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Expression and Role of Connexin-43 in Brain Tissue of Hypoxic-Ischemic Neonatal Rats

Author: LinJieYing
Tutor: LinNiYang
School: Shantou University
Course: Pediatrics
Keywords: Gap junctions Connexin 43 Cerebral hypoxia Cerebral ischemia Newborn Rats Apoptosis
CLC: R722.1
Type: Master's thesis
Year: 2007
Downloads: 50
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Abstract


Objective To study the expression of Cx43 in neonatal rats with hypoxic-ischemic brain damage (HIBD), to explore the role and significance in HIBD. Method 7-day-old healthy SD rats 128 randomly divided into a control group and hypoxia-ischemia (HI) group, n = 64. Two groups of rats killed time points for each group were randomly divided into 6h, 24h, 48h, 72h four group (n = 16). The HI rats advance ligation of the left common carotid artery to restore two hours and then placed in 8% of the oxygen concentration in the anoxic tank for 2 hours, and then continue to feed back into the maternal side, the establishment of neonatal rat HIBD model, in missing oxygen process and hypoxia observed within 2h the HI rats behavior activities; control rats only separation of the left common carotid artery ligation, without hypoxic continue feeding back into the maternal side. Each group of eight rats were used HE staining, DNA in situ end labeling method (TUNEL), immunohistochemical staining was observed in each group neonatal rat brain tissue pathological changes, apoptosis and the expression of Cx43 distribution; each group In addition, eight of protein immunoblotting (Western-blot) quantitative analysis of Cx43 protein expression in the cortex and hippocampus. Results 1 HI group behavior observation: hypoxia for about 10 minutes, all rats irritability, lack of oxygen for about 20 minutes, all rats cyanosis, breathing deeply and fast, standing firm after 30 minutes, crawling right hind limb was dragged step , 40 to 60 minutes activity was significantly reduced drowsiness after one hour, two hours after reoxygenation fixed rotation to the left. 2 HE staining pathological changes: the control group at each time phase point under a microscope brain tissue structure, the level of clear cell arrangement rules, the outline of the normal cells, and nuclear center, clear nucleoli. HI 6h were observed in a small part of the swelling of the nerve cells, focal nuclear pyknosis; 24h group visible nerve cell degeneration, necrosis, visible apoptosis, pyknosis of some nuclei, interstitial edema; 48h group in addition to visible above changes disordered arrangement of nerve cells, poloidal unclear, significant edema of the nerve cell degeneration and necrosis, vacuolation, nuclear structure is unclear; the 72h group of the above-mentioned phenomenon is further aggravated the lesion center shows a large number of pyknotic nuclei and nuclear debris seen around than multi-apoptotic cells. 3 TUNEL detection of apoptotic cells: the control group at each time phase point cortex, hippocampus no TUNEL-positive cells; different levels of the HI group in addition to the the 6h group no TUNEL-positive cells express, 24h, 48h, 72h group cortex, hippocampus are visible TUNEL-positive cells, HI 24h, 48h, 72h group apoptotic cells were: (15.12 ± 2.68,30.24 ± 3.65,68.72 ± 5.60). With the passage of time after hypoxia-ischemia, TUNEL-positive cells increased significantly, the group differences were significant, p <0.01. 4 of Cx43 protein expression by immunohistochemical staining results (1) the control group at different time points hippocampus, cortex have positive expression of Cx43 distribution in astrocyte cell membrane and around protrusions, 6h, 24h, 48h, 72h group of Cx43 staining positive area (8.38 ± 0.16,8.38 ± 0.19,8.39 ± 0.03,8.35 ± 0.15)%, as age increases, at each time point group of Cx43 expression differences not significant (p> 0.05). (2) HI group at each time phase points hippocampus, cortex, both of Cx43 expression, mainly distributed in the astrocyte cell membrane and around protrusions, 6h, 24h, 48h, 72h group Cx43 staining positive area were (18.64 ± 0.30,29.65 ± 0.26,35.34 ± 0.21,42.05 ± 0.31)%, as hypoxic ischemic time goes on, Cx43 positive expression gradually increased groups compared, the difference was significant (p <0.01); comparison of the HI group and the control group, the HI group each phase point of Cx43 expression was higher than that in the control group, p <0.01 5 Western-blot results: groups electrophoresis can be displayed with the density ratio of the control group in the 43-46KD range the bands, Cx43/GAPDH electrophoresis strips 6h, 24h, 48h, 72h phase were (1.00 ± 0.01,0.99 ± 0.02,1.01 ± 0.01,1.01 ± 0.01); HI group 6h, 24h, 48h, 72h phase point Cx43/GAPDH electrophoresis strip ratio HI group compared with the control group (1.11 ± 0.04,1.25 ± 0.02,1.37 ± 0.01,1.50 ± 0.01); same time phase point Cx43 protein expression compared with the control group, p <0.01; HI6h group Cx43 expression has begun increase over time in the hypoxic-ischemic gradually increase to 72h group is the most obvious, HI among the three groups, the difference was significant (p <0.01); the control group at each time point comparison of Cx43 protein expression was no difference The passage of time is gradually increased significant, p> 0.05; conclusion of a hypoxic-ischemic model is simple, reliable results, and behavioral changes in rats obvious pathological changes of brain tissue; brain injury with hypoxia ischemia 72 hours, the most obvious. 24 hours after 2 HI neuronal apoptosis appears to 48h 72h apoptosis gradually increased, and its variation with the light microscope to the severity of the pathological changes of the brain tissue to coincide. 3 Cx43 in neonatal rat cortex, the hippocampus has been a marked expression distribution in astrocyte cell membrane and around protrusions Cx43 expression after birth has been relatively stable, within 72 hours of its expression, no significant change. 4 Cx43 expression in neonatal hypoxic-ischemic brain tissue; 24h, 48h, 72h became clear, and its variation with neuronal apoptosis and brain tissue pathological changes in the severity of the time frame coincides with the passage of time, and occurred early in apoptosis. These results suggest that the increase in expression of Cx43 involved in the the neonatal rat HIBD pathological formation process, and speculated that connect through the gap to enhance the propagation and amplification of cell injury, sisters killing effect in HIBD early play, cause and aggravate brain damage.

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CLC: > Medicine, health > Pediatrics > Newborns, premature children disease > Neonatal disease
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