Sheila M. Brooke

Metyrapone, an Inhibitor of Glucocorticoid Production, Reduces Brain Injury Induced by Focal and Global Ischemia and Seizures

Increasing evidence indicates that glucocorticoids (GCs), produced in response to physical/emotional stressors, can exacerbate brain damage resulting from cerebral ischemia and severe seizure activity. However, much of the supporting evidence has come from studies employing nonphysiological paradigms in which adrenalectomized rats were compared with those exposed to constant GC concentrations in the upper physiological range. Cerebral ischemia and seizures can induce considerable GC secretion. We now present data from experiments using metyrapone (an 11-β-hydroxylase inhibitor of GC production), which demonstrate that the GC stress-response worsens subsequent brain damage induced by ischemia and seizures in rats. Three different paradigms of brain injury were employed: middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia; four-vessel occlusion (4VO) model of transient global forebrain ischemia; and kainic acid (KA)-induced (seizure-mediated) excitotoxic damage to hippocampal CA3 and CA1 neurons. Metyrapone (200 mg/kg body wt) was administered systemically in a single i.p. bolus 30 min prior to each insult. In the MCAO model, metyrapone treatment significantly reduced infarct volume and also preserved cells within the infarct. In the 4VO model, neuronal loss in region CA1 of the hippocampus was significantly reduced in rats administered metyrapone. Seizure-induced damage to hippocampal pyramidal neurons (assessed by cell counts and immunochemical analyses of cytoskeletal alterations) was significantly reduced in rats administered metyrapone. Measurement of plasma levels of corticosterone (the species-typical GC of rats) after each insult showed that metyrapone significantly suppressed the injury-induced rise in levels of circulating corticosterone. These findings indicate that endogenous corticosterone contributes to the basal level of brain injury resulting from cerebral ischemia and excitotoxic seizure activity and suggest that drugs that suppress glucocorticoid production may be effective in reducing brain damage in stroke and epilepsy patients.

Memory retrieval impairment induced by hippocampal CA3 lesions is blocked by adrenocortical suppression.

There is evidence that in rats, partial hippocampal lesions or selective ablation of the CA3 subfield can disrupt retrieval of spatial memory and that hippocampal damage disinhibits hypothalamic-pituitary-adrenocortical (HPA)-axis activity, thereby elevating plasma levels of adrenocorticotropin and corticosterone. Here we report evidence that attenuation of CA3 lesion-induced increases in circulating corticosterone levels with the synthesis inhibitor metyrapone, administered shortly before water-maze retention testing, blocks the impairing effects of the lesion on memory retrieval. These findings suggest that elevated adrenocortical activity is critical in mediating memory retrieval deficits induced by hippocampal damage.

Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage

Overexpression of bcl‐2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl‐2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl‐2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron‐enriched primary hippocampal cultures, with overexpression of bcl‐2 driven by a herpes simplex virus amplicon: (i) Bcl‐2 protected strongly against glutamate, whose toxicity is at least partially ROS‐dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl‐2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate‐induced oxidative damage; (ii) Bcl‐2 was also mildly protective against the pro‐oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl‐2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl‐2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl‐2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.

Disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures

Glucocorticoids (GCs), the adrenal steroids secreted during stress, compromise the ability of hippocampal neurons to survive various necrotic insults. We have previously observed that GCs enhance the hippocampal neurotoxicity of reactive oxygen species and, as a potential contributor to this, decrease the activity of the antioxidant enzyme, glutathione peroxidase (GSPx). In this report, we have studied the possible mechanisms underlying this GC effect upon GSPx in primary hippocampal cultures and have observed several results. (i) Corticosterone (the GC of rats) decreased glutathione levels; this was predominately a result of a decrease in levels of reduced glutathione (GSH), the form of glutathione which facilitates GSPx activity. (ii) Corticosterone also decreased levels of NADPH; this may help explain the effect on GSH as NADPH is required for regeneration of GSH from oxidized glutathione. (iii) However, the corticosterone effect on total glutathione levels could not just be caused by the NADPH effect, as there were also reduced levels of oxidized glutathione. (iv) Corticosterone caused a small but significant decrease in GSPx activity over a range of glucose concentrations; this occurred under circumstances of an excess of glutathione as a substrate, suggesting a direct effect of corticosterone on GSPx activity. (v) This corticosterone effect was likely to have functional implications, in that enhancement of GSPx activity (to the same magnitude as activity was inhibited by corticosterone) by GSPx overexpression protected against an excitotoxin. Thus, GCs have various effects, both energetic and non‐energetic in nature, upon steps in GSPx biochemistry that, collectively, may impair hippocampal antioxidant capacity.

Over-expression of antioxidant enzymes protects cultured hippocampal and cortical neurons from necrotic insults.

There is now considerable knowledge concerning neuron death following necrotic insults, and it is believed that the generation of reactive oxygen species (ROS) and oxidative damage play a pivotal role in the neuron death. Prompted by this, we have generated herpes simplex virus‐1 amplicon vectors over‐expressing the genes for the antioxidant enzymes catalase (CAT) or glutathione peroxidase (GPX), both of which catalyze the degradation of hydrogen peroxide. Over‐expression of each of these genes in primary hippocampal or cortical cultures resulted in increased enzymatic activity of the cognate protein. Moreover, each enzyme potently decreased the neurotoxicity induced by kainic acid, glutamate, sodium cyanide and oxygen/glucose deprivation. Finally, these protective effects were accompanied by parallel decreases in hydrogen peroxide accumulation and the extent of lipid peroxidation. These studies not only underline the key role played by ROS in the neurotoxicity of necrotic insults, but also suggest potential gene therapy approaches.

Dexamethasone resistance among nonhuman primates associated with a selective decrease of glucocorticoid receptors in the hippocampus and a history of social instability.

We have studied some of the neuroendocrine and social correlates of dexamethasone resistance in a nonhuman primate population. Subjects were 51 male Macaca fascicularis monkeys with known behavioral histories and who had been given dexamethasone (DEX) suppression tests a week prior to killing. We compared the subset of monkeys who were most DEX responsive (post-DEX cortisol values of 3.1 +/- 0.5 micrograms/dl) versus a DEX-resistant subset (cortisol values of 9.2 +/- 2.0 micrograms/dl); we found two features that distinguished these groups: (a) DEX-resistant monkeys had significantly fewer available glucocorticoid receptor (GR) binding sites in the hippocampus; they did not differ in numbers of mineralocorticoid receptor (MR) sites in the hippocampus, nor in numbers for either receptor in the cortex or hypothalamus as a whole. (b) Animals had resided for a number of years in social groups that were either stable or were repeatedly destabilized by changing of group membership; the latter has been shown to constitute a sustained stressor. DEX-resistant animals were more than twice as likely to have come from an unstable group as were DEX-responsive monkeys. Rodent studies have shown that sustained stress can cause a selective downregulatory decrease in the numbers of hippocampal corticosteroid receptors, and that such a loss is associated with DEX resistance. The present data suggest similar associations in the primate, and may be of relevance to the DEX resistance observed in a subset of human depressives.

Endocrine modulators of necrotic neuron death.

The order of the first nine authors was determined randomly, and reflects equivalent contributions to this manuscript. Studies were made possible by support from the Adler Foundation. In recent years, there has been extraordinary progress in understanding the cellular and molecular cascades that mediate neuron death following necrotic insults. With this knowledge has come the recognition of ways in which these cascades can be modulated by extrinsic factors, altering the likelihood of subsequent neuron death. In this review, we consider the ability of a variety of hormones to modulate necrotic death cascades. Specifically, we will examine the ability of the stress hormones glucocorticoids and corticotropin‐releasing factor, of thyroid hormone, and of pre‐ischemic exposure to catecholamines to augment necrotic neuron death. In contrast, estrogen, insulin and post‐ischemic exposure to catecholamines appear to decrease necrotic neuron death. We review the heterogeneous mechanisms that are likely to mediate these hormone effects, some possible clinical implications and the therapeutic potentials of these findings.

Sleep deprivation elevates plasma corticosterone levels in neonatal rats.

Plasma corticosterone (CORT) levels were measured after short periods of sleep deprivation in rats at postnatal days 12, 16, 20, and 24. There was an age-dependent increase in basal CORT levels and sleep deprivation significantly elevated CORT at all ages compared to non-sleep deprived controls. The levels of CORT after sleep deprivation in P16, P20 and P24 animals were similar, resulting in an age-dependent decrease of the magnitude of the response. Sleep deprived P12 animals had lower levels of CORT. However, the observed response to sleep deprivation suggests that sleep loss is a significant stressor at this age. These observations suggest that younger animals are more sensitive to the effects of mild sleep deprivation than older ones.

Mechanisms of estrogenic protection against gp120-induced neurotoxicity

gp120, an HIV coat glycoprotein that may play a role in AIDS-related dementia complex (ADC), induces neuronal toxicity characterized by NMDA receptor activation, accumulation of intracellular calcium, and downstream degenerative events including generation of reactive oxygen species and lipid peroxidation. We have previously demonstrated estrogenic protection against gp120 neurotoxicity in primary hippocampal cultures. We here characterize the mechanism of protection by blocking the classical cytosolic estrogen receptors and by measuring oxidative end points including accumulation of extracellular superoxide and lipid peroxidation. Despite blocking ERalpha and ERbeta with 1 microM tamoxifen, we do not see a decrease in the protection afforded by 100 nM 17 beta-estradiol against 200 pM gp120. Additionally, 17alpha-estradiol, which does not activate estrogen receptors, protects to the same extent as 17beta-estradiol. 17beta-Estradiol does, however, decrease gp120-induced lipid peroxidation and accumulation of superoxide. Together the data suggest an antioxidant mechanism of estrogen protection that is independent of receptor binding.

Glucocorticoids exacerbate the deleterious effects of gp120 in hippocampal and cortical explants.

Abstract: Glucocorticoids (GCs), the adrenal steroids secreted during stress, can compromise the ability of hippocampal neurons to survive numerous necrotic insults. We have previously observed that GCs worsen the deleterious effects of gp120, the glycoprotein of the acquired immune deficiency syndrome virus, which can indirectly damage neurons and which is thought to play a role in the neuropathological features of human immuno‐deficiency virus infection. Specifically, GCs augment gp120‐induced calcium mobilization, ATP depletion, decline in mitochondrial potential, and neurotoxicity in fetal monolayer cultures from a number of brain regions. In the present report, we demonstrate a similar gp120/GC synergy in adult hippocampal and cortical explants. We generated explants from rats that were either adrenalectomized, adrenally intact, or intact and treated with corticosterone to produce levels seen in response to major stressors. Metabolic rates in explants were then indirectly assessed with silicon microphysiometry, and cytosolic calcium concentrations were assessed with fura‐2 fluorescent microscopy. We observed that basal levels of GCs tonically augment the disruptive effects of gp120 on metabolism in the CA1 cell field of the hippocampus and in the cortex. Moreover, raising GC concentrations into the stress range exacerbated the ability of gp120 to mobilize cytosolic calcium in a number of hippocampal cell fields. Finally, we observed that the synthetic GC prednisone had similarly exacerbating effects on gp120. Thus, GCs can worsen the deleterious effects of gp120 in a system that is more physiologically relevant than the fetal monolayer culture and in a region‐specific manner.