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Biomedical subjects

H M Schipper

Publications and source records attributed to H M Schipper.

At least 73 records · Page 4Linked to original sources
PubMed1993

The prevention of oxyhemoglobin-induced endothelial and smooth muscle cytoskeletal injury by deferoxamine.

The oxidized breakdown products of hemoglobin are important in the pathogenesis of cerebral vasospasm because of their effects on the endothelium and the smooth muscle of the arterial wall. Cytoskeletal changes in cultured vascular cells are sensitive indicators of oxidative injury. Cultured endothelial cells and smooth muscle cells showed a dose-related disruption of the cytoskeleton, particularly the F-actin and vimentin filaments, when exposed to 10(-5) M oxyhemoglobin. The cytoskeletal injury was prevented by the addition of 10(-3) M deferoxamine or 1% albumin. These experiments support a role for deferoxamine in the pharmacological treatment of vasospasm. Furthermore, cytoskeletal studies of cultured arterial endothelial and smooth muscle cells provide a novel in vitro approach by which to study the cellular mechanisms of oxidant injury initiated by the breakdown products of hemoglobin.

Actins↗
PubMed1993

Cysteamine gliopathy in situ: a cellular stress model for the biogenesis of astrocytic inclusions.

In cultured astroglia, cysteamine induces the accumulation of peroxidase-positive cytoplasmic inclusions in the context of a generalized cellular stress response. In the present study, systemic cysteamine administration over a 3 week period induced HSP27, 72, 90, and GRP94 (stress proteins) in astrocytes and significantly increased numbers of peroxidase-positive astrocytic inclusions in the various brain regions relative to controls. Similar patterns of HSP expression were also observed at 24 hours following cysteamine treatment indicating that cellular stress may be a very proximal event in the biogenesis of the astrocytic inclusions. The topography of glial peroxidase activity may provide a "map" of central nervous system regions particularly prone to oxidative stress during normal aging and under pathologic conditions.

Animals↗
PubMed1992

Expression of the non-proliferation-specific protein, statin, in grey matter neuroglia of the aging rat brain.

The monoclonal antibody, S-44, identifies statin, a 57 kDa nuclear protein which appears to be expressed exclusively in non-proliferating cells. We previously demonstrated that in the aging rat corpus callosum approximately one third of neuroglia are statin-negative, suggesting the existence of an unexpectedly large cycling glial compartment. In the present study, double-labeling of individual cultured astroglia with [3H]thymidine and the S-44 antibody provided direct evidence for the non-proliferative status of statin-positive cells. The S-44 antibody was used to immuno-localize statin and thereby determine growth fractions for neuroglia in various grey matter regions of 3-, 18-, and 33-month-old rats. The proportion of statin-negative (cycling) cells for the three ages combined ranged from about 24% in the molecular layer of the dentate gyrus to 38% in the molecular layer of the parietal cortex. In most regions surveyed total glial counts and proportions of statin-positive and -negative cells did not vary significantly as a function of advancing age. These results suggest that (i) as in corpus callosum, pools of cycling neuroglia in various grey matter regions are far in excess of those previously predicted by S-phase labeling with [3H]thymidine or BUdR, and (ii) ratios of proliferating-to-quiescent neuroglia are tightly regulated over much of the animal's adult life span. These conserved ratios may be used as markers of normal CNS senescence, and deviations thereof may indicate the presence and extent of intervening neuropathologic processes.

Aging↗
PubMed1992

Iron content correlates with peroxidase activity in cysteamine-induced astroglial organelles.

A subpopulation of astrocytes in periventricular brain regions and in cysteamine-treated neuroglial cultures contains cytoplasmic granules that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. The autofluorescence and pseudoperoxidase activity are consistent with the presence of porphyrins and heme iron, respectively. In the present study, we employed diaminobenzidine cytochemistry, transmission electron microscopy, and energy-dispersive X-ray microanalysis (electron microprobe) in an attempt to correlate fine structure with the peroxidase activity and elemental composition of the cysteamine-induced inclusions in cultured astrocytes. In osmicated preparations, these membrane-bound inclusions varied greatly in size, were round or ovoid in shape, and exhibited an intensely electron-dense granular matrix. In non-osmicated preparations, many inclusions exhibited internal membranous partitions producing complex subcompartmentalization. Diaminobenzidine reaction product, indicative of endogenous peroxidase activity, was occasionally observed distributed diffusely throughout the granule matrix. More commonly, peroxidase activity was restricted to specific intraorganellar compartments. Elemental iron was detected in the inclusions by electron microprobe analysis. The presence and concentration of iron in these organelles correlated closely with the presence and intensity of diaminobenzidine staining, suggesting that redox-active iron mediates the pseudoperoxidase reactions in these cells. Cysteamine-induced derangements of porphyrin-heme biosynthesis may be responsible for the proliferation of iron-containing gliosomes in these astrocytes.

3,3'-Diaminobenzidine↗
PubMed1992

Vitamin E protects hypothalamic beta-endorphin neurons from estradiol neurotoxicity.

Estradiol valerate (EV) treatment has been shown to result in the destruction of 60% of beta-endorphin neurons in the hypothalamic arcuate nucleus. Evidence suggests that the mechanism of EV-induced neurotoxicity involves the conversion of estradiol to catechol estrogen and subsequent oxidation to free radicals in local peroxidase-positive astrocytes. In this study, we examined whether treatment with the antioxidant, vitamin E, protects beta-endorphin neurons from the neurotoxic action of estradiol. Our results demonstrate that chronic vitamin E treatment prevents the decrement in hypothalamic beta-endorphin concentrations resulting from arcuate beta-endorphin cell loss, suggesting that the latter is mediated by free radicals. Vitamin E treatment also prevented the onset of persistent vaginal cornification and polycystic ovarian condition which have been shown to result from the EV-induced hypothalamic pathology.

Animals↗
PubMed1991

Region-specific immunolocalization of atrial natriuretic peptide in mixed fetal rat brain cell cultures.

In adult rodent CNS, atrial natriuretic peptide (ANP) has been localized by immunolabeling and nucleic acid hybridization techniques primarily to hypothalamic neurons and, to a lesser extent, to neurons of the telencephalon and brainstem. In canine brain, ANP immunoreactivity has been reported in neocortical and brainstem astrocytes. Yet, in a recent study using fetal rat tissue, ANP was detected by radioimmunoassay in predominantly neuronal, but not glial, cultures. In the present study ANP was detected by radioimmunoassay on in vitro day 8 in media derived from fetal rat diencephalic and rhombencephalic, but not telencephalic, cultures and in cell homogenates from all 3 regions surveyed. Using indirect immunofluorescence less than one cell per 400x field stained for ANP in the telencephalic cultures and ANP immunopositivity colocalized exclusively to neurons by concomitant anti-neurofilament immunolabeling. In diencephalic monolayers, approximately 1-3 cells per 400x field were ANP-positive; although most of these cells were neurons, small numbers of ANP-positive astrocytes were also detected using anti-glial fibrillary acidic protein (GFAP) immunolabeling. ANP-positive cells were most numerous in rhombencephalic cultures (5-10 cells per 400x field); as in diencephalic cultures, ANP immunoreactivity colocalized to both neurons and astrocytes in this region. In diencephalon and rhombencephalon, less than 1% of all ANP-positive cells were astrocytes and less than 1% of GFAP-positive astrocytes exhibited immunoreactive ANP. In fetal brain, neuronal and astrocytic ANP may play a role in fluid and electrolyte homeostasis. Alternatively, fetal brain cell ANP may subserve entirely different functions (e.g. as trophic factors) as has been suggested for other neuropeptides in the developing nervous system.

Animals↗
PubMed1991

Statin immunolocalization in human brain tumors. Detection of noncycling cells using a novel marker of cell quiescence.

Surgical specimens of 35 human brain tumors were examined with a novel monoclonal antibody, S-44, immunoreactive to statin, a nuclear protein specifically expressed in quiescent (noncycling) G0-phase cells. Benign tumors typically were statin positive with labeling indices (LI) between 22% and 96%: acoustic schwannomas (n = 3, mean = 29.9 +/- 19.4%); meningiomas (n = 4, mean = 59.0 +/- 15.1%); pituitary adenomas (n = 3, mean = 79.9 +/- 28.2%), and an epidermoid cyst (41.0%). By contrast, the statin LI of 18 of 24 (75%) malignant brain tumors was less than or equal to 2%: medulloblastomas (n = 7, mean = 0.3 +/- 0.2%); anaplastic astrocytomas (n = 3, mean = 1.6 +/- 2.7%); glioblastomas (n = 10, mean = 10.3 +/- 14.4%); metastatic carcinomas (n = 3, mean = 3.0 +/- 4.6); and a germinoma (0.2%). The vascular endothelium among diverse tumors typically was statin positive. All 21 tumors with a statin LI less than 10% were malignant, and all nine tumors with a statin LI greater than 40% were benign. The statin LI of benign tumors (n = 11, mean = 55.1 +/- 26.7%) was significantly higher than that of the malignant tumors (n = 24, mean = 5.2 +/- 10.5%, P less than 0.001). The absence of statin expression is a new way to determine the malignancy of human brain tumors. The statin LI may be useful to guide the prognosis and treatment of individual patients. The mechanisms that control statin expression are important in therapy seeking to shift the proliferating, cycling cells to the quiescent, G0 compartment.

Adolescent↗
PubMed1991

Gomori-positive astrocytes: biological properties and implications for neurologic and neuroendocrine disorders.

Granule laden astrocytes exhibiting an affinity for chrome alum hematoxylin and aldehyde fuchsin (Gomori stains) have been described in the periventricular brain of all terrestrial vertebrate species examined to date including humans. The astrocytic inclusions are rich in sulfhydryl groups, emit an orange-red autofluorescence, and stain intensely with diaminobenzidine, a marker of endogenous peroxidase activity. The distinct autofluorescence pattern and the absence of neutral lipid, acid phosphatase, and beta-glucuronidase activity exclude lipofuscin or lysosomes as components of these astrocytic granules. The emission of orange-red autofluorescence and the nonenzymatic nature of the peroxidase activity implicate the presence of porphyrins and metalloporphyrins such as heme as major constituents of these cytoplasmic gliosomes. The role of Gomori-positive astrocytes under normal and pathologic conditions is incompletely understood. In vivo, numbers of astrocytic granules increase as a function of advancing age, in response to chronic estrogen stimulation, and following X-irradiation. In vitro, these cells accumulate with increasing time in culture and following exposure to the sulfhydryl agent, cysteamine. Gomori-positive astrocytes may supply heme to neurons for the synthesis of cytochromes, catalases, and other heme enzymes. They may play a role in photostimulation of sexual cyclicity, the promotion of neuritic development, the degradation of toxic lipoperoxides, and the metabolism of various neurotransmitters. Conversely, these cells may contribute to the pathogenesis of several neurologic and neuroendocrine disorders. Examples of the latter include a) augmentation of goldthioglucose neurotoxicity, b) induction of hypothalamic anovulation and reproductive failure, c) exacerbation of porphyric encephalopathy, and d) potentiation of parkinsonism and other free radical-related neurodegenerations.

Animals↗
PubMed1991

Rumenectomy-induced proliferation in duodenal villous epithelium is mechanistically related to the disappearance of statin, a non-proliferation-specific nuclear protein.

We undertook this study to determine the effect of rumenectomy (a known cause of duodenal crypt cell hyperplasia) on the epithelial growth kinetics of the crypt-villus axis in rat duodenum. Ten rats were randomly assigned to control (gastrotomy) and experimental (rumenectomy) groups. After 14 days rats were sacrificed and representative sections were stained with the monoclonal antibody to statin, a non-proliferation-specific protein, by the immunoperoxidase procedure. In the control group, the mean percentages of statin-positive cells in the proximal duodenum, distal duodenum, proximal jejunum, and distal jejunum were 79 +/- 8.5, 79.5 +/- 5.7, 85 +/- 1.4, and 83.5 +/- 0.7, respectively. In the rumenectomy group, statin-positive nuclei were found in the region of the villous apices only, and the corresponding values for the above four areas were 26.2 +/- 4.9, 24.5 +/- 3.5, 31.7 +/- 4.5, and 80.5 +/- 2.1. Except for distal jejunum, the differences in statin expression in the control and experimental groups were significant (p less than 0.001). Rumenectomy leads to the disappearance of statin from the villous column cells of the duodenum and proximal small bowel. The lack of expression of statin in the rumenectomy group documents the potential usefulness of this measure in future studies in neoplasia were understanding of the proliferative status is of crucial importance.

Animals↗
PubMed1991

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.

Animals↗
PubMed1991

Catechol oxidation by peroxidase-positive astrocytes in primary culture: an electron spin resonance study.

In rodents, chronic estrogenization has been shown to induce degeneration of dendrites and myelin figures in the hypothalamic arcuate nucleus adjacent to peroxidase-positive astrocyte processes. Because in this brain region estradiol is metabolized to 2-hydroxyestradiol (catecholestrogen), we hypothesized that the latter may be oxidized by the astrocytic peroxidase activity to cytotoxic ortho-semiquinones as occurs in peripheral tissues. Cysteamine induces nonenzymatic peroxidase activity in cultured astroglia identical to that observed in vivo. Using electron spin resonance, we demonstrate robust peroxidase-catalyzed oxidation of 2-hydroxyestradiol and dopamine by cysteamine-pretreated astrocyte cultures relative to untreated controls. These results implicate the peroxidase-positive astrocytes in the pathogenesis of estradiol-related hypothalamic damage, parkinsonism, and other free-radical-related neurologic disorders.

3,3'-Diaminobenzidine↗
PubMed1990

Expression of statin, a non-proliferation-dependent nuclear protein, in the postnatal rat brain: evidence for substantial retention of neuroglial proliferative capacity with aging.

Statin is a 57 kDa protein expressed in nuclei of reversibly and irreversibly growth-arrested (Go-phase) cells. In this report, immunohistochemical localization of statin in the developing and aging rat brain was achieved using the monoclonal antibody, S-44. On postnatal day 2, post-migratory neurons in the developing cerebral cortex were statin-positive. Many statin-negative cells were observed in the lateral subependymal zone of the lateral ventricle. By postnatal day 10, most neuronal nuclei were statin-positive although small numbers of statin-negative neurons were still encountered in the lateral subependymal zone and hippocampal dentate gyrus. At 3, 18 and 33 months, all neuronal nuclei surveyed were statin-positive. These results support the contention that, save for the postnatal persistence of 'germinal zones' such as the subependymal region and dentate gyrus, neuronal proliferation in the rat is largely completed by the time of birth. In striking contrast to neuronal statin expression, a significant fraction of neuroglia in both grey and white matter remains statin-negative at all ages examined. In the corpus callosum, 33.2%, 34.0% and 34.7% of glial nuclei were statin-negative at 3, 18 and 33 months, respectively. These findings indicate that: (i) even in senescent brain, the cycling (statin-negative) glial pool is substantially larger than previously surmised from S-phase labeling experiments; and (ii) during aging, the ratio of noncycling-to-cycling neuroglia remains very tightly regulated. Examination of other non-neuronal cell types revealed that most, if not all, ependymal and choroid plexus epithelial cells were statin-positive in the neonatal and adult brains in keeping with the predominantly prenatal proliferation of these tissues. Our results indicate that statin immunolabeling using the S-44 antibody is a powerful technique for the in situ identification of non-proliferating cells in the developing and aging nervous system.

Aging↗
PubMed1990

Effects of progestins on the estradiol-related accumulation of astrocytic granules in the hypothalamic arcuate nucleus.

Adult female rats treated with a single 2 mg injection of estradiol valerate (EV) develop anovulatory sterility (persistent estrus) and neuropathologic changes in the hypothalamic arcuate nucleus. A prominent feature of the latter is the accumulation of peroxidase-positive cytoplasmic inclusions in arcuate astrocytes which results from aberrant patterns of chronic, ovarian E2 secretion in this model. In the present study, we tested an hypothesis that progesterone may antagonize the pathologic effects of E2 within the medial basal hypothalamus analogous to effects in peripheral steroid target tissues. In intact EV-treated rats, we observed complete suppression of the astrocytic reaction by subsequent injections of medroxyprogesterone acetate (MPA). However, chronic progesterone exposure did not significantly antagonize this estrogenic affect in ovariectomized rats implanted subcutaneously with steroid-releasing Silastic capsules. Taken together, these results suggest that progestins may block the development of an E2-related arcuate lesion by suppressing the pituitary-ovarian axis rather than by directly antagonizing the dystrophic effects of E2 at the hypothalamic level.

Animals↗
PubMed1990

Gomori-positive astrocytes in primary culture: effects of in vitro age and cysteamine exposure.

Gomori-positive astrocytes have been identified in the periventricular brain in situ and in diencephalic explants on the basis of their endogenous peroxidase activity, affinity for chrom alum hematoxylin, and orange-red autofluorescence. To facilitate analyses of their functional properties, we sought to identify these cells in dissociated fetal rat brain cultures. Astrocytes containing cytoplasmic inclusions with the above tinctorial and fluorescent properties represented less than 1% of cultured astrocytes at day 10 in vitro (DIV). There was a marked increase in the fraction of Gomori-positive astrocytes and their granule content between 10 and 46 DIV. As in situ, the peroxidase activity appeared to be non-enzyme-mediated insofar as it catalyzed diaminobenzidine oxidation over a wide range of pH (3-11) and could not be inhibited by tissue preheating or the catalase inhibitor, aminotriazole. Metalloporphyrins probably mediate both the pseudoperoxidase activity and autofluorescence in these cells. Cysteamine and cystamine, but not ethanolamine or L-cysteine, induced a massive accumulation of Gomori-positive astrocytes when administered from DIV 6-18. Alterations of the redox microenvironment or induction of porphyrin/heme biosynthetic enzymes may be the mechanisms responsible for this cyst(e)amine effect. Dissociated rat brain culture enriched for Gomori astroglia should provide ample opportunity to investigate the functional properties of these cells.

Animals↗
PubMed1990

Glial peroxidase activity in the hypothalamic arcuate nucleus: effects of estradiol valerate-induced persistent estrus.

To test the hypothesis that stimulation of glial peroxidase activity by estrogens may play a role in the pathogenesis of the previously reported degenerative changes in the hypothalamic arcuate nucleus that occur in female rats treated with a long-acting estrogen preparation, the cellular localization and number of peroxidase-positive granules in the hypothalamus were determined in adult female rats treated with a single intramuscular injection of 2 mg of estradiol valerate. The persistent estrus state, manifested as persistent vaginal cornification and polycystic ovaries, was induced in 80% of the animals. In comparison with normally cycling controls, the arcuate nuclei of persistent estrus rats exhibited a 3- and 2.8-fold increase in numbers of diaminobenzidine-positive granules and granule clusters, respectively (P less than 0.01 for both comparisons). These peroxidase-positive granules were identified in astrocytes by double-label immunohistochemistry utilizing antiserum to glial fibrillary acidic protein. Diaminobenzidine staining occurred over a pH range of 4-10.5 and was resistant to the catalase inhibitor, aminotriazole, and to tissue pre-heating, indicating that the histochemical reaction was not due to tissue enzyme activity. Rather, these findings indicate that a non-enzymatic, pseudoperoxidation reaction has been induced in these cells by estrogen administration. Possible mediators of this reaction are metallo-porphyrins known to be present in rodent hypothalamus. The mechanisms by which astrocyte peroxidase activity may play a role in estrogen-related neural damage are discussed.

Animals↗
PubMed1990

Statin expression in the untreated and SarCNU-exposed human glioma cell line, SK-MG-1.

Cytokinetic analyses of gliomas and other neoplasms rely exclusively on the use of proliferation-dependent markers such as [3H]-thymidine and BuDR incorporation and the detection of growth-dependent proteins such as proliferating cell nuclear antigen (PCNA) and Ki-67. In normal tissues, the monoclonal antibody S-44 recognizes statin, a nuclear protein expressed only in nonproliferating cells. In the present study, indirect immunofluorescence microscopy using S-44 identified nuclear statin in 5.9% of a population of untreated human SK-MG-1 glioma cells in vitro. Incremental doses of the alkylating agent sarcosinamide chloroethylnitrosourea (SarCNU) induced a linear increase in the fraction of statin-positive SK-MG-1 cells. Labeling of nuclear statin with the monoclonal antibody S-44 may be a potentially useful marker of the cytotoxic effects of anticancer drugs in gliomas and other neoplastic tissues.

Antibodies, Monoclonal↗