Vitali Vassiljev

Medium transitory oxygen-glucose deprivation induced both apoptosis and necrosis in cerebellar granule cells

Recent experiments have shown that an ischemic insult can induce both necrosis and apoptosis. A series of experiments were designed to examine the potential induction of apoptosis by oxygen-glucose deprivation (OGD) in cerebellar granule cell culture. A medium OGD (90 min) induced apoptosis in cell culture, with maximal effect 12 h after exposure, as indicated by following morphological (TdT-mediated dUTP-biotin nick end-labeling) and biochemical markers (DNA oligonucleosomal fragmentation). Mitochondrial injury (MTT assay) was among the early effects we detected during and after OGD and it was correlated with the dynamics of TUNEL positive cells. The amount of LDH release from damaged cells, associated with necrosis was increased significantly 12 h after exposure. These results indicate that medium OGD induced a rapid (<12 h) mixture of apoptosis and necrosis, followed by mainly secondary necrosis.

Neuroprotective action of group I metabotropic glutamate receptor agonists against oxygen-glucose deprivation-induced neuronal death.

The metabotropic glutamate receptor (mGluR) non-selective agonist (1S,3R)-1-aminocycloheptane-trans-1,3-dicarboxylic acid [(1S, 3R)ACPD] and group I selective receptor agonist 3, 5-dihydrophenylglycine (DHPG) effectively attenuated oxygen-glucose deprivation (OGD)-induced death of the cultured cerebellar granule cells. Furthermore, (1S,3R)ACPD (100 microM) reduced the number of apoptotic cells. Antiapoptotic action of (1S,3R)ACPD was prevented by the group I selective antagonist (RS)-1-aminoindan-1, 5-dicarboxylic acid (AIDA, 100 microM) and protein kinase C (PKC) inhibitor bisindolylmaleimide (BMI, 1 microM).

Do nuclear condensation or fragmentation and DNA fragmentation reflect the mode of neuronal death

It is generally believed that nuclear condensation and fragmentation as well as DNA fragmentation reflect the events related to the neuronal apoptosis. Our report demonstrates that severe oxygen-glucose deprivation (OGD) induced condensation and fragmentation of nuclear chromatin of neurones in primary cultures of cerebellar granule cells without intemucleosomal DNA fragmentation. DNA fragmentation detected by TUNEL assay was seen only after mild OGD or after addition of colchicine but not after severe OGD. Thus, at least in primary cerebellar granule cell cultures, the chromatin condensation and fragmentation cannot be considered as a hallmark of apoptosis but rather reflect the neuronal death despite of its form.

Small platform stress increases [3H]Ro 15-4513 binding in the cerebellum of mice

Abstract— Small platform stress was induced in male BALB/c mice by placing them on small platforms (d = 3.5 cm) surrounded by water for 24 or 72 h. This experimental model contains several factors of stress, like rapid eye movement (REM) sleep deprivation, isolation, immobilization and falling into the water. After 24 h small platform stress exposure latency to sleep was measured after the administration of the benzodiazepine receptor agonist diazepam (1.0 and 2.5 mg/kg, i.p.) and the benzodiazepine receptor inverse agonist Ro 15–4513 (1.0 mg/kg, i.p.). As could be expected, diazepam significantly shortened the latency to sleep. Surprisingly the administration of Ro 15–4513 also shortened the latency to sleep. In addition [3H]Ro 15–4513 binding was measured in the cerebellum of control and small platform stressed mice. Small platform stress for 24 h did not alter the maximal number of [3H]Ro 15–4513 binding sites (Bmax) and decreased their affinity (KD). Small platform stress for 72 h significantly increased the number of [3H]Ro 15–4513 binding sites and decreased their affinity. These effects were due to changes in diazepam‐sensitive binding. In conclusion, it could be supposed that exposure of mice to small platform stress causes changes in the function of the [3H]Ro 15–4513 binding sites, probably a shift of binding sites toward agonist conformation, that leads to changes in the effects of Ro 15–4513.

The influence of the nitric oxide synthase inhibitor L-NAME on the effects of ethanol in rats after acute ethanol administration

The aim of this work was to study the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) on the sedative and toxic effects of ethanol in rats. Ethanol at a dose of 3 g/kg, intraperitoneally induced sleep in rats (sleep time: 111.2+/-10.3 min.). Administration of the nitric oxide synthase inhibitor L-NOARG (20 and 40 mg/kg, intraperitoneally) 30 min. before ethanol significantly increased the duration of ethanol-induced sleep. L-NOARG at doses of 20 and 40 mg/kg reduced the exploratory activity of rats in the open-field test and significantly enhanced the sedative effect of ethanol in this test. It is possible that this effect is not caused by the interaction of ethanol with nitric oxide pathways but by synergistic CNS depression caused by ethanol and L-NOARG. L-NOARG (20 and 40 mg/kg) had no effect on ethanol concentrations in blood after acute ethanol administration (2 and 3 g/kg). Moreover, the combined administration of ethanol (2 g/kg) and L-NOARG (20 and 40 mg/kg) caused a decrease in the body weight of animals, observed for 14 days. Also, livers of these rats were studied for necrosis and connective tissue reaction. In histological studies L-NOARG at a dose of 40 mg/kg had no effect on hepatic necrosis caused by the acute administration of ethanol but strengthened connective tissue reaction. L-NOARG is widely used in pharmacological studies, including those concerning the effects of ethanol. However, on the basis of our data the possibility of toxic interactions with ethanol should be considered.