| Objective Hypoxic-ischemic brain damage (HIBD) caused by perinatal asphyxia often leads to the death of neonates or severe neurological sequelae.lt has been the single most problem in neonatal period. Hypoxic-ischemic brain damage is an evolving process, which begins during the period of primary injury and extends to that of secondary injury (reperfusion injury).There is increasing evidence that nitric oxide (NO) plays an important role in the pathogenesis of neonate hypoxic-ischemic brain damage. NO is formed by nitric oxide synthase (NOS) from L-arginine. There are three NOS isoforms that are neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). In cerebral hypoxia and ischemia, nNOS mediates early neuronal injury. We study the expression of nNOS gene in neonatal rat model of hypoxic-ischemic brain damage, and investigate the change and role of nNOS during HIBD. Methods 1. The establishment of neonatal rat model of hypoxic-ischemic brain damage.Neonatal seven-day-old Wistar rats were under a middle anterior neck incision. The left common carotid artery was isolated from vein and nerve and ligated. The pups were allowed to recover from surgery for 2 hours. Then they were placed in a 2.5-L airtight glass container submerged in a 37.0癈 water bath and flushed for 2h with a humidified mixture of 8% oxygen (O?) and 92% nitrogen (N?) delivered at 1.5-2.0L/min.2. Experimental groupsFifty-six animals were randomly assigned to one of two groups, the HIBD or the control group. In control group, the left common carotid artery was isolated but not ligated and pups were not exposed to hypoxic environment. Animals of HIBD group were killed at Oh, Ih ( during hypoxia ), 2h ( end of hypoxia ) and 6h after the start of hypoxia. Those of control one were killed at the same time point. In each time point, five rats were used to the expression study of nNOS gene, and two used to the histological study.3. Histological studyAnimals were perfused through the left ventricle with cold normal saline followed by 4癈4% paraformaldehyde in 0.1 M phosphate buffer ( pH 7.4 ). The left cerebral hemisphere ( the ligated side ) was removed and the coronal brain section ( 5 mm thick ) cut at the level of the hippocampus and thalamus was post-fixed in the same fixative for five days. Serial 4- y m paraffin-embedded coronal sections were stained with hematoxylin and eosin and examined under the light microscope.4. RNA extractionRats were killed by decapitation and the left cerebral hemisphere ( the ligated side ) was rapidly removed for dissection of selected regions of the cortex and hippocampus. Samples were quick-frozen on liquid nitrogen and stored at -80癈 until used. Total cellular RNA was extracted from samples by using TRIzol as per the manufacturer's instructions. 5.RT-PCRThe reaction mixture for the synthesis of cDNA by RT reaction was in a 20 u 1 total volume. The mixture was incubated at 25 癈 for 10 min, 42 癈 for 50 min, 70 癈 for 15 min and 4癈 for 1 min.The PCR mixture was in a 25 u 1 total volume. The mixture was incubated at 94 癈 for 3 min (once ) , 94癈 for 40 s, 63癈 for 40 s, and 72癈 for 30s ( 56cycles ), 94 癈 for 40 s, 61 癈 for 40 s, and 72 癈 for 30s ( 23 cycles ), and 72 癈 for 7 min. PCR product was visualized by ethidium bromide staining after 1% agarose gel electrophoresis and photographed. Results l.Histological study?Control group : In every time point, there was no obvious sign of neuronal damage and with cells of apparently normal appearance in the brain tissue. Throughout layers I-VI of cerebral cortex, neurons were of clear, well-defined somata and with obvious nuclei. The subregions of the hippocampus. CA1.CA2,CA3.CA4 and dentate gyrus, did not show significant evidence of neuronal injury and loss of cells.?Hoh and HIH group: These two groups did not show different histological appearance compared to control group.(3) H:H group: Degenerated neurons were scattered in the cortex and hippocampus, defined as those with mildly... |
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