Study Of Neuroprotective Effect Of Cromakalim In Different Brain Area Of Neonatal Rats With Hypoxic-ischemic Encephalopathy

Background and Objective:Neonatal hypoxic-ischemic encephalopathy (HIE), which results from hypoxia in perinatal period and is the cause of hypoxic ischemic brain damage. It displays on clinical is the brain illness. Some patients can leave behind all kinds of the nervous system sequelaes. It occupies the important position in the perinatal neurous system disease and is the main reason of the children's brain paralysis.Recent years studies discovered that the opening of the ATP-sensitive K⁺ channels has the protective function to many kinds of tissue cells under the damage of hypoxia and ischemia.The K_ATP channel is activated to open in pathological states and so on brain under oxygen deficit ischemia , causes the massive K⁺ outflow, cell membrane hyperpolarizing, thus causes the Na⁺, Ca²⁺ internal flow to reduce, avoids or reduces the calcium overload, the reduced energy consumption, to reduce the excitability amino acid and the oxygen free radical release, protects the brain cell. Cromakalim, as a putative mitochondrial K_ATP channel opener (KCO), has been implicated in neuroprotection after cerebral hypoxic-ischemic injury. Administered as pretreatment, cromakalim can attenuate hypoxia-ischemia-related neuronal injury, but the mechanisms of its protective effect are still not clear.In this study we set out to observe the expression of K_ATP channel gene (Kir6.1, Kir6.2, SUR1, SUR2B) in the different brain area (cerebral cortex, hippocampus, cerebellum, cerebral ganglion, brain stem) of neonatal rats with HIE, and the different protective function of cromakalim in different brain area so as to investigate the molecular mechanism of the brain's protective function of the KCOs. These results provides scientific basis for in-depth study on the different expression of K_ATP channel gene in different brain area, and affords us academic basis for the higher specific KCOs to treat the newborn's HIE.Materials and Methods: A total of 250 newborn Sprague-Dawley (SD) rats were randomly divided into five groups: Normal control group (Control), hypoxia-ischemia group (HI), cromakalim treatment group (Cro), solvents control group (NaOH) and cromakalim⁺ glibenclamide group (Cro+Gli). In HI group, Cro group, NaOH group and Cro+Gli group hypoxic-ischemic brain injury model was produced. Medicine was given at the time of 0h, 24h, 48h, 72h after HIBD. The rats in Cro group were treated with cromakalim (100ug/kg, peritoneal injection, i.p.) The rats in Cro+Gli group were treated with glibenclamide (300ug/kg, peritoneal injection, i.p.) after the injection of cromakalim at the same time to restrain the opening of the channel. The same volume of solvents(0.1M NaOH)was administrated with the rats in the NaOH group.SD rats of every group were given neurological scores after HIBD. The neonatal rats were decapitated and the brain tissues were taken. HE staining method was used to observe the pathological changes and given microscopic damage grading. TTC staining method was used to measure cerebral infarction. RT-PCR was used to detect the expression of K_ATP channel gene (Kir6.1, Kir6.2, SUR1, SUR2B).The data were analyzed by repeated measures, one-way analysis of variance (ANOVA), LSD, SNK, Dunnett T3, and using EXCEL2003 and SPSS13.0 for Windows. The results were expressed in mean±standard deviation((x|-)±s).Correlation analysis was done by Pearson correlation. Values of P<0.05 were considered as statistically significant.Results:1. The behavior changes and the neurological scores of the rats after HIBD :Rats in the Control group could not be seen the obvious nerve function flaw. The rats in HI, and NaOH group became dysphoric, tachypnic and cyanotic and even drowsy lately. Some of them stood unsteadily and even twitched or went into a coma. Compared to those in HI and NaOH groups, the behavior and the neurological scores of the rats in Cro group after HIBD were improved. The scores of Cro+Gli group were higher than the Cro group(P <0.05).The HI group and NaOH group have no statistical significance (P > 0.05).The HI group and NaOH group> Cro+Gli group >Control group, P<0.01. The difference has statistical significance.2. The pathological changes of the brain tissues on HE staining and microscopic damage grading:The brain tissue slices were observed under light microscope. In Control group, the Construction of brain tissue was clear and the cells were arranged well with complete and clear cell outline and nucleoli. The number of cells was normal and interstitial tissue was not swollen. The cortical neurons in HI and NaOH group were disorderly arranged with cell edema and unclear nuclear structure or pycnosis,and the number of neurons decreased following degenerating and necrosis of the cells. Apoptosis could be observed and interstitial edema was obvious. The number of neurons in Cro group was slightly decreased and the majority of cells were regularly arranged. Interstitial edema, degeneration and necrosis were observed, but it was obviously improved compared to those in HI and NaOH group. The morphological characteristic of cells in Cro+Gli group was serious compared to Cro group. The results of microscopic damage grading: Every group in each brain area was compared. The HI group and NaOH group have no statistical significance (P>0.05), The HI group and NaOH group> Cro+Gli group> Control group, P<0.05 or P<0.01.The difference has statistical significance. Compared with the HI groups in each brain area, the scores of cerebral cortex, hippocampus >cerebellum, cerebral ganglion, brain stem. And the difference has statistical significance.3. TTC staining method was used to measure cerebral infarction:All the brain tissue was red in the control group. The cerebral infarction of the HI group and NaOH group was the biggest, In the Cro group it was the smallest. And the cerebral infarction of the Cro+Gli group was bigger than that of the Cro group. Compared with every group, the HI group and NaOH group have no statistical significance (P>0.05).The HI group and NaOH group> Cro+Gli group>Control group, P<0.01. The difference has statistical significance. 4. RT– PCR detects the expression of K_ATP channel gene in different brain areas:Compared with Kir6.1 or Kir6.2 in different brain area (the HI group, NaOH group and the Cro group, Cro+Gli group, Control group), P<0.01. The difference has statistical significance. Compared to the HI group and the NaOH group, the difference has no statistical significance, P>0.05.In the cerebral cortex and in the hippocampus, Kir6.2>kir6.1, P<0.05, the difference have statistical significance. Compared with Kir6.2 in different brain area (the Cro group and the Control group),in the cerebral cortex, the expression was close,P=0.44. And the difference has no statistical significance. In the hippocampus, P=0.29, the difference has also no statistical significance. The P value in the cerebellum, cerebral ganglion and brain stem were lower than 0.05. So it has statistical significance. Compared with SUR1 or SUR2B in different brain area (the HI group, NaOH group and the Cro group, Cro+Gli group, Control group), P<0.01. The difference has statistical significance. Compared to the HI group and the NaOH group, the difference has no statistical significance, P>0.05. In the cerebral cortex, SUR2B> SUR1 and in the hippocampus, SUR1> SUR2B, P<0.05, the difference have statistical significance.Conclusion:1.K_ATP opener cromakalim can significantly improve neurological function deficits and brain cells'nterstitial edema, degeneration and necrosis, decrease the infarct area. This result indicates that K_ATP openers have a protective effect on cerebral ischemia/reperfusion.2.After hypoxic ischemic brain damage of neonatal rats, the scores of cerebral cortex and hippocampus were highest by microscopic damage grading.That shows the damage degrees of them are higest.3. The different parts of the same brain have different K_ATP channel. Cromakalim has good protective function on the cerebral cortex and the hippocampus which were damaged mostly. In addition,glibenclamide can suppress cromakalim's brain protective function.