Biomedical subjects

J R Brawer

Publications and source records attributed to J R Brawer.

At least 19 recordsLinked to original sources

PubMed1998

Astrocyte mitochondria: a substrate for iron deposition in the aging rat substantia nigra.

Little is currently known concerning the cellular substrates for, and the mechanisms mediating the pathological deposition of, redox-active brain iron in Parkinson's disease. In various subcortical brain regions, populations of astroglia progressively accumulate peroxidase-positive cytoplasmic inclusions derived from effete, iron-laden mitochondria. In the present study, histochemical, ultrastructural, and elemental microanalytical techniques were used to demonstrate the existence of peroxidase-positive astroglia in the substantia nigra of adult rats. At 4 months of age and earlier, few GFAP-positive nigral astroglia contained small, electron-dense cytoplasmic inclusions which exhibited faint endogenous peroxidase activity (diaminobenzidine reaction product) and no detectable iron by microprobe analysis. In contrast, by 14-18 months of age, there was a significant, fourfold increase in numbers of peroxidase-positive astrocyte inclusions in the substantia nigra. The nigral gliosomes in the older animals were heterogeneously electron dense, immunoreactive for ubiquitin and a mitochondrial epitope, and often exhibited X-ray emission peaks for iron. Copper peaks were also detected in a minority of nigral gliosomes. Previous in vitro work indicated that the iron-mediated peroxidase activity in these cells promotes the bioactivation of dopamine and other catechols to neurotoxic free radical intermediates. Thus, mitochondrial sequestration of redox-active iron in aging nigral astroglia may be one factor predisposing the senescent nervous system to parkinsonism and other neurodegenerative disorders.

Aging↗

PubMed1998

Mercuric chloride induces a stress response in cultured astrocytes characterized by mitochondrial uptake of iron.

Mercury exerts a variety of toxic effects on both neurons and glia. Mercury induces aberrations in microtubules, ion channels and mitochondria presumably by binding to sulfhydryl groups. Indirect evidence further suggests that mercury targeted to mitochondria may induce iron-catalyzed oxygen radical production. We have previously shown that the mitochondria of astrocytes subjected to oxidative stress accumulate redox active transition metals that may catalyze the formation of cytotoxic oxygen free radicals. In the present study we have investigated the effect of mercuric chloride on astrocytes in monolayer culture in order to determine whether mercury accumulates in astrocytic mitochondria and whether mercury exposure triggers a stress response-associated uptake of iron. Our results indicate that mercuric chloride exposure initiates the constellation of changes in mitochondrial structure that typifies the response of these cells to oxidative stress. Energy dispersive Xray microspectroscopy demonstrates that these altered mitochondria concentrate both mercury and iron. Concurrent with these changes, mercuric chloride treatment activates transcription of the heme oxygenase-1 (HO-1) gene in a dose dependent manner, further indicating an oxidative stress response. Thus, mercury-induced stress may transform innocuous astrocytes into potentially lethal sources of cytotoxic oxygen free radicals.

Animals↗

PubMed1997

The effect of estradiol-induced hypothalamic pathology on sulfated glycoprotein-2 (clusterin) expression in the hypothalamus.

Sulfated glycoprotein-2 (SGP-2 or clusterin) is a complex multifunctional molecule that has been recently been implicated in neuronal degeneration and remodeling. We have shown that estradiol treatment results in a selective destruction of beta-endorphin neurons in the hypothalamic arcuate nucleus. We have used immunocytochemistry to determine the distribution of SGP-2 immunoreactivity in the rat hypothalamus and to assess the effects of the estradiol-induced destruction of beta-endorphin neurons on SGP-2 expression. We have found that SGP-2-immunopositive neurons normally occur in the medial preoptic area (MPOA), supraoptic nucleus (SON), paraventricular nucleus (PVN), dorsomedial nucleus (DM), and the lateral hypothalamic area (LHA) in both males and females. The neuropil appears free of label. Treatment with estradiol valerate results in the appearance of immunopositive punctate deposits in the neuropil in the MPOA, PVN and DM. The number and distribution of SGP-2-positive neurons are unaffected by estradiol treatment except in the MPOA, where there are twice as many SGP-2-positive neurons as in controls. These effects are precluded by treatment with vitamin E, with blocks the cytotoxic action of estradiol on beta-endorphin neurons. Thus, we interpret these changes as responses to the loss of beta-endorphin afferents.

Animals↗

PubMed1995

Uptake and subcellular distribution of 51Cr in Gomori-positive astrocytes in primary culture.

An unusual population of astrocytes containing Gomori-positive inclusions occurs in periventricular regions of the brain in all mammalian species. The inclusions are autofluorescent and exhibit non-enzymatic peroxidase activity. Estradiol treatment in vivo and cysteamine treatment in vitro have been shown to increase the number and size of these inclusions. Recent studies indicate that the Gomori inclusions are accumulations of autophagocytized abnormal mitochondria. The mitochondrial changes initiating Gomori inclusion formation begin with a loss of cristae. Energy dispersive X-ray microanalysis also reveals small emission peaks indicative of chromium. The appearance of chromium peaks in the initial stages of mitochondrial transformation suggests that enhanced permeability to chromium could play a causal role in generating Gomori inclusions. In the present study, we have examined the uptake and intracellular distribution of chromium during Gomori inclusion formation in cysteamine-treated cultured astrocytes. 51Cr was added to the media of glial cultures 24 hours prior to the initiation of the formation of Gomori inclusions by the addition of cysteamine. Cultures were fixed and prepared for EM radioautography at 12, 24, and 72 hours following the addition of cysteamine. 51Cr was added to control cells but they were not treated with cysteamine, and, they did not, therefore, develop Gomori inclusions. Cysteamine exposure resulted in a rapid sustained increase in radiolabel over the astrocytes. Much of the label was concentrated over mitochondria. At the late time points, label concentrated progressively over developing Gomori inclusions. These results confirm that the onset of Gomori inclusion formation coincides with increase cellular permeability to chromium and they indicate that uptake of chromium by mitochondria may play an important role in initiating development of these structures.

Animals↗

PubMed1994

The 21-aminosteroid antioxidant, U74389F, prevents estradiol-induced depletion of hypothalamic beta-endorphin in adult female rats.

A single intramuscular injection of 2 mg estradiol valerate (EV) results in neuronal degeneration and beta-endorphin depletion in the hypothalamic arcuate nucleus of adult female rats. We have hypothesized that peroxidase-positive astrocytes in this brain region oxidize estrogens and catecholestrogens to semiquinone radicals which mediate oxidative neuronal injury. In the present study, dietary administration of the potent antioxidant 21-aminosteroid, U-74389F, completely blocked EV-induced beta-endorphin depletion in the hypothalami of adult female rats. Neither EV nor 21-aminosteroid treatment had any effect on hypothalamic concentrations of neuropeptide Y and Met-enkephalin, confirming that the estradiol lesion is fairly selective for the beta-endorphin cell population. The present findings support the hypothesis that the toxic effect of estradiol on hypothalamic beta-endorphin neurons is mediated by free radicals.

Animals↗

PubMed1994

The origin and composition of peroxidase-positive granules in cysteamine-treated astrocytes in culture.

Gomori astrocytes, which are prominent in periventricular regions of the brain, contain inclusions that stain with Gomori dyes, and exhibit an orange-red autofluorescence and a non-enzymatic peroxidase activity. Recently, such astrocytes have been induced in dispersed glial cultures by exposure to cysteamine. Using these cells, we have shown that the peroxidase-positive inclusions (Gomori bodies) are multicompartmental, that iron co-localizes with the peroxidase activity, and that the iron is often segregated in one of the compartments of the body. The goal of the present study was to determine the origin and process of formation of these bodies. The results indicate that cysteamine induces aberrations in mitochondrial structure associated with the acquisition of iron and the associated peroxidase activity. Mitochondria thus transformed appear to initiate an autophagic process in which they, and adjacent structures, are sequestered. The presence of acid phosphatase activity in a number of mature Gomori bodies attests to the participation of lysosomal elements in this process. These results indicate, therefore, that the Gomori body is a complex autophagosome in which the iron-containing compartments, putatively responsible for the peroxidase activity, represent undegraded transformed mitochondria.

Acid Phosphatase↗

PubMed1994

Composition of Gomori-positive inclusions in astrocytes of the hypothalamic arcuate nucleus.

BACKGROUND: Astrocytes within the hypothalamic arcuate nucleus contain Gomori-positive inclusions that exhibit a nonenzymatic peroxidase activity. The source and composition of these Gomori-positive inclusions are currently unknown. Recent evidence, derived from cultured astrocytes, suggests that Gomori-positive inclusions may consist of autophagocytized accumulations of altered mitochondria and that the peroxidase activity is generated by iron or other metals which accumulate in these mitochondria. METHODS: The present study applies electron microscopy, energy dispersive X-ray microanalysis, and immunocytochemistry in conjunction with confocal microscopy to determine the structure and composition of Gomori-positive inclusions in vivo. RESULTS: The results indicate that Gomori-positive inclusions are heterogeneous structures often associated with microtubules and that they contain conspicuous mitochondrial components. Gomori-positive inclusions exhibit X-ray emission peaks for copper and, less often, chromium, either of which could account for the peroxidase activity. CONCLUSIONS: These results support the hypothesis that Gomori-positive inclusions are autophagosomes in which mitochondria are prominent.

Animals↗

PubMed1993

Pathologic effect of estradiol on the hypothalamus.

Estradiol provides physiological signals to the brain throughout life that are indispensable for the development and regulation of reproductive function. In addition to its multiple physiological actions, we have shown that estradiol is also selectively cytotoxic to beta-endorphin neurons in the hypothalamic arcuate nucleus. The mechanism underlying this neurotoxic action appears to involve the conversion of estradiol to catechol estrogen and subsequent oxidation to o-semiquinone free radicals. The estradiol-induced loss of beta-endorphin neurons engenders a compensatory increment in mu opioid binding in the medial preoptic area rendering this region supersensitive to residual beta-endorphin or to other endogenous opioids. The consequent persistent opioid inhibition results in a cascade of neuroendocrine deficits that are ultimately expressed as a chronically attenuated plasma LH pattern to which the ovaries respond by becoming anovulatory and polycystic. This neurotoxic action of estradiol may contribute to a number of reproductive disorders in humans and in animals in which aberrant hypothalamic function is a major component.

Animals↗

PubMed1993

Estradiol is selectively neurotoxic to hypothalamic beta-endorphin neurons.

The neurotoxic effects of estradiol on hypothalamic arcuate neurons were examined in a model of chronic estrogenization induced by means of a single injection of estradiol valerate (EV). Eight weeks after EV treatment, a 60% decrease in the total number of beta-endorphin-immunoreactive neurons was detected in the arcuate nucleus. In contrast, the numbers of neurotensin-, somatostatin-, and tyrosine hydroxylase-immunoreactive neurons were unchanged, suggesting that the effects of estradiol were selective for beta-endorphin neurons. Further evidence for the selectivity of estradiol's actions was provided by RIAs indicating decreases in hypothalamic beta-endorphin concentrations, but not in Metenkephalin or neuropeptide-Y concentrations. Cell counts performed in Nissl-stained material using unbiased stereological methods revealed a reduction in the total number of neurons in the EV-treated group compared to that in the controls. The estimated number of neurons lost (approximately 3500) corresponded precisely with the total number of beta-endorphin neurons lost (approximately 3600), as estimated using quantitative immunocytochemistry. These results confirm the selectivity of estradiol's effect on the beta-endorphin cell population and demonstrate that the observed decrease in beta-endorphin immunoreactivity reflects actual cell loss. The evidence indicates that the selective neurotoxic effect of estradiol on hypothalamic beta-endorphin neurons contributes to reproductive senescence, suggesting that steroids may participate in disruption of the biological functions that they normally facilitate.

Animals↗

PubMed1992

Monosodium glutamate-induced reductions in hypothalamic beta-endorphin content result in mu-opioid receptor upregulation in the medial preoptic area.

Estradiol valerate (EV) treatment in the rat induces a lesion of the hypothalamic arcuate nucleus, resulting in significant decreases in hypothalamic beta-endorphin. In addition, the EV treatment causes a selective increase in mu-opioid binding in the medial preoptic area (MPOA). Since beta-endorphin neurons located in the arcuate nucleus project extensively to the MPOA, we have hypothesized that the EV-induced loss of these afferents induces a compensatory upregulation of mu-opioid receptors in opioid target neurons. In order to test this hypothesis, we have utilized monosodium glutamate (MSG) treated animals as a model of beta-endorphin cell loss and hence of beta-endorphin deafferentation of the MPOA. Neonatal MSG treatment has been shown to result in the destruction of 80-90% of arcuate neurons accompanied by pronounced decreases in beta-endorphin concentrations in both arcuate nucleus and MPOA. mu-Opioid binding sites were radioautographically labeled in sections from the MPOA of sham- and MSG-injected animals using the methionine enkephalin analogue 125I-FK 33-824 and quantitated by computer-assisted densitometry. The remainder of the hypothalamus of these same animals was utilized for the determination of the beta-endorphin concentration. The hypothalami of rats treated with MSG exhibited 62% (p < 0.01) less beta-endorphin than saline-injected controls. In addition, the mean mu-opioid-binding densities in the MPOA were 24% (p < 0.05) above controls in the MSG-treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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

Thecal and interstitial cells in polycystic ovaries (PCO) in the rat.

Two different types of experimentally-induced polycystic ovaries (PCO) have been examined. A macrocystic ovarian condition is induced by estradiol valerate (EV) injection, whereas a microcystic ovarian condition is engendered with subcutaneous estradiol implants. In both of these models thecal and secondary interstitial cells were characterized using three functionally significant indices. Expression of alkaline phosphatase was evaluated immunohistochemically, hCG/LH-binding capacity was assessed by means of EM radioautography, and the size and percent cytoplasmic area of intracytoplasmic lipid were determined, in the same cells, by morphometry. In both types of ovary, thecal cells of healthy and atretic follicles stained heavily for alkaline phosphatase whereas cystic theca exhibited little or no staining. Intermittent faintly stained patches of secondary interstitial cells, as well as intensely stained spheroidal cell clusters, were most numerous in the microcystic ovary and occurred less frequently in the macrocystic ovary. Cystic thecal cells in both conditions exhibited large lipid droplets and minimal hCG binding. Lipid droplet area was minimal and hCG binding maximal in secondary interstitial cells of both types of ovary. It is concluded that specific clusters of secondary interstitial cells are important steroidogenic elements in PCO, whereas cystic theca is relatively inert.

Alkaline Phosphatase↗

PubMed1991

Alterations in gonadotropin-releasing hormone-dependent gonadotropin secretion in rats with polycystic ovaries.

A single injection of estradiol valerate (EV) to adult female rats induces a persistent anovulatory polycystic ovarian (PCO) condition. During the 8-20-wk interval following EV treatment, this condition is associated with a selective compromise of LH release, decreased pituitary content of LH, and decreased GnRH-stimulated LH secretion. A marked increase in mean plasma concentrations of LH and enhanced LH response to GnRH occur after 20 wk post-EV treatment. Despite this apparent improvement, the PCO condition remains unchanged. The present study was undertaken to elucidate the underlying causes for these spontaneous improvements in LH parameters. We reasoned that these changes may be the result of alterations in 1) pituitary GnRH receptor levels; or 2) the mode of LH secretion, i.e. GnRH-dependent versus GnRH-independent; or 3) post-GnRH receptor events. Hence, we assessed pituitary GnRH receptor concentration as well as the pituitary content of LH and FSH in rats with PCO of 9 wk and 22 wk duration. To examine the possibility of a change in the mode of LH secretion, we examined the effects of in vivo suppression of LH secretion by treatment with a GnRH antagonist [N-Ac-D-Nal1, D-Phe2,3, D-Arg6, Phe7, D-Ala10]-GnRH (GnRH-ANTAG) in the same groups of animals. Mean pituitary weights were greater in the 9-wk-PCO than in the 22-wk-PCO animals. The pituitary concentration of GnRH receptors (on either a weight or milligram pituitary-membrane protein basis) was similar in the 9-wk- and 22-wk-PCO animals. Pituitary LH and FSH contents, however, were significantly higher (5-fold and 2-fold, respectively) in 22-wk-PCO rats compared to the 9-wk-PCO animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Distribution of mu, delta, and kappa opioid receptors in the hypothalamus of the rat.

The radioautographic distribution of mu, delta and kappa opioid binding sites was examined by in vitro radioautography in the rat hypothalamus using the highly selective ligands [125I]-FK 33-824, [125I]azidoDTLET and [125I]DPDYN, respectively. Levels of mu opioid binding sites varied considerably amongst hypothalamic nuclei. mu Opioid labeling was dense in the medial preoptic area, medial preoptic nucleus, suprachiasmatic nucleus and ventromedial nucleus, whilst the supraoptic nucleus, paraventricular nucleus, arcuate nucleus and dorsomedial nucleus were devoid of labeling. Delta opioid labeling was sparse throughout most of the hypothalamus; however, moderate binding densities were detected in the suprachiasmatic and ventromedial nucleus. kappa Opioid labeling was also scant throughout the hypothalamus with the exception of the suprachiasmatic nucleus which was very densely labeled. Our results indicate that the 3 opioid receptors types are differentially distributed within the hypothalamus, although a significant overlap exists. In general, the distribution of hypothalamic opioid receptors correlates well with that of opioid-containing terminal fibers and may represent the anatomical substrate for opioid involvement in the hypothalamic regulation of autonomic, behavioral and neuroendocrine functions.

Animals↗

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

Remission of the polycystic ovarian condition (PCO) in the rat following hemiovariectomy.

The estradiol valerate-induced polycystic ovarian condition in the rat represents a normal ovarian response to aberrant endocrine stimuli. Although we have shown that removal of one polycystic ovary (hemiovariectomy) results in restoration of cyclicity and normal morphology in the remaining ovary by 1 week, nothing is known about the process of recovery or about the role of the hypothalamo-pituitary unit in initiating recovery. We have therefore examined ovaries at 3, 12, 24, 48, and 120 hours following removal of the contralateral polycystic ovaries. The ovarian content and size distribution of healthy and atretic follicles was determined, as well as the occurrence of follicular cysts, type III large follicular structures, and corpora lutea. The plasma LH pattern was also examined at a short postoperative interval. At 3 hours, there was a significant increase in mean ovarian weight that coincided with the emergence of healthy large secondary follicles. By 12 hours, there was a significant sustained diminution in the number of atretic follicles of all sizes, but the total number of healthy follicles did not increase significantly until 120 hours. The cystic follicles had all but disappeared by 120 hours because of mechanical compression by newly developing ovarian tissue. Ovarian recovery is, therefore, biphasic, consisting of a very early diminution in atresia coincident with, and perhaps caused by, a major alteration in the plasma LH pattern. The second phase is characterized by a wave of follicular recruitment and development.

Animals↗

PubMed1990

Induction of Stein-Leventhal-like polycystic ovaries (PCO) in the rat: a new model for cystic ovarian disease.

An injection of estradiol valerate (EV) in the rat produces an anovulatory polycystic ovarian (PCO) condition. Chronic estrogen exposure, produced by subcutaneously implanted, estradiol (E2)-containing chronic release capsules, results in acyclicity and in hypothalamic changes similar to those seen in the EV-injected rat. We, therefore, examined the ovarian histology and plasma gonadotropin patterns in the E2-implanted rat and found that this model exhibits a polycystic ovarian condition and a plasma gonadotropin pattern very different from those in the EV-treated model. The plasma patterns of LH and FSH are bimodal consisting of small frequent pulses as well as less frequent large episodes of long duration. The ovaries contain multiple small cysts, characterized by an extensively hypertrophied theca interna, and vast chords of hypertrophied secondary interstitial cells. In contrast, cystic follicles in EV-treated rats are fewer in number, but much larger than those in the E2-implanted animals. The cystic theca and the secondary interstitial cell clusters are also far less extensive in the EV-induced polycystic ovary. These and other differences between the two types of PCO indicate that they are produced by fundamentally different morphogenic mechanisms. The cystic ovary produced by the E2 implants is similar in appearance to that seen in the human Stein-Leventhal condition, and thus provides a new model for the study of cystic ovarian disease.

Animals↗