Sunita Rajdev

Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction

Increased expression of heat shock protein 70 (HSP70) in the brain has been extensively documented in association with a variety of insults, including ischemia, and is suggested to play a role in cell survival and recovery after ischemic injury. To more directly assess the protective role of HSP70 during ischemic brain damage, we used transgenic mice overexpressing the rat HSP70 (HSP70tg mice). In contrast to wild‐type (wt) littermates, high levels of HSP70 messenger RNA and protein were detected in brains of HSP70tg mice under normal conditions, and immunohistochemical analysis revealed primarily neuronal expression of HSP70. Heterozygous HSP70tg mice and their wt littermates were subjected to permanent focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Cerebral infarction after 6 hours of ischemia, as evaluated by Nissl staining, was significantly less in HSP70tg mice compared with wt mice. This reduction in infarction volume in HSP70tg mice was not attributable to an altered cardiovascular anatomy or to initial differences in body temperature or hemodynamic parameters. The HSP70tg mice were still protected against cerebral infarction 24 hours after permanent focal ischemia. The data suggest that HSP70 can markedly protect the brain against ischemic damage and that approaches aimed at inducing HSP70 may lead to new therapeutic interventions in cerebrovascular injuries. Ann Neurol 2000;47:782–791

Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons

Intracellular free Mg2+ concentrations ([Mg2+]i) in single rat brain neurons were measured with the Mg(2+)-sensitive fluorescent dye magfura-2. Addition of glutamate with glycine raised [Mg2+]i from 1 to more than 11 mM compared with the resting concentration of 0.5 mM, an effect mediated by N-methyl-D-aspartate receptors. Most of the increase in [Mg2+]i was independent of extracellular Mg2+, but was dependent on extracellular Ca2+. The second component of the increase induced by glutamate was independent of extracellular Ca2+, but required extracellular Mg2+ and was amplified by extracellular Na+ removal. These results indicate that regulation of [Mg2+]i by neurotransmitters such as glutamate may be important in controlling neuronal excitability.

Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons.

We describe here the properties of calcium green-5N (CG5N), a novel Ca(2+)-sensitive fluorescent probe. CG5N binds Ca2+ with an affinity of 4.29 +/- 0.67 microM. Ca2+ binding is associated with a 14.7-fold increase in fluorescence. Mg2+ binds to CG5N with an affinity of 5 mM but produces a very small change in fluorescence. Using CG5N [Ca2+]i was recorded in single cultured neurons from fetal rat forebrain. Glutamate together with glycine increased [Ca2+]i by 1.17 +/- 0.15 microM. Pretreating cells with 5 mM potassium cyanide significantly potentiated the response to glutamate, resulting in a mean increase of 8.8 +/- 2 microM. These results demonstrate that CG5N is a useful indicator for measuring very large changes in [Ca2+]i in neurons.

NMDA receptor-mediated neurotoxicity: a paradoxical requirement for extracellular Mg2+ in Na+/Ca2+-free solutions in rat cortical neurons in vitro.

Abstract: Accumulation of intracellular Ca2+ is known to be critically important for the expression of NMDA receptor‐mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg2+ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg2+ content independently of Ca2+ to investigate the potential role of Mg2+ in excitotoxicity in rat cortical neurons in vitro. In Ca2+‐free solutions in which the Na+ was replaced by N‐methyl‐d‐glucamine or Tris (but not choline), which also contained 9 mM Mg2+, exposure to 100 µM glutamate or 200 µM NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg2+ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg2+‐dependent NMDA receptor‐mediated toxicity. Thus, the increase in intracellular Mg2+ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.

Stress proteins as molecular markers of neurotoxicity

In response to many environmental and pathophysiologic stressful stimuli, cells undergo a stress response characterized by induction of a variety of proteins, including the heat shock protein family. The inducible heat shock protein 70 (hsp70) is believed to participate in an array of cellular activities, including cytoprotection. Normal brain cells have little detectable hsp70 RNA or protein. However, following a stressful condition hsp70 mRNA and protein are induced in different cell types depending on the severity and the nature of the stimulus. The induction of hsp70 protein correlates with the regional and cellular vulnerability to a particular injury as identified by standard histologic methods. The pattern of hsp70 expression differs in response to various neurotoxic stimuli, including hyperthermia, ischemia, seizures, hemorrhage, and N-methyl-D-aspartate receptor antagonist administration. Hsp70 expression is a useful marker of cellular injury and may help to identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus. Hsp70 may also play a neuroprotective role in the brain.

Acute phencyclidine neurotoxicity in rat forebrain: induction of haem oxygenase-1 and attenuation by the antioxidant dimethylthiourea

Phencyclidine and other N‐methyl‐d‐aspartate receptor antagonists are toxic to pyramidal neurons in the posterior cingulate/retrosplenial cortex of rat brain. Previous studies have shown induction of heat shock protein 70 in affected neurons. In this study, expression of haem oxygenase‐1, a heat shock protein induced by oxidative stress, was examined in rat forebrain after administration of a single intraperitoneal dose of phencyclidine (50 mg/kg). Northern and Western blot analyses of brain tissue extracts from phencyclidine‐treated rats revealed a marked induction of haem oxygenase‐1 mRNA and protein, respectively. Immunohistochemistry studies revealed that phencyclidine increased haem oxygenase‐1 immunoreactivity primarily in posterior cingulate/retrosplenial, piriform and entorhinal cortices, striatum and hippocampus. Haem oxygenase‐1 protein was induced in non‐neuronal cells, mainly astrocytes. Some microglia expressing haem oxygenase‐1 protein were also found in the posterior cingulate/retrosplenial cortex. Haem oxygenase‐1 immunoreactive astrocytes and microglia were present in close proximity to the heat shock protein 70‐positive neurons in the posterior cingulate/retrosplenial cortex following phencyclidine. Pretreatment of rats with 1,3‐dimethylthiourea, an antioxidant, significantly reduced haem oxygenase‐1 protein induction by phencyclidine. Thus, induction of haem oxygenase‐1 in glia by phencyclidine appears to be mediated mostly by oxidative stress. Experiments with the amino cupric silver stain for neuronal degeneration revealed phencyclidine‐induced neurotoxicity in the posterior cingulate/retrosplenial cortex. The number of affected neurons was significantly reduced after 1,3‐dimethylthiourea pretreatment. This suggests that the neurotoxicity of N‐methyl‐d‐aspartate antagonists is due in part to the oxidative stress and may be amenable to therapeutic interventions.

Cyclothiazide modulates AMPA receptor-mediated increases in intracellular free Ca2+ and Mg2+ in cultured neurons from rat brain

Abstract: We investigated the modulation of (±)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐induced increases in intracellular free Ca2+ ([Ca2+]i) and intracellular free Mg2+ ([Mg2+]i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA‐induced changes in [Ca2+]i were increased by 0.3–100 µM cyclothiazide, with an EC50 value of 2.40 µM and a maximum potentiation of 428% of control values. [Ca2+]i responses to glutamate in the presence of N‐methyl‐d‐aspartate (NMDA) receptor antagonists were also potentiated by 10 µM cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non‐NMDA receptors. Almost all neurons responded with an increase in [Ca2+]i to both kainate and AMPA in the absence of extracellular Na+, and these Na+‐free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC50 value of 12.0 µM. Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA‐ and glutamate‐induced increases in [Mg2+]i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non‐NMDA glutamate receptors and may provide the basis for the increase in non‐NMDA receptor‐mediated excitotoxicity produced by cyclothiazide.

Differential cerebral protein synthesis and heat shock protein 70 expression in the core and penumbra of rat brain after transient focal ischemia

OBJECTIVEThe purpose of this study was to correlate the cerebral protein synthesis (CPS) reductions in the ischemic core and penumbra with the metabolic stress response indicated by heat shock protein 70 (HSP70) synthesis. METHODSRats were subjected to 90 minutes of temporary focal cerebral ischemia produced by occlusion of the middle cerebral artery, using the endovascular suture model. Regional CPS was qualitatively evaluated, with [35S]methionine autoradiography, after reperfusion for 2 to 72 hours. The observed changes were correlated with HSP70 immunoreactivity, as assessed in the same brain sections. The ischemic core in the striatum was characterized by HSP70 expression only in endothelial and/or glial cells, with an absence of expression in neurons. The penumbra was delineated as the cortical middle cerebral artery territory region in which HSP70 was also expressed in metabolically stressed neurons. RESULTSAfter 2 hours of reperfusion, CPS was reduced to 30 ± 16% of the homologous contralateral hemisphere value in the core and to 75 ± 22% in the penumbra (P < 0.05). This difference was still present at 72 hours, when CPS values were 62 ± 21% and 98 ± 29% of the nonischemic contralateral hemisphere values in the core and penumbra, respectively (P < 0.05). CONCLUSIONPersistent inhibition of CPS in regions in which neuronal HSP70 expression is absent may distinguish core areas of infarction from penumbral regions in which neuronal HSP70 is present, which eventually recover from sublethal metabolic stress during reperfusion after temporary focal ischemia.

Fluoxetine prevents PCP- and MK801-induced HSP70 expression in injured limbic cortical neurons of rats

BACKGROUND N-Methyl-D-aspartate (NMDA) receptor antagonists, including phencyclidine (PCP) and dizocilpine (MK801), cause schizophrenialike psychosis in humans, and produce vacuolated neurons in the cingulate and retrosplenial cortices of the rat brain. Since psychotically depressed patients and schizophrenic depressed patients may require treatment with selective serotonin reuptake inhibitors (SSRIs), it is of interest to examine the relationship between SSRIs and NMDA antagonist neurotoxicity. METHODS The neurotoxicity of PCP and MK801 was assessed using heat shock protein (HSP70) immunocytochemistry and HSP70 Western blots because HSP70 is expressed in the injured, vacuolated neurons. Female rats were given fluoxetine (0, 5, 10, and 20 mg/kg IP) followed 1 hour later by MK801 (1 mg/kg IP) or PCP (50 mg/kg IP). RESULTS Pretreatment with fluoxetine (20 mg/kg IP) 1 hour before MK801 prevented the induction of HSP70 by MK801 in the cingulate and retrosplenial cortices. Pretreatment with fluoxetine (10 or 20 mg/kg IP) 1 hour before PCP also prevented the HSP70 induction by PCP. CONCLUSIONS Fluoxetine prevents the neurotoxicity of NMDA receptor antagonists in rat brain. This suggests the possibility that SSRIs could modulate psychosis, and may provide a model for examining the link between the hallucinogenic properties of PCP and lysergic acid diethylamide.

Effects of Monovalent and Divalent Cations on 3‐(+)[125I]Iododizocilpine Binding to the N‐Methyl‐d‐Aspartate Receptor of Rat Brain Membranes

Abstract: We have investigated the binding of 3‐[125I]iododizocilpine ([125I]iodo‐MK‐801) to the N‐methyl‐d‐aspartate (NMDA) receptor in well‐washed rat brain membranes. [125I]Iododizocipline binding was displaced by the following: dizocilpine > thienylphencyclidine > phencyclidine > ketamine. Binding of [125I]iododizocilpine was enhanced by glutamate, glycine, and spermidine, whose actions could be reversed by CGS‐19755, 7‐chlorokynurenate, and arcaine, respectively. [125I]Iododizocilpine binding was also enhanced by a number of divalent cations, including Ba2+, Ca2+, Mg2+, Mn2+, and Sr2+, and several monovalent cations, including Na+ and K+. These cations enhanced [125I]iododizocilpine binding by an action at the polyamine site. In addition, the inhibitory effects associated with high concentrations of these cations was markedly reduced compared to those found in previous studies with [3H]dizocilpine. Analysis of the ability of spermidine, Mg2+, and Sr2+ to alter the inhibition of [125I]iododizocilpine by arcaine gave pA2 values of 5.41, 4.47, and 4.93, corresponding to EC50 concentrations of 3.9, 34.7, and 12.0 μM, respectively, suggesting that physiological concentrations of Mg2+ may occupy the polyamine site. These results demonstrate that [125I]‐iododizocilpine is a useful probe for the NMDA receptor. Moreover, its high specific activity and relative insensitivity to the inhibitory actions of divalent cations should make [125I]iododizocilpine a valuable ligand for the study of NMDA receptors in intact cellular systems.