Interleukin-10 and PD150606 modulate expression of AMPA receptor GluA1 and GluA2 subunits under hypoxic conditions.

The goal of this study was to evaluate the effects of anti-inflammatory cytokine, interleukin-10 (IL-10), and calpain inhibitor, PD150606, on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits in rat hippocampal slices exposed to repeated brief hypoxic episodes. We studied both individual and combinatory effects of PD150606 and IL-10 on the expression of AMPA receptor subunits under hypoxic conditions for GluA1 and GluA2 as well as their phosphorylated forms - pSer831-GluA1 and pSer880-GluA2. Additionally, we studied whether brief hypoxic episodes and IL-10 may affect mRNA expression of transcriptional factors such as hypoxia-inducible factor-1α and nuclear factor κB (NF-κB). Western blotting analysis of hippocampal slice homogenates revealed that IL-10 and PD150606, both individually and in combination, ameliorate hypoxia-induced decrease in the expression of GluA1 and pSer831-GluA1, with different level of efficiency measured at 10, 50, and 90 min after hypoxia induction. Interestingly, brief hypoxic episodes did not induce any changes in the expression of GluA2 and pSer880-GluA2 subunits, whereas PD150606 showed biphasic effect, decreasing the expression of GluA2 and pSer880-GluA2 at 10 min and potentiating it at 90 min after hypoxia induction. IL-10 alone did not show any effect but was able to reverse PD150606 action on the expression of pSer880-GluA2 at 10 min and further potentiated it for GluA2 at 90 min after hypoxia. Finally, PCR analysis revealed that modulation of GluA1 and GluA2 expressions by hypoxia, and IL-10 was not associated with changes in the expression of hypoxia-inducible factor-1α and nuclear factor-κB (NF-κB) transcriptional factors.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca²âº 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.

Interleukin-10 prevents the hypoxia-induced decreases in expressions of AMPA receptor subunit GluA1 and alpha subunit of Ca(2+)/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 ß-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 ß-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.

Interleukin-10 modulates [Ca2+]i response induced by repeated NMDA receptor activation with brief hypoxia through inhibition of InsP(3)-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.

Anti-inflammatory cytokines, TGF-ß1 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 ß1 (TGF-ß1) 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-ß1 reduces the depressive effect of brief hypoxia on the population spike amplitude more effectively than IL-10. 2. During TGF-ß1 exposure (in contrast to IL-10), three 3-min hypoxic episodes do not induce the rapid hypoxic preconditioning. 3. TGF-ß1 and IL-10 equally abolish posthypoxic hyperexcitability induced by repeated brief episodes of hypoxia in CA1 pyramidal neurons. These findings indicated that TGF-ß1 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-ß1 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.

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.

Apamin, a selective blocker of SK(Ca) 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.

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.

Neuroprotective effects of interleukin-10 and tumor necrosis factor-alpha 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.

Oxygen-induced DNA damage in freshly isolated brain cells compared with cultured astrocytes in the Comet assay.

Brain cells are continuously exposed to reactive oxygen species generated by oxidative metabolism, and in certain pathological conditions defence mechanisms against oxygen radicals may be weakened and/or overwhelmed. DNA is a potential target for oxidative damage, and genomic damage can contribute to neuropathogenesis. It is important, therefore, to identify tools for the quantitative analysis of DNA damage in models of neurological disorders. The aim of this study was to compare the susceptibility of DNA to oxidative stress in cells freshly dissociated from the mouse brain, to that in cultured brain cells. Both primary cultures and a continuous cell line of astrocytes were considered. All cells were treated by xanthine/xanthine oxidase, a superoxide generator or hydrogen peroxide, applied alone or in the presence of the oxygen radical scavengers, superoxide dismutase, catalase, or ascorbic acid. DNA damage, quantified with the Comet assay, was consistent in all the different cell preparations exposed to oxidative stress, and was attenuated in similar ways by superoxide dismutase and catalase, scavengers of superoxide anion and hydrogen peroxide, respectively. The results with ascorbic acid were more variable, presumably because this compound may switch from anti- to pro-oxidant status depending on its concentration and other experimental conditions. Overall, similar responses were found in freshly dissociated and cultured brain cells. These results suggest that the Comet assay can be directly applied to cells freshly dissociated from the brain of rodents, including models of neurological disorders, such as stroke models and animals with targeted mutations that mimic human diseases.