrus was reduced whereas the interneuron staining in the hilus was increased. In the FF-lesioned hippocampus, anti-PKC(alpha) and anti-PKC(beta II) labeled reactive glial cells, whereas anti-PKC(beta I) and -(gamma) did not. Quantitative Western blot analysis revealed a dramatic increase in the particulate/total PKC for all isozyme forms, although the total levels of PKC, except PKC(gamma), did not change following FF lesions. The PKC(gamma) concentration doubled after FF lesions. Perforant path lesions resulted in a marked alteration in the neuronal staining in dentate gyrus with anti-PKC(alpha) and -(beta I) and in increased numbers of anti-PKC(alpha)- and anti-PKC(beta II)-positive glial cells. Anti-PKC(gamma) staining did not change noticeably. The total PKC concentration did not change for isozymes alpha, beta I, and gamma, but PKC (beta II) concentration increased by 48% following perforant path lesions as detected by Western blot analysis. The particulate/total PKC decreased for all four isozymes although the reduction in PKC(beta I) concentration was not statistically significant. This change in PKC compartmentalization is in marked contrast to an increased level of particulate PKC following FF lesions. Thus, the effects of deafferentation and deafferentation for each PKC isoform were different.

Animals↗
PubMed1993

In vivo production and release of acetylcholine from primary fibroblasts genetically modified to express choline acetyltransferase.

Primary rat fibroblasts genetically modified to express Drosophila choline acetyltransferase (dChAT) synthesize and release acetylcholine (ACh) in vitro. The ACh produced from the transduced fibroblasts was found to be enhanced by increasing amounts of choline chloride in the culture media. These dChAT-expressing cells were then implanted into the intact hippocampus of adult rats and in vivo microdialysis was performed 7-10 days after grafting to assess the ability of the cells to produce ACh and respond to exogenous choline in vivo. Samples collected from anesthetized rats revealed fourfold higher levels of ACh around dChAT grafts than from either non-grafted or control-grafted hippocampi. Localized choline infusion (200 microM) through the dialysis probes was found to induce a selective twofold increase in ACh release only from the dChAT-expressing fibroblasts. These results indicate not only that dChAT-expressing fibroblasts continue to synthesize and secrete ACh for at least 10 days after intracerebral grafting, but that the levels of ACh can be manipulated in vivo. The ability to regulate products within genetically modified cells in vivo may provide a powerful avenue for exploring the role of discrete substances within the CNS.

Acetylcholine↗
PubMed1993

Nerve growth factor reverses neuronal atrophy in a Down syndrome model of age-related neurodegeneration.

Atrophy and dysfunction of certain neurons, including cholinergic neurons in the basal forebrain, are key features of the neuropathology of Alzheimer's disease (AD). Since all individuals with Down syndrome (DS) develop AD neuropathology by the 4th decade, we reasoned that a genetic model of DS, the trisomy 16 (Ts 16) mouse, may provide an animal model to study the neurodegeneration in AD. Ts 16 mice fail to survive birth; to evaluate neurons for long periods in vivo required transplantation of fetal tissue. We previously demonstrated that Ts 16 basal forebrain cholinergic neurons (BFCNs) undergo age-related atrophy similar to DS and AD, and now show that a specific neurotrophic factor, nerve growth factor (NGF), acts to reverse Ts 16-induced atrophy of BFCNs and stimulates hypertrophy of these cells. As NGF levels were not decreased in the host, abnormalities intrinsic to Ts 16 BFCNs presumably caused the atrophy. Our results suggest that NGF may be useful in reversing cholinergic neurodegeneration in DS and AD.

Aging↗
PubMed1993

Thymidine kinase-mediated killing of rat brain tumors.

Gene therapy has many potential applications in central nervous system (CNS) disorders, including the selective killing of tumor cells in the brain. A rat brain tumor model was used to test the herpes simplex virus (HSV)-thymidine kinase (TK) gene for its ability to selectively kill C6 and 9L tumor cells in the brain following systemic administration of the nucleoside analog ganciclovir. The HSV-TK gene was introduced in vitro into tumor cells (C6-TK and 9L-TK), then these modified tumor cells were evaluated for their sensitivity to cell killing by ganciclovir. In a dose-response assay, both C6-TK and 9L-TK cells were 100 times more sensitive to killing by ganciclovir (median lethal dose: C6-TK, 0.1 microgram ganciclovir/ml; C6, 5.0 micrograms ganciclovir/ml) than unmodified wild-type tumor cells or cultured fibroblasts. In vivo studies confirmed the ability of intraperitoneal ganciclovir administration to kill established brain tumors in rats as quantified by both stereological assessment of brain tumor volumes and studies of animal survival over 90 days. Rats with brain tumors established by intracerebral injection of wild-type or HSV-TK modified tumor cells or by a combination of wild-type and HSV-TK-modified cells were studied with and without ganciclovir treatments. Stereological methods determined that ganciclovir treatment eliminated tumors composed of HSV-TK-modified cells while control tumors grew as expected (p < 0.001). In survival studies, all 10 rats with 9L-TK tumors treated with ganciclovir survived 90 days while all untreated rats died within 25 days. Curiously, tumors composed of combinations of 9L and 9L-TK cells could be eliminated by ganciclovir treatments even when only one-half of the tumor cells carried the HSV-TK gene. While not completely understood, this additional tumor cell killing appears to be both tumor selective and local in nature. It is concluded that HSV-TK gene therapy with ganciclovir treatment does selectively kill tumor cells in the brain and has many potential applications in CNS disorders, including the treatment of cancer.

Animals↗
PubMed1993

Regulation of dopamine production by genetically modified primary fibroblasts.

Primary skin fibroblasts were genetically modified with catecholamine-synthesizing enzyme genes and studied as potential syngeneic donor cells to supply catecholamines in animal models of Parkinson's disease. Primary skin fibroblasts obtained from inbred Fischer 344 rats were transduced with tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC) cDNAs using retroviral vector system. The transduced cells were characterized in vitro by enzymatic assay, immunocytochemistry, and HPLC analysis of catecholamine production and release. Accumulation of high levels of dopamine was detected in the media in a time-dependent manner. Secretion of dopamine and its metabolites appeared to be constitutive without significant storage capacity in vesicles or regulation at the level of secretion. The feasibility of regulating the final dopamine production by the AADC-transduced cells was explored in two ways. First, administration of various doses of the precursor, L-dopa, resulted in a controlled production of dopamine by these cells. Second, coculturing AADC-transduced cells with TH-transduced cells in various proportions allowed control of dopamine production. TH-transduced cells served as an endogenous source of precursor. We propose the use of these cells to study the role of AADC in restoring the dopamine-deficient behavior and to compare the effect of dopamine-producing cells with L-dopa-producing cells either by cografting TH-transduced cells with AADC-transduced cells or by grafting TH-transduced cells alone. The role of AADC in vivo will be assessed in future experiments involving animal models of Parkinson's disease.

Animals↗
PubMed1992

Morphological and neurochemical features of cultured primary skin fibroblasts of Fischer 344 rats following striatal implantation.

In order to assess the feasibility of using primary skin fibroblasts as a donor population for genetic modification and subsequent intracerebral grafting, the present study examines the structural and neurochemical characteristics of intrastriatal grafts of isogeneic primary fibroblasts over a period of 6 months. In culture, primary skin fibroblasts obtained from a female Fischer 344 rat display robust growth, but once confluent these cells exhibit contact inhibition. Following the implantation of cultured primary cells within the striatum of other adult rats from the same inbred strain, isologous grafts stain immunohistochemically for fibronectin at 1 week, and this immunostaining persists up to 6 months. Immunoreactivity for laminin is intense within the grafts from 1 to 8 weeks, but decreases by 6 months. Astrocytes within the striatum respond dramatically to the implantation of primary fibroblasts, such that immunohistochemical staining for glial fibrillary acidic protein increases markedly from 1 to 8 weeks after implantation. Although the intensity of immunostaining for glial fibrillary acidic protein diminishes among striatal astrocytes between 8 weeks and 6 months, the astrocytic border between the grafts and striatal neuropil remains intensely immunoreactive. Capillaries within the grafts stain immunohistochemically for glucose transporter (a facilitated glucose uptake carrier) as early as 3 weeks after implantation. Following intravenous infusions of peroxidase, capillaries within fibroblast grafts do not permit the extravasation of this macromolecule at 8 weeks and 6 months. Thus, capillaries formed within intracerebral grafts of primary skin fibroblasts exhibit a functional impermeable barrier to macromolecules similar to those capillaries of the host striatum. At the ultrastructural level, grafts possess numerous fibroblasts and have an extracellular matrix filled with collagen. Reactive astrocytic processes filled with intermediate filaments are found throughout the grafts. Hypertrophied astrocytes and their processes also appear to form a continuous border between the grafts and striatal neuropil. Grafts of primary fibroblasts also possess an extensive vasculature that is composed of capillaries with nonfenestrated endothelial cells; the occurrence of reactive astrocytic processes closely associated with or enveloping capillaries is variable. These results provide direct morphological and neurochemical evidence for the long-term survival of isologous fibroblasts after implantation within the rat striatum. From these data, we propose that isologous skin fibroblasts can be considered as donor candidates for successful intracerebral grafting following gene transfer.

Animals↗
PubMed1992

Mouse model of neurodegeneration: atrophy of basal forebrain cholinergic neurons in trisomy 16 transplants.

Vulnerability of specific brain regions and neuronal populations is a characteristic feature of Alzheimer disease and Down syndrome. Cholinergic neurons of the basal forebrain degenerate in both disorders. The basis for neuronal degeneration is unknown. Mouse trisomy 16 (Ts 16) is an animal model of Down syndrome. We sought an experimental system in which the survival and development of Ts 16 basal forebrain cholinergic neurons could be examined beyond the fetal period. As Ts 16 mice do not survive birth, we transplanted fetal Ts 16 and control basal forebrain into the hippocampus of young adult mice. Transplanted neurons survived and grew neurites in all grafts. Over time, we observed selective atrophy of cholinergic neurons in Ts 16 grafts. Denervation of the hippocampus produced a significant increase in the size of Ts 16 cholinergic neurons. This suggests that hippocampal-derived neurotrophic factors acted to prevent degeneration. beta/A4-amyloid-containing plaques were not seen. Ts 16 provides a model of spontaneous, genetically determined neurodegeneration that may be used to understand better the molecular pathogenesis of neuronal dysfunction in Alzheimer disease and Down syndrome.

Alzheimer Disease↗
PubMed1992

Cooperative regulation of nerve growth factor synthesis and secretion in fibroblasts and astrocytes by fibroblast growth factor and other cytokines.

Acidic fibroblast growth factor (aFGF) enhances nerve growth factor (NGF) synthesis by astrocytes obtained from various brain regions. NGF secretion by fibrous-shaped astrocytes transformed by dibutyryl-cAMP (db-cAMP) pretreatment was less than that by untreated astrocytes. However, aFGF also enhanced NGF secretion by fibrous-shaped astrocytes. The effects of various kinds of intracellular signaling modulators on NGF synthesis were examined. None of the following second messenger effectors had an effect on NGF synthesis: protein kinase C (PKC) agonist (phorbol myristate acetate (PMA)) or antagonist (sphingosine (SP)). LiCl, and ionomycin (Iono). Further, increases of intracellular cAMP by forskolin (FK) or db-cAMP have no significant effect on NGF synthesis in astrocytes under a standard culture condition. However, NGF synthesis by astrocytes in the presence of aFGF was significantly enhanced by db-cAMP, but not by FK or sodium butyrate. These results indicate that an excessive amount of cAMP enhances the effect of aFGF on NGF synthesis in astrocytes. NGF synthesis in astrocytes was not affected by treatment with anti-aFGF or anti-bFGF neutralizing antibodies, indicating that FGFs are not involved in the autocrine regulation of NGF synthesis in astrocytes. Transforming growth factor-beta 1 (TGF-beta 1), which inhibits some effects of FGFs, increased NGF synthesis in concert with aFGF. Furthermore, the highest NGF synthesis was observed when astrocytes were stimulated by all of the following cytokines: aFGF, interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha) and TGF-beta 1. The mechanism regulating NGF synthesis in fibroblasts obtained from prenatal rat skin was also investigated. Acidic FGF, basic FGF (bFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-alpha), TGF-beta 1, IL-1 beta, and TNF-alpha were found to be regulators of NGF synthesis in skin fibroblasts. Among these cytokines, aFGF is the most potent regulator of NGF synthesis in fibroblasts. NGF synthesis by skin fibroblasts, either in the presence or absence of aFGF, was not modified by any of the following: FK, PMA, SP, LiCl, and Iono. However, db-cAMP significantly enhanced NGF synthesis in both conditions. Sodium butyrate enhanced NGF synthesis in the presence of aFGF, but not in the absence of aFGF. These results suggest that an excessive amount of cAMP and butyrate moiety regulate NGF synthesis in skin fibroblasts in different ways.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗
PubMed1992

Quantitative effects of cerebral infarction on spatial learning in rats.

Outcome following stroke is difficult to measure because the behavioral response to infarction is variable. We hypothesized that cognitive function, such as spatial learning, may be a reproducible and sensitive outcome variable. We developed an animal model of multifocal cerebral ischemia in order to study the effects of infarction on learning. To cause ischemia, several hundred microspheres were injected into the internal carotid arteries of rats. After ischemia, behavior was measured using a global rating and a Morris water maze. Postmortem serial brain sections were stained and the size of the infarctions was measured. We found that intracerebral microspheres caused cortical infarction and an impairment of spatial learning. This impairment was not due to occlusion of the internal carotid artery and was not found in animals who received a sham injection of saline. The degree of learning impairment was not correlated with the volume density of the infarctions or with the volume density of the remaining cerebral hemisphere. The learning impairment clearly differentiated normal from lesioned animals, and the impairment was probably due to a delay in acquisition of spatial information rather than a defect in retention or retrieval. Measurement of learning deficit after cerebral ischemia is an efficient and sensitive method for evaluating new stroke treatments and possibly for exploring structure function relationships.

Animals↗
PubMed1992

Effects of gene transfer into cultured CNS neurons with a replication-defective herpes simplex virus type 1 vector.

Vectors derived from herpes simplex virus type 1 (HSV-1) may provide useful tools for gene transfer to cells of the mammalian nervous system. We have studied the infection of cultured CNS neurons using a vector derived from an HSV-1 mutant deleted for the major HSV-1 transcriptional regulatory protein-encoding gene, IE 3. This vector, denoted Cgal delta 3, contains the E. coli lacZ gene driven by the strong promotor of the human cytomegalovirus major immediate-early gene inserted into a non-coding portion of the mutant viral genome. We studied the efficiency of Cgal delta 3 infection of rat CNS neurons at various times after cell preparation from embryonic rats, the effect of vector infection on the glia subpopulation of the neuronal cultures, and the stability of lacZ expression in infected neurons cultured under conditions optimized for neuronal differentiation and survival using an astrocyte feeder layer. Under these conditions, an HSV-derived vector is a highly efficient vehicle in vitro for short-term gene transfer to cells of the CNS. Despite the fact that this vector cannot undergo a lytic cycle, it was toxic to cultured CNS neurons and glia. Even with the use of an astrocyte feeder layer to support infected neurons, we have detected only transient expression of the lacZ gene, due either to loss of the infected cells and/or to shut off of transgene expression. Further improvements will be needed in the design of HSV vectors to allow long-term gene transfer to cultured neurons.

Animals↗
PubMed1992

Delayed treatment with nerve growth factor improves acquisition of a spatial task in rats with lesions of the nucleus basalis magnocellularis: evaluation of the involvement of different neurotransmitter systems.

Rats received bilateral lesions of the nucleus basalis magnocellularis by infusion of ibotenic acid. Fourteen days later, osmotic minipumps releasing human recombinant nerve growth factor (0.3 micrograms/day) were implanted subcutaneously. Starting one month after the lesion, spatial learning of the animals was tested using the Morris water maze. Acquisition of the task was impaired by the lesion, but treatment with nerve growth factor reduced the average latency to find the platform by approximately 9 s, which represents 28% of the lesion-induced behavioral deficit. Retention of this task and spatial acuity, tested in a trial in which the platform was not present, did not show a statistically significant improvement. Lesions of the nucleus basalis magnocellularis reduced the choline acetyltransferase activity in the neocortex, but not in the hippocampus. Treatment with nerve growth factor increased the choline acetyltransferase activity in the neocortex but not in the hippocampus. There was no significant difference in the levels of norepinephrine, dopamine, serotonin or their metabolites in the cortex or hippocampus between nerve growth factor-treated animals and lesioned control animals. There was no significant correlation between any of these neurochemical changes and behavioral performance (acquisition and spatial acuity). Treatment with nerve growth factor did not increase the number or the size of nerve growth factor receptor-immunoreactive neurons in the nucleus basalis magnocellularis. These data suggest that delayed treatment with nerve growth factor results in an improvement of spatial learning in rats with lesions of the nucleus basalis magnocellularis. A possible role for cholinergic mechanisms in this effect is discussed.

3,4-Dihydroxyphenylacetic Acid↗