| Obejective:Ischemic stroke, caused by blockage (thrombosis or arterial embolism), involves the rapid loss of brain functions because of disruption of the blood supply to the brain. Ischemic stroke is a serious global health problem. It is triggered by a complex series of biochemical and molecular mechanisms that initiate the ischemic cascade, including excitotoxic glutamatergic signaling, ionic imbalance, free-radical reactions, and so on. The duration of ischemia is an important determinant of subsequent damage. Rats subjected to20min to30min of four-vessel occlusion (4-VO) develop ischemic neuronal damage. Therefore, all cells are sensitive to oxygen alteration, and sustained hypoxia results in cell death. The vulnerability of brain cells, particularly neurons, has usually been ascribed to their functional and metabolic characteristics. The hippocampal CA1subfield displays a selective vulnerability to ischemic injury. The transient global ischemia induced by10min of4-VO destroys71.6%of CA1neurons, and causes spatial learning/memory dysfunction. Acute and chronic cognitive impairments continue to be a problem for many stroke survivors following hypoxic-ischemic brain injury.Piperphentonamine hydrochloride ((E)-5-{(3,4-Methylenedioxyphenylethane) methylamino}-1-p-hydroxyphenyl-1-penten-3-one hydrochloride, PPTA), which is similar with levosimendan, is a new cardiovascular drug used for treating acute and decompensated heart failure. Recently, levosimendan has been reported as a promising new pharmacological tool for neuroprotection in an in vitro model of traumatic brain injury. Previous studies have shown that PPTA reduces reperfusion injury in in vivo ischemic models in cats, rats, and dogs. On the other hand, PPTA is a recently synthesized positive inotropic drug that improves myocardial contraction without increasing calcium concentration. It also has a vasodilating effect by opening calcium-sensitive potassium channels. PPTA confers cardioprotection and possible neuronal protection during ischemic injury. Therefore, the study investigated the protective effects of PPTA on the hippocampal damage and memory deficit in rats subjected global cerebral ischemia/reperfusion injury. Meanwhile, we also observed the effect of PPTA on the viability and morphology of neuron by establishing the oxygen-glucose deprivation (OGD) model in cultured hippocampal neuron and PC12cells.Methods:(1) The global cerebral ischemia/reperfusion injury model was made by four-vessel occlusion in rats. Then the Sprague-Dawley rats were randomly divided into control group, I/R group, PPTA groups (2.5,5and10mg/kg) and PPTA was administrated at2h after the onset of reperfusion. Morris water maze and step-though training were used to evaluate the learning and memory abilities. The histological changes of hippocampus were detected by Nissl staining. LDH activity and iNOS, caspase-3, bax/bcl-2mRNA expression were measured by colorimetric Assay Kit and real-time PCR.(2) An in vitro oxygen-glucose deprivation (OGD) experiments were performed in primary neurons and rat pheochromocytoma PC12cells induced by physical or chemical injury. The neuroprotective effect of PPTA was further confirmed by MTT assay and morphological observation. LDH and NO activity were measured by colorimetric Assay Kit. The iNOS, caspase-3, bax/bcl-2mRNA expression were detected by Real-time PCR and the expression in iNOS was measured by western blot.Results:1. Experiments on global cerebral ischemia/reperfusion injury in rats(1) During the5-d acquisition trials in the water maze test, all the animals swam progressively shorter distances over time. Compared to the sham-operated controls, I/R rats swam longer distances in search of the platform and this was attenuated by repeated treatment with PPTA at doses of5and10mg/kg. In the place navigation test, the escape latency of PPTA (5and10mg/kg) shown a significantly increase compared with the ischemia-induced rats. There was no significant difference in the velocity of the rats in Morris water maze test, suggesting that the memory deficit resulted from the ischemia but not rats themselves and this was blocked by PPTA. In the step-through passive avoidance test, latency of the animals did not differ in crossing from the illuminated compartment to the darkened side during training. In contrast,24h after initial training, the sham-operated rats exhibited an increase in latency following footshocks, suggesting that the animals had acquired memory of the aversive stimulation associated with the darkened compartment. The latency of the ischemic animals was significant shorter than that of sham groups in the retention test, suggesting that ischemia impaired long-term memory of rats. This was reversed by PPTA (5or10mg/kg), in particular at the dose of10mg/kg.(2) Cresyl violet staining was performed at16days to examine the loss of neuronal cells in hippocampal CA1region after ischemia. CA1pyramidal cells of sham-operated rats showed round and pale stained nuclei, in contrast, the cells were shrunken with pyknotic nuclei under sublethal ischemia insult. Interestingly, the injection of PPTA at a dose of5or10mg/kg attenuated ischemia induced neuronal degeneration compared to the ischemic rats.(3) After injection of PPTA, the release of LDH in hippocampus significantly decreased compared to the I/R group. Also, in the Real time PCR testing, PPTA up-regulate bcl-2mRNA expression and down-regulate the expression of caspase-3, bax and iNOS.2. Experiments on oxygen-glucose deprivation in neuronal cells(1) The PC12cells exposed to20mmol/L Na2S2O4with free-glucose DMEM for4h and reoxygenation for24h, and the cells were assayed by MTT. As the results shown, cell viability was markedly decreased after the cell was exposed to OGD insult, but when posttreatment with PPTA (3,6and12μmol/L), OGD-induced cell toxicity was significantly attenuated in a dose-dependently manner.(2) LDH and NO leakage were increased in PC12cells and primary hippocampal neuron after treatment with OGD/OGD-R, whereas addition of PPTA to neuronal cells exposed to OGD injury led to a decrease compared with the OGD-group.(3) Morphological observation indicated that a majority of neuronal cells exposed to OGD/OGD-R exhibited swelling in their shape, with most cells losing their neurites and showing a marked decrease in the cell number. In contrast, cells posttreated with different concentrations of PPTA (3,6,12μmol/L) inhibited the morphological changes to some extent in a concentration-dependent manner.(4) Real-time PCR results showed that PC12cells extract from the OGD had a decrease in the expression of bcl-2mRNA, a significantly increase in caspase-3, bax and iNOS compared with the control, these were altered by treatment of PPTA (3,6,12μmol/L).(5) Treatment of PPTA in PC12cells after OGD insult also attenuated the level of protein of iNOS.Conclusion:In the present study, data showed treatment with PPTA could ameliorate the memory deficit and suppressed the neuronal damage in rats following global cerebral ischemia injury. The antiapoptosis effect of PPTA was observed not only in rats but also in in vivo neuron; and it could inhibit the activation of iNOS-NO pathway. We hypothesis that PPTA reverse the memory deficit associating with reduce neuronal damage. Therefore, as a new Ca2+sensitizer, PPTA protect neuron from injury induced by ischemia/hypoxia; leading to a potential candidate for the treatment of stroke in the future. |
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