Neonatal Proinflammatory Stress Induces Accumulation of Corticosterone and Interleukin-6 in the Hippocampus of Juvenile Rats: Potential Mechanism of Synaptic Plasticity Impairments.

Infectious diseases in early postnatal ontogenesis can induce neuroinflammation, disrupt normal central nervous system development, and contribute to pathogenesis of cerebral pathologies in adults. To study long-term consequences of such early stress, we induced neonatal proinflammatory stress (NPS) by injecting bacterial lipopolysaccharide into rat pups on postnatal days 3 and 5 and then assessed the levels of corticosterone, proinflammatory cytokines and their mRNAs, and neurotrophins and their mRNAs in the hippocampus and neocortex of the one-month-old animals. Long-term potentiation (LTP) was studied in hippocampal slices as an index of synaptic plasticity. NPS-induced impairments of LTP were accompanied by the accumulation of corticosterone and IL-6 in the hippocampus. In the neocortex, a decrease in exon IV BDNF mRNA was detected. We suggest that excessive corticosterone delivery to hippocampal receptors and proinflammatory changes persisting during brain maturation are among the principal molecular mechanisms responsible for NPS-induced neuroplasticity impairments.

Content of mRNA for NMDA glutamate receptor subunits in the frontal cortex and striatum of rats after morphine withdrawal is related to the degree of abstinence.

We studied the expression of mRNA for genes, whose protein products regulate the glutamate/NO/cGMP signal cascade in the frontal cortex, striatum, midbrain, and hippocampus of rats with various degrees of spontaneous morphine withdrawal syndrome. The concentration of Grin2a mRNA (subunit of the NMDA glutamate receptor) in the frontal cortex increased mainly in animals with severe abstinence. By contrast, the expression of mRNA for the Grin2b subunit in the striatum decreased in animals with mild abstinence. Variations in the content of mRNA for other products of the cascade (isoforms of NO-dependent guanylate cyclase and cGMP-dependent protein kinase) after morphine withdrawal were not related to the degree of abstinence. Our results suggest that the region-specific expression of mRNA for certain subunits of the NMDA glutamate receptor after morphine withdrawal can determine the degree of abstinence.

[Evaluation of antiepileptic effects of cortexin in a model of convulsions].

We studied antiepileptic effects of cortexin administered in doses 0,015, 0,15 and 1,0 mg/kg intraperitoneally in solution or intranasally in the complex with nanoparticles in a model of acute and chronic convulsions in rats induced by pentylenetetrazole. In the model of epileptic status, the long-term preliminary administration of cortexin had no effect on convulsions while in the model of chronic convulsions (temporal epilepsy), cortexin had a marked dose-dependent antiepileptic effect. The influence of cortexin on neuroplasticity and its clinical potential are discussed.

Activities of some caspase cascade enzymes and myocardial contractility in experimental left ventricular focal ischemia.

Focal left ventricular ischemia was modeled in male Chinchilla rabbits. Activities of caspase-3 and caspase-8 in the left and right ventricular myocardium and myocardial contractility were studied after 1, 3, and 5 days. Caspase-3 activity increased significantly in the left ventricular peri-infarction zone and right ventricular myocardium, while caspase-8 activity did not differ from the control. Left ventricular contractility decreased significantly and the hemodynamic load of the right ventricle sharply increased. These results attest to induction of the internal (mitochondrial) pathway of apoptosis in myocardial cells most likely caused by left ventricular hypoxia and right ventricular overload.

Effects of selective anxiolytic afobazole on active caspase-3.

We studied the effects of afobazole on apoptosis through active caspase-3. Afobazole in a final concentration of 10(-8) M inhibits hyperactivation of effector apoptotic caspase-3 in HT-22 cell culture under conditions of glutamate toxicity.

[Assessment of caspase-3 activity in rabbit myocardial tissue during experimental hemodynamic overload of the left ventricle of the heart].

It's well known that chronic overload of the cardiac left ventricle is accompanied by an increase in the cardiomyocyte apoptosis rate. However direction and extent of programmed cell death changes under an acute overload of the left ventricle still requires detailed investigation. Caspase-3 activity has been investigated in myocardium of rabbits on the 1, 3 and 5 days after modeling of left ventricle hemodynamic overload caused by surgical narrrowing of the ascending aorta. Control group included intact animals. It was found that caspase-3 activity significantly increased in both ventricles on day 1; it increased more than twofold above controls on day 3; it began to decrease by day 5. On the basis of the obtained data it was concluded that: an acute hemodynamic overload of the left ventricle is a cause of apoptosis acceleration in the myocardial tissue of both cardiac ventricles during first days of the investigated process.

Changes in cell proliferation in the subventricular zone of the brain in adult rats given beta-amyloid peptide (25-35).

The effects of intracerebroventricular administration of fragment (25-35) of beta-amyloid peptide [Abeta(25-35)] on cell proliferation in the subventricular zone of the dentate gyrus of the hippocampus in adult rats were analyzed. Animals received doses of 15 nmol of pre-aggregated Abeta(25-35) or the Abeta(35-25) control peptide, or solvent (sterile water) into the lateral ventricles. On post-injection days 1-5, rats received intraperitoneal injections of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU). BrdU incorporated into DNA was detected immunohistochemically on frontal brain sections six and 12 days after peptide administration. At six days, the numbers of BrdU-containing cells in the subventricular zone showed no differences between the study groups. At 12 days, the total number of BrdU-positive cells decreased significantly in all study groups. At the same time, the number of labeled cells in rats given Abeta(25-35) was significantly greater in this brain zone than in animals given water or the control peptide. Thus, Abeta(25-35) significantly increased cell proliferation in the subventricular zone after intracerebroventricular administration.

A secreted caspase-3-substrate-cleaving activity at low pH belongs to cathepsin B: a study on primary brain cell cultures.

The cysteine proteases caspase-3 and cathepsins are involved in both neuronal plasticity and neuropathology. Using primary neuroglial and glial cerebellar cultures, the pH dependence of cleavage of a synthetic caspase-3 substrate, Ac-DEVD-AMC, was studied. At acidic pH, cathepsin B cleaved Ac-DEVD, this activity being significantly higher than that of caspase-3 at pH 7.4. This activity is blocked by peptide inhibitors of both caspase-3 and cathepsin B. Substitution of culture medium for balanced salt solution stimulated cathepsin B secretion in both types of cultures. Ischemia (oxygen-glucose deprivation) significantly decreased secretion of cathepsin B activities into the culture medium.

Caspase-3 activity in hippocampal slices reflects changes in synaptic plasticity.

Electrophysiological measures of the functional activity of neurons in field CA1 in conditions of paired-pulse stimulation of Schäffer collaterals were performed in relation to the involvement of caspase-3 in mediating neuroplasticity; the relationship between functional activity and caspase-3 activity in hippocampal slices from Wistar rats was addressed. Enzyme activity was assessed in each individual slice at the end of the electrophysiological experiment. The results obtained here showed that the highest level of enzyme activity was seen when the efficiency of interneuronal interactions decreased. Nerve cell excitability showed no changes; interactions increasing synaptic efficiency, particularly in paired-pulse stimulation, produced normal response amplitudes. Further deterioration of the functional state of slices and impairments in spike generation were accompanied by increases in caspase-3 activity to the normal level. Increases in the activity of another proteinase, cathepsin B, were generally seen in any deviation from normal functioning, though there was no correlation with any of the electrophysiological parameters. It is suggested that high caspase-3 activity in slices is linked with neuroplastic processes in synapses and has no direct relationship to nerve cell apoptosis.

Contractile function of the heart and the state of some stages of lipid metabolism during acute diphtheritic intoxication.

Acute diphtheritic intoxication was modeled in rabbits by intravenous administration of native diphtherin (0.3 minimal lethal dose per 1 kg body weight). The contractile function of the left and right ventricles (peak systolic pressure under conditions of basal hemodynamics and during 5-sec occlusion of the aorta and pulmonary artery, respectively) was estimated 1, 3 and 5 days after the start of the pathological process, the intensity of lipid peroxidation was evaluated by measuring the content of TBA- reactive products in the myocardium. Impairment of the contractile function of both ventricles was observed in the course of intoxication. The level of TBA-reactive products in the left ventricle significantly decreased on day 1, but then returned to normal. Thus, impairment of the contractile function of the left ventricle at the early stages of diphtheritic intoxication is not mediated by activation of lipid peroxidation in cardiomyocytes.

[Caspase-3 activity in the rat hippocampal slices reflects changes in synaptic plasticity].

Considering the involvement of caspase-3 in neuronal plasticity, we studied caspase-3 activity in the rat hippocampal slices, and electrophysiological characteristics of extracellular responses to paired-pulse stimulation of Schaffer's collaterals in the CA1 subfield of hippocampus. Caspase-3 activity was measured after electrophysiological recording in each slice separately. Maximal caspase-3 activity was observed in the slices with low responsiveness to single afferent stimulation indicative of decreased efficacy of interneuronal interaction. This phenomenon is unrelated to depression of neuronal excitability since paired-pulse stimulation increases the synaptic efficacy to second stimulus thus restoring population spike amplitudes to normal values. In "damaged" slices with impaired spike generation up to disappearing spikes to both stimuli, caspase-3 activity was close to the normal level of the "healthy" slices. The activity of another proteinase, cathepsin B, was increased in the "damaged" slices, no correlation with the modifications of electrophysiological indices being detected. Our data suggest that high caspase-3 activity in hippocampal slices is involved in maintenance of synaptic plasticity but not necessarily related to apoptosis.

Brain cathepsin B cleaves a caspase substrate.

We show that an enzyme exists in rat brain capable of cleaving the caspase-3 specific peptide substrate Ac-DEVD-AMC at low pH. The enzyme shows properties of a cysteine protease and is localized, predominantly, in lysosomes. We have purified this enzyme from rat brain and identified it by MALDI-TOF MS. The enzyme possessing "acidic" DEVDase activity in rat brain appears to be cathepsin B. It remains obscure, whether cathepsin B participates in cleavage of caspase-3 substrates in vivo. We suggest that under certain conditions (e.g. in hypoxia) cathepsin B participates in cleavage of caspase-3 substrates in brain cells.

[Nitric oxide synthase activity and nitrates/nitrites level in brain regions during the spontaneous morphine withdrawal in rats].

Nitric oxide synthase (NOS) activity and nitrate/nitrites (NO(x)-) concentrations were measured in brain regions of rats during the spontaneous morphine withdrawal. Male Wistar rats were injected intraperotoneally with morphine hydrochloride at increasing doses (10-100 mg/kg) during 6 days twice a day. Thirty six hours after the last injection the severity of the spontaneous morphine withdrawal syndrome was determined by specific autonomic and locomotor indices. Both NOS activity and NO(x)- levels increased in the midbrain and the hippocampus, decreased in the striatum and the hypothalamus, and did not change in the cerebral cortex and the brain stem. NO(x)- concentrations in the cerebellum did not change, while NOS activity decreased. Both NOS activities and NO(x)- concentrations in the cerebral cortex, striatum, midbrain, and cerebellum correlated with withdrawal syndrome severity on the whole, and with the specific signs of abstinence.

Caspase-like activity is essential for long-term synaptic plasticity in the terrestrial snail Helix.

Although caspase activity in the nervous system of mollusks has not been described before, we suggested that these cysteine proteases might be involved in the phenomena of neuroplasticity in mollusks. We directly measured caspase-3 (DEVDase) activity in the Helix lucorum central nervous system (CNS) using a fluorometrical approach and showed that the caspase-3-like immunoreactivity is present in the central neurons of Helix. Western blots revealed the presence of caspase-3-immunoreactive proteins with a molecular mass of 29 kDa. Staurosporin application, routinely used to induce apoptosis in mammalian neurons through the activating cleavage of caspase-3, did not result in the appearance of a smaller subunit corresponding to the active caspase in the snail. However, it did increase the enzyme activity in the snail CNS. This suggests differences in the regulation of caspase-3 activity in mammals and snails. In the snail CNS, the caspase homolog seems to possess an active center without activating cleavage typical for mammals. In electrophysiological experiments with identified snail neurons, selective blockade of the caspase-3 with the irreversible and cell-permeable inhibitor of caspase-3 N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone prevented development of the long-term stage of synaptic input sensitization, suggesting that caspase is necessary for normal synaptic plasticity in snails. The results of our study give the first direct evidence that the caspase-3-like activity is essential for long-term plasticity in the invertebrate neurons. This activity is presumably involved in removing inhibitory constraints on the storage of long-term memory.

Central administration of a caspase inhibitor impairs shuttle-box performance in rats.

Recent studies suggest that caspase-3-mediated mechanisms are essential for neuronal plasticity. N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val- Asp(OMe)-fluoromethyl ketone (z-DEVD-fmk), a caspase inhibitor with predominant specificity toward caspase-3, has been shown to block long-term potentiation in hippocampal slices. Intrahippocampal infusion of a caspase-3 inhibitor to rats has been shown to significantly impair spatial memory in the water maze. The present work was designed to study whether i.c.v. administration of a caspase-3 inhibitor z-DEVD-fmk impairs learning in other tasks related to specific forms of memory in rats. The rats received bilateral injections of z-DEVD-fmk or N-benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (z-FA-fmk) ("control" peptide) at a dose of 3 nmol. Administration of z-DEVD-fmk significantly decreased the number of avoidance reactions in some blocks of trials in the active avoidance (shuttle box) learning, while z-FA-fmk had no effect as compared with intact rats. However, only a slight effect of the caspase inhibitor across the session was found. z-DEVD-fmk impaired development of some essential components of the two-way active avoidance performance, such as escape reaction, conditioned fear reaction, and inter-trial crossings. Measurement of caspase-3 activity in rat brain regions involved in active avoidance learning revealed most expressed z-DEVD-fmk-related inhibition of the enzyme activity (about 30%) in the fronto-parietal cortex. A similar effect was close to significant in the hippocampus, but not in the other cerebral structures studied. In primary cultures of cerebellar neurons z-DEVD-fmk (2-50 microM) inhibited caspase-3 activity by 60-87%. We suggest that moderate inhibition of caspase-3 resulting from the central administration of z-DEVD-fmk to rats may impair active avoidance learning. Taking into account previous data on the involvement of neuronal caspase-3 in neuroplasticity phenomena we assume that the enzyme may be important for selected forms of learning.

[Effects of morphine withdrawal on the indices of free radical homeostasis and nitric oxide system in rat liver and thymus]. / Vliianie sindroma otmeny morfina na pokazateli svobodnoradikal'nogo gomeostaza i sistemu oksida azota v pecheni i timuse krys.

Indices of oxidative stress, nitric oxide (NO) metabolism as well as the activity of caspase-3, an important enzyme of apoptotic cell death, were measured during the morphine withdrawal syndrome in liver and thymus of rats. Male Wistar rats were administered with morphine hydrochloride (i.p., at increasing doses from 10 to 100 mg/kg, twice a day, for 6 days). Thirty-six hours after the last administration the withdrawal syndrome was monitored using the specific autonomic and locomotor indices. During this period, weights of body and thymus significantly decreased. Oxidative stress in liver was accompanied by an increase in aspartate aminotransferase and gamma-glutamyl transferase in blood serum. No signs of oxidative stress could be demonstrated in thymus. The activity of the Ca(2+)-dependent isoform of nitric oxide synthase (NOS) in liver increased, while, the activity of the Ca(2+)-independent NOS diminished, the total activity of NOS in liver and thymus remained unchanged. The concentration of nitrates/nitrites in blood was decreased, in thymus increased, and in liver unchanged. Caspase-3 activity changed neither in liver, nor in thymus. The results are discussed from the perspective of possible antioxidant and antiapoptotic role of NO during morphine withdrawal syndrome.

Nitric oxide synthase activity in Fasciola hepatica: a radiometric study.

The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(omega)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica.

Single intracerebroventricular administration of amyloid-beta (25-35) peptide induces impairment in short-term rather than long-term memory in rats.

Ample experimental evidence indicates that intracerebral injection or infusion of amyloid-beta peptides (Abeta) to rodents induces learning and memory impairments as well as neurodegeneration in brain areas related to cognitive function. In the present study, we assessed the effects of a single intracerebroventricular (i.c.v.) injection of aggregated Abeta fragment (25-35) at a dose of 15nmol/rat on short-term and long-term memory in rats during the 6-month post-surgery period. The results demonstrate that Abeta(25-35)-induced memory impairments in spontaneous alternation behavior in a Y-maze at 17, 36, and 180 days after the surgery as well as in a social recognition task 110 days post-surgery. Abeta(25-35) also impaired spatial memory in an 8-arm radial maze, but did not influence performance of the step-down passive avoidance task. These results suggest that Abeta(25-35) preferably induces impairments of spatial and non-spatial short-term (working) memory rather than long-term memory in rats.

Administration of aggregated beta-amyloid peptide (25-35) induces changes in long-term potentiation in the hippocampus in vivo.

Intracereroventricular administration of aggregated beta-amyloid protein fragment (25-35) (7.5 nmol/ventricle) was followed one month later by significant changes in the dynamics of long-term potentiation in the hippocampus in vivo, expressed as powerful and stable increases in the amplitude of evoked potentials. This phenomenon may be associated with oxidative stress in the hippocampus, which has previously been demonstrated in this model, and, thus, with disturbances in ion homeostasis.