N. A. Andreeva

Inhibition of glutamate-induced intensification of free radical reactions by gangliosides: possible role in their protective effect in rat cerebellar granule cells and brain synaptosomes

The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (I00 μM) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na+, K+-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10–100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.

On the origin of a sustained increase in cytosolic Ca2+ concentration after a toxic glutamate treatment of the nerve cell culture

A sustained increase of cytosolic Ca2+ concentration, [Ca2+]i, (Ca2+ plateau) was induced by a 15‐min treatment with 50 μM glutamate of cultured cerebellar granule cells and hippocampal neurons in a Mg2+‐free solution. Plateau proved to be insensitive to inhibition of Na+ o/Ca2+ i exchange caused by removal external Na+ in the post‐glutamate period. A ~ 105‐fold reduction of [Ca2+]o (from 1.5 mM to 20 nM) in the post‐glutamate period caused in most cells only a slow and small decrease in [Ca2+]i, although the same low‐Ca2+ trial before glutamate treatment caused in hippocampal cells very quick blockade of spontaneous [Ca2+]i oscillation and a decrease in the basal [Ca2+]i. The results suggest that the Ca2+ plateau is due to a suppression of the Ca2+ extrusion from the cell (in particular via Na+/Ca2+ exchange) rather than from a persistent increase in Ca2+ permeability of neuronal membrane.

Neuroprotective effects of the antifungal drug clotrimazole

Pretreatment with 10 microM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 microM clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca(2+) overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca(2+) overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.

5-(N-ethyl-N-isopropyl)amiloride and mild acidosis protect cultured cerebellar granule cells against glutamate-induced delayed neuronal death

In the experiments on the primary cerebellar granule cell cultures, delayed neuronal death was induced by 15 min treatment of the cells with 50 microM glutamate. 5-(N-ethyl-N-isopropyl)amiloride (10 microM) known as a potent inhibitor of the Na+/H+ exchanger, when added to the glutamate-containing Mg(2+)-free solution caused a considerable (approximately by 40%) decrease in the number of dead cells counted 4 h after the termination of glutamate treatment. Patch-clamp experiments with freshly isolated rat hippocampal neurons have shown that the neuroprotective effect of 5-(N-ethyl-N-isopropyl)amiloride can be explained by its ability to block N-methyl-D-aspartate channels (receptors) at micromolar concentrations. A similar mechanism apparently underlies neuroprotective effect of external acidosis (reduction of pH from 7.6-7.8 to 6.7-6.8) during glutamate application. 5-(N-ethyl-N-isopropyl)amiloride (10 microM) and low pH (6.7) also proved capable of exhibiting neuroprotective effects upon application during the post-glutamate period. In this instance, however, the number of dead cells was decreased by no more than 20%. This neuroprotective effect of 5-(N-ethyl-N-isopropyl)amiloride and low pH is interpreted as resulting from inhibition of Na+/H+ exchange, since a direct blockade of N-methyl-D-aspartate receptors by 1 mM DL-2-amino-5-phosphonovalerate after termination of glutamate treatment did not attenuate the delayed neuronal death. Finally, we have established that the addition of 10 microM 5-(N-ethyl-N-isopropyl)amiloride to the cultures both during glutamate treatment and after its termination results in a complete protection of cultured cerebellar granule cells.

Expression of hypoxia-inducible factor-1 in the cochlea of newborn rats.

Hypoxia/ischemia is a major pathogenetic factor in the development of hearing loss. An important transcription factor involved in the signaling and adaptation to hypoxia/ischemia is the hypoxia-inducible factor-1 (HIF-1). To study HIF-1 expression we used an in vitro hypoxia model of explant and dissociated cultures of the stria vascularis, the organ of Corti with limbus and the modiolus from the cochlea of 3-5-day-old Wistar rats. Hypoxia differentially increased HIF-1 activity as measured by a reporter gene. Twenty-four hour hypoxia increased HIF-1 activity 14.1+/-3.5-fold in the modiolus, 9.4+/-3.0-fold in the organ of Corti with limbus, and 6.4+/-1.5-fold in the stria vascularis. The HIF-1alpha mRNA level was measured by quantitative reverse transcription polymerase chain reaction and showed a lower expression in the modiolus (1.3+/-0.2 pg/microg RNA) than in both the organ of Corti with limbus and the stria vascularis (2.7-3.2+/-1.3, P<0.01). Hypoxia had no effect on the HIF-1alpha mRNA levels. The region-specific regulation of HIF-1 expression on the transcriptional and posttranslational levels may expand the possibilities for adaptation of the cochlea to hypoxia.

Recombinant human erythropoietin prevents ischemia-induced apoptosis and necrosis in explant cultures of the rat organ of Corti.

This study was designed to evaluate the effect of recombinant human erythropoietin (rhEPO), insulin-like growth factor-1 (rhIGF-1) and epidermal growth factor (rhEGF) on ischemia-induced hair cell loss in an organotypic cochlea culture. The apical, middle and basal parts of the organs of Corti (newborn rat, postnatal days 3-5) were exposed to ischemia (3.5 h) in glucose-free artificial perilymph (pO2 10-20 mmHg) with or without growth factors. Controls were exposed to normoxia. Twenty-four hours after the onset of ischemia, the cultures were stained using tetramethyl rhodamine isothiocyanate (TRITC) phalloidin (hair cells), propidium iodide (membrane integrity) and apoptosis detection kit (DNA-fragmentation). Ischemia (3.5 h) induced a hair cell loss of 20 and 40% in the middle and basal cochlear parts, respectively, and an increase of the numbers of PI-stained and DNA-fragmented nuclei (controls 0-1, ischemia 4-7 nuclei/100 microm). The basal part was more affected than the apical one. rhEPO and rhIGF-1 significantly attenuated the ischemia-induced hair cell loss by reducing processes involved in apoptosis and necrosis. rhEPO has been in clinical use for more than a decade and found to be well tolerated. Therefore, rhEPO could be an effective drug for the prevention of hearing loss via a hair cell protective mechanism.

Expression of apoptosis-related genes in the organ of Corti, modiolus and stria vascularis of newborn rats

Cell death in the inner ear tissues is an important mechanism leading to hearing impairment. Here, using microarrays and real-time RT-PCR we analyzed expression of selected apoptosis-related genes in rats inner ear. We determined the gene expression in tissues freshly isolated from neonatal rats (3-5 days old) and compared it to that of explants cultured for 24 h under normoxic or hypoxic conditions. For the analyses, we used pooled samples of the organ of Corti (OC), modiolus (MOD) and stria vascularis (SV), respectively. We observed region-specific changes in gene expression between the fresh tissues and the normoxic culture. In the OC, expression of the proapoptotic genes caspase-2, caspase-3, caspase-6 and calpain-1 was downregulated. In the MOD, the antioxidative defense SOD-2 and SOD-3 were upregulated. In the SV, caspase-2, caspase-6, calpain-1 and SOD-3 were downregulated and SOD-2 upregulated. We speculate that these changes could reflect survival shift in transcriptome of inner ear explants tissues under in vitro conditions. With the exception of SOD-2, hypoxic culture conditions induced the same changes in gene expression as the normoxic conditions indicating that culture preparation is likely the dominating factor, which modifies the gene expression pattern. We conclude that various culture conditions induce different expression pattern of apoptosis-related genes in the organotypic cochlear cultures, as compared to fresh tissues. This transcriptional pattern may reflect the survival ability of specific tissues and could become a tempting target for a pharmacological intervention in inner ear diseases.

Menadione reduces rotenone-induced cell death in cerebellar granule neurons.

Oxidative stress has been implicated in neuronal death caused by cerebral ischemia or some neurologic disorders. Chemical hypoxia (term defining the simulation by using respiratory inhibitors) chosen as in vitro ischemic model, was induced in primary cultures of rat cerebellar granule neurons by inhibitors of mitochondrial electron transport such as rotenone or paraquat (complex I), 3-nitropropionic acid (3-NPA, complex II), antimycin A (complex III), or sodium azide (complex IV). All compounds caused neuronal death determined by trypan blue staining and MTT-test. On the other hand, neurotoxicity of rotenone and paraquat but not of 3-NPA, antimycin or azide was significantly abolished by menadione (vitamin K3, 2-methyl-1,4-naphthoquinone). This neuroprotective effect of menadione was associated with a decrease of rotenone-induced free radical production.

Dramatic effects of external alkalinity on neuronal calcium recovery following a short-duration glutamate challenge: the role of the plasma membrane Ca2+/H+ pump.

Alkalinization of the external medium has been shown to suppress Ca2+ extrusion from neurons due to inhibition of the plasmalemmal Ca2+/H+ pump. In our experiments on fura‐2‐loaded rat cerebellar granule cells and mouse hippocampal neurons, an increase in pHo from 7.4 to 8.5 following a 1‐min glutamate or NMDA challenge caused a dramatic delay in [Ca2+]i recovery which in some cases was accompanied by an additional increase in [Ca2+]i. Normalization of pHo, or removal of Ca2+ from the alkaline solution allowed [Ca2+]i to decrease rapidly again. External alkalinity did not affect the initial rapid decline in [Ca2+]i following a 25 mM K+ pulse. In cerebellar granule cells, the alkaline pHo considerably increased the 45Ca2+ uptake both at rest and following a 2‐min GLU pulse. A comparison of these effects of alkaline pHo with those produced by removal of the external Na+ led us to conclude that the Ca2+/H+ pump plays a dominant role in the mechanism of the fast Ca2+ extrusion from glutamate‐ or NMDA‐treated neurons.

Role of mitochondria in the mechanisms of glutamate toxicity

Current data on glutamate-induced functional and morphological changes in mitochondria correlating with or being a result of their membrane potential changes are reviewed. The important role of Ca2+, Na+, and H+ in the potentiation of such changes is considered. It is assumed that glutamate-induced loss of mitochondrial potential is mediated by Ca2+ overload resulting in the induction of nonspecific permeability of the inner mitochondrial membrane.