| Objective To investigate the expression of connexin-43(Cx43) in brain tissue of hypoxic-ischemic neonatal rats and explore its role in hypoxic-ischemic brain damage (HIBD).Methods Totally 128 seven-day-old neonatal rats were randomly divided into two groups: control group and hypoxic-ischemia(HI) group with 64 rats in each. According to the time of sacrefice,' 64 rats of every group were further randomly divided into four groups including six hour(6h), twenty-four hour(24h), forty-eight hour(48h) and seventy-two hour(72h), with 16 rats in each group. The left common carotid artery of rats were ligated firstly and then the rats were put into the hypoxic cabin with the mixed gases of N2 and O2 for 2 hours. The rats in control group were cut the skin of neck without ligating the left common carotid artery. The concentration of O2 which was kept between 7％-9％on average-in hypoxic cabin was monitored by oxygen meter. In each group, 8 rats were used for observing pathologic changes, neural cell apoptosis, and the expression of Cx43 through the techniques of HE staining, TUNEL staining and immunohistochemical staining, and the brains another 8 rats was used for measuring the expression levels of Cx43 protein of rat cortex and hippocampus by Western-blot analysis.Result 1 The behavior of rats after hypoxic-ischemia(HI):All rats became dysphoria about ten minutes after HI, and then emerged cyanosis and dyspnea after ten minutes. After thirty minutes, the rats stood unsteadily and appeared limp with right posterior limb, following all rat's activities decreased obviously and fell into a drowsiness. Most of rats behaved turning to the left stably 2 hour after normal concentration of oxygen was given.2 The pathological changes by HE staining:Observation by light microscope: The structures of nerve cells in control group were normal and clear. In HI group, some nerve cells were degenerated, necrosis and broken. The structure of tissue was not clear. Histological study showed that the degenerated and necrotic neurons increased progressively at 6h to 72h after HI. The injuries in HI 72 h group were most severe.3 Apoptotic cells were observed by TUNEL staining:In control group, apoptotic cells were not observed by TUNEL staining. In HI 6 h group, apoptotic cells also were not observed. After HI, the level of apoptotic cells increased progressively at 24 h to 72 h(24h: 15.12±2.68, 48h: 30.24±3.65, 72h: 68.72±5.60). There was a highly, significant difference among them (p＜0.01).4 The expression of Cx43 by immunohistochemical staining:The positive cells of Cx 43 existed in all the control and HI groups. Cx43 distributed in cellular membrane and ecptoma of astrocytes. In control group, the expression of Cx43 has no difference at 6h to 72h (6h 8.38±0.16, 24h 8.38±0.19, 48h 8.39±0.03, 72h 8.35±0.15)％. There was no significant difference among them(p＞0.05). After HI, the expression of Cx43 increased progressively at 6h to 72h(6h 18.64±0.30, 24h 29.65±0.26, 48h 35.34±0.21, 72h 42.05±0.31)％. There was a highly significant difference among them (p＜0.01). At 6h, 24h, 48h and 72h, there was a highly significant difference between HI and control group (p＜0.01).5 The expression of Cx43 by Western-blot analysis:Proteins of Cx43 are extracted at 43-46KD position in all the control and HI groups. In control group, the expression of Cx43 has no difference at 6h to 72h(6h 1.00±0.01, 24h 0.99±0.02, 48h 1.01±0.01, 72h 1.01±0.01). There was no significant difference among them (p＞0.05). After HI, the expression of Cx43 increased progressively at 6h to 72h(6h 1.11±0.04, 24h 1.25±0.02, 48h 1.37±0.01, 72h 1.50±0.01). There was a highly significant difference among them (p＜0.01). At 6h, 24h, 48h and 72h, there was a highly significant difference between HBID and control group (p＜0.01).Conclusion1 The neonatal rat model of HIBD was successfully established. The method is easy and simple. The injuries of rat brain increased progressively at 6h to 72h after HI, and at 72h injuries were most severe.2 The level of apoptotic cells increased progressively after HI, regularity of which was consistent with the time frame for development of brain damage.3 Cx43 had expressed in the neonatal rat cortes and hippocampus. They distributed in cellular membrane and ecptoma of astrocytes. The level of Cx43 protein was constancy in neonatal rat brain during earlier period after birth. There was no significant difference within 72 hours in the neonatal rat.4 The expression of Cx43 protein increased progressively after HI, regularity of which was consistent with the time frame for development of brain damage and apoptosis. The change of Cx43 is earlier than apoptosis.The results implied Cx43 may play important roles in the pathogenesis of HIBD in neonatal rtas through enhancing gap junction intercellular communication which killed the adjacent cells, resulted in and increased brain damage after hypoxic-ischemia. |
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