Sergei G. Levin

Neuroprotective effects of interleukin-10 and tumor necrosis factor-α against hypoxia-induced hyperexcitability in hippocampal slice neurons

In our previous experiments we have demonstrated that repeated exposures of rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked epileptiform discharges in CA1 pyramidal neurons. The aim of this study was to investigate the comparative effects of interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-alpha) on the hyperexcitability of CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The method of field potentials measurement in CA1 region of hippocampal slices have been described in our previous work [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466]. The principal results of our work are summarized as follow. Pro-inflammatory cytokine TNF-alpha (0.8, 4 and 20 ng/ml) and anti-inflammatory cytokine IL-10 (1 and 10 ng/ml) significantly reduced the hyperexcitability in CA1 pyramidal neurons induced by brief episodes of hypoxia in the rat hippocampal slices. The neuroprotective effects of IL-10 and TNF-alpha against the hypoxia-induced hyperexcitability were mediated by anti-hypoxic actions of these cytokines through, possibly, mechanism of preconditioning.

Interleukin-10 modulates [Ca2+]i response induced by repeated NMDA receptor activation with brief hypoxia through inhibition of InsP3-sensitive internal stores in hippocampal neurons

The goal of this study was to evaluate an effect of interleukin-10 (IL-10) on the Ca(2+) response induced by repeated NMDA receptor activation with brief hypoxia in cultured hippocampal neurons. We focused on the importance of internal Ca(2+) stores in the modulation of this Ca(2+) response by IL-10. To test this, we compared roles of InsP(3)- and ryanodine-sensitive internal stores in the effects of IL-10. Measurements of intracellular cytosolic calcium concentration ([Ca(2+)](i)) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. Repeated episodes of NMDA receptor activation with brief hypoxia induced the spontaneous (s) [Ca(2+)](i) increases about 3 min after each hypoxic episode. The amplitude of the s[Ca(2+)](i) increases was progressively enhanced from the first hypoxic episode to the third one. IL-10 (1 ng/ml) abolished these s[Ca(2+)](i) increases. Exposure of cultured hippocampal neurons with thapsigargin (1 μM) or an inhibitor of phospholipase C (U73122, 1 μM) for 10 min also abolished the s[Ca(2+)](i) increases. On the other hand, antagonist of ryanodine receptors (ryanodine, 1 μM) did not affect this Ca(2+) response. These studies appear to provide the first evidence that Ca(2+) release from internal stores is affected by anti-inflammatory cytokine IL-10 in brain neurons. It is suggested that these data increase our understanding of the neuroprotective mechanisms of IL-10 in the early phase of hypoxia.

Repeated brief episodes of hypoxia modulate the calcium responses of ionotropic glutamate receptors in hippocampal neurons

The aim of this study was to evaluate the intracellular cytosolic calcium concentration ([Ca(2+)](i)) changes induced by activation of ionotropic glutamate receptors in cultured hippocampal neurons after repeated brief episodes of hypoxia. To investigate what kinds of ionotropic glutamate receptors are involved we used specific agonists for AMPA- and NMDA-type glutamate receptors. Measurements of [Ca(2+)](i) in cultured hippocampal neurons were made by imaging Fura-2AM loaded hippocampal cells. In the rat hippocampal slice method, field potential measurements in CA1 pyramidal neurons were used. The main result of our study is that brief hypoxic episodes progressively depress the [Ca(2+)](i) increases induced by agonists of AMPA and NMDA glutamate receptors in cultured hippocampal neurons. An effectiveness of this depression is increased from the first hypoxic episode to the third one. Hypoxic preconditioning effect is observed during 10-20 min after termination of hypoxic episode and depends on [Ca(2+)](i) response amplitudes to agonists before hypoxia. In contrast to AMPA receptor activation, NMDA receptor activation before hypoxia induce the spontaneous [Ca(2+)](i) increase about 3 min after each hypoxic episode. These spontaneous [Ca(2+)](i) increases may be an indicator of the development of posthypoxic hyperexcitability in hippocampal neurons. Our results suggest that brief hypoxia-induced depression of the glutamate receptor-mediated [Ca(2+)](i) responses contributes to the development of rapid hypoxic preconditioning in hippocampal CA1 neurons.

Anti-inflammatory cytokines, TGF-β1 and IL-10, exert anti-hypoxic action and abolish posthypoxic hyperexcitability in hippocampal slice neurons: Comparative aspects

The aim of this study was to investigate the comparative effects of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) on the repeated brief hypoxia-induced alterations in the activity of hippocampal slice CA1 pyramidal neurons. The method of field potentials measurement in CA1 region of hippocampal slices was used. The principal results of our work are summarized as follow. 1. TGF-β1 reduces the depressive effect of brief hypoxia on the population spike amplitude more effectively than IL-10. 2. During TGF-β1 exposure (in contrast to IL-10), three 3-min hypoxic episodes do not induce the rapid hypoxic preconditioning. 3. TGF-β1 and IL-10 equally abolish posthypoxic hyperexcitability induced by repeated brief episodes of hypoxia in CA1 pyramidal neurons. These findings indicated that TGF-β1 and IL-10 are able to evoke anti-hypoxic effect and abolish the development of posthypoxic hyperexcitability induced by repeated brief hypoxic episodes in hippocampal CA1 pyramidal neurons. Our results also demonstrated that TGF-β1 reduced the effectiveness of hypoxia to depress neuronal activity more effectively than IL-10. We suggest that the present findings allow to explain the certain neuroprotective mechanisms of IL-10 and TGF-beta1 in the early phase of hypoxia and indicate that a therapeutic anti-inflammatory approach using these substances can provide neuroprotection in the brain hypoxic conditions.

Comparative roles of ATP-sensitive K+ channels and Ca2+-activated BK+ channels in posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro.

The aim of this study was to investigate the comparative effects of glibenclamide (GC), a selective blocker of K(+)(ATP) channels, and iberiotoxin (IbTX), a selective blocker of BK(+)(Ca) channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. In contrast to GC (10 microM), IbTX (10nM) significantly abolished both posthypoxic hyperexcitability and rapid hypoxic preconditioning induced by brief hypoxic episodes. These effects of IbTX did not depend on its ability to reduce the hypoxia-induced decrease of population spike (PS) amplitude during hypoxic episodes since GC (10 microM), comparatively with IbTX (10nM), significantly reduced the depressive effect of hypoxia on the PS amplitude during hypoxic episodes but did not abolish both posthypoxic hyperexcitability and rapid hypoxic preconditioning in CA1 pyramidal neurons. Our results indicated that BK(+)(Ca) channels, in comparison with K(+)(ATP) channels, play a more important role in such repeated brief hypoxia-induced forms of neuroplasticity in hippocampal CA1 pyramidal neurons as posthypoxic hyperexcitability and rapid hypoxic preconditioning.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca2+ response

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.

Apamin, a selective blocker of SKCa channels, inhibits posthypoxic hyperexcitability but does not affect rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the effects of apamin, a selective blocker of SK(Ca) channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. Application of apamin (50nM) to the hippocampal slices during hypoxic episodes significantly abolished posthypoxic hyperexcitability induced by brief hypoxic episodes. However, in contrast to our previous results with iberiotoxin, a selective blocker of BK(Ca) channels, apamin significantly enhanced the depressive effect of brief hypoxia on the PS amplitude during hypoxic episode and did not abolish the rapid hypoxic preconditioning in CA1 pyramidal neurons. Present results indicate that SK(Ca) channels, along with previously implicated BK(Ca) channels, play an important role in the development of posthypoxic hyperexcitability induced by brief hypoxic episodes in CA1 pyramidal neurons. However, SK(Ca) channels, in contrast to the BK(Ca) channels, are not involved in the rapid hypoxic preconditioning in CA1 hippocampal region in vitro.

The Effects of Interleukin-10 on the Development of Epileptiform Activity in the Hippocampus Induced by Transient Hypoxia, Bicuculline, and Electrical Kindling

The comparative effects of the anti-inflammatory cytokine interleukin-10 on the development of epileptiform activity were studied in hippocampal field CA1 neurons in different models of epileptogenesis not accompanied by visible morphological lesions in brain cells: 1) a model of hypoxic kindling in rat hippocampal slices; 2) a disinhibitory model of epileptogenesis in rat hippocampal slices using the GABAA receptor blocker bicuculline; and 3) a partial electrical kindling model in intact rats. Interleukin-10 (1 ng/ml) blocked the development of post-hypoxic hyperexcitability of field CA1 pyramidal neurons in hippocampal slices, decreasing the effectiveness of hypoxia in suppressing neuron activity during the hypoxic episode. Interleukin-10 had no effect on the initiation of epileptiform activity in pyramidal neurons induced by the proconvulsant bicuculline. Single intrahippocampal injections of interleukin-10 at a dose of 1 ng in 5 μl suppressed the development of focal convulsions (“ictal” discharges) at the stimulation site in partial kindling in freely moving animals for several hours after administration. However, this cytokine had no effect on the duration of the “interictal” component of focal afterdischarges or on the severity of behavioral seizures. These results show that the anti-inflammatory cytokine interleukin-10, at the concentrations used here, has not only antihypoxic activity, but also a protective effect in relation to the initiation of the “ictal,” but not the “interictal” component of epileptiform activity in hippocampal neurons.

Interleukin-10 prevents the hypoxia-induced decreases in expressions of AMPA receptor subunit GluA1 and alpha subunit of Ca2+/calmodulin-dependent protein kinase II in hippocampal neurons

The goal of this study is to evaluate the effects of anti-inflammatory cytokine interleukin-10 (IL-10) on the repeated brief hypoxia-induced changes in expressions of AMPA receptor subunit GluA1 and α- and β-subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). The hypoxia-induced changes in the rat hippocampal slice CA1 neuronal activities were investigated by the method of field potentials recording. Subunit-specific antibodies staining of Western blots of hippocampal slice homogenates to characterize the receptor subunit GluA1 and α- and β-subunit of CaMKII were used. IL-10 (1ng/ml) abolished the development of posthypoxic hyperexcitability in the CA1 pyramidal neurons induced by repeated brief hypoxia. This neuroprotective effect of IL-10 was rapidly developed within 10min after hypoxic episodes and accompanied by reversions of the hypoxia-induced decreases in expressions of AMPA receptor subunit GluA1 and α-subunit of CaMKII. These findings provide some evidence about existence of the novel mechanism underlying the rapid neuroprotective effect of anti-inflammatory cytokine IL-10 against hypoxia-induced neurological deteriorations.

Developmental changes in hyperexcitability of CA1 pyramidal neurons induced by repeated brief episodes of hypoxia in the rat hippocampal slices

We have previously demonstrated that repeated exposure of adult rat hippocampal slices to brief episodes of hypoxia induce a sustained decrease in the threshold of stimulus-evoked population spike discharges in CA1 pyramidal neurons [O. Godukhin, A. Savin, S. Kalemenev, S. Levin, Neuronal hyperexcitability induced by repeated brief episodes of hypoxia in rat hippocampal slices: involvement of ionotropic glutamate receptors and L-type Ca2+ channels, Neuropharmacology 42 (2002) 459-466, S.V. Kalemenev, A.V. Savin, S.G. Levin, O.V. Godukhin, Long-term potentiation and epileptiform activity induced by brief hypoxic episodes in CA1 area of the rat hippocampal slices. Russ. Physiol. J. 86 (2000) 1676-1681]. In the present study, using the above-mentioned in vitro model of epileptogenesis, we compared the developmental changes in hypoxia-induced hyperexcitability of CA1 neuronal network in the rat hippocampal slices prepared from three age rat groups: postnatal days (P) 13-14 (young), P60-70 (adult) and P600-650 (old). Furthermore, we were interested in learning about an age dependence of the hypoxia-induced changes in the efficacies of glutamatergic transmission and paired-pulse inhibition in CA3-CA1 synapses that may underlie ontogenetic differences in seizure susceptibility in hippocampal network. The principal results of this work are summarized as follow. In comparison with P60-70 hippocampal slices, CA1 pyramidal neurons in P13-14 and P600-650 slices showed intrinsically (without repeated brief hypoxa) an increased propensity to generate epileptiform stimulus-evoked population spike discharges. However, in contrast to adult and old animals, repeated brief episodes of hypoxia are incapable to induce a sustained decrease in the threshold of stimulus-evoked population spike discharges in CA1 pyramidal neurons of hippocampal slices prepared from of P13-14 rats, though they transform paired-pulse inhibition to paired-pulse facilitation and induce hypoxic LTP in CA3-CA1 synapses. The role of some other factors in the developmental changes in hyperexcitability of CA1 pyramidal neurons in response to repeated brief episodes of hypoxia is discussed.