| Objective: To replicate the Marmarou model of severe brain injury in rats, and to explore neuronal nitric oxide synthase(nNOS) and inducible nitric oxide synthase(iNOS) expression following traumatic brain injury(TBI) in rats and the cytotoxic effect of NO , meanwhile, to study the mechanism of nerve cell necrosis and apoptosis in the hippocampal CA1 region, the relationship between NO and NOS with the neuron loss in the hippocampal CA1 region, and the effect of NO and NOS on spatical learning and memory. In the end, to investigate the cerebralprotective role of AG and 7-NI is in order to offer theoretic evidences for clinical therapy and new prospect of therapy.Methods: 250 male Wistar rats, weighing 350-400g, were randomly divided into: 1 control group, 2 trauma group, 3 AG treatment group, 4 7-NI treatment group, and 5 AG and 7-NI combined treatment group, each of which was divided into 8 subgroups according to time phase after brain injury, that was 1h, 3h, 6h, 12h, 1d, 3d, 7d and 14d. Each subgroup of control group had 4 rats and the other subgroup had 5 or 6rats. According to Marmarou, severe closed brain injury was made by dropping 450g copper rod from a height of 1.5m, 18mm in diameter. And then the rats were killed at each time point, which were fixed through anteriothora and then the removed brain was fixed in 0.1M PBS of 4% paraformaldehyde at 4℃ for 24h. After regular dehydrating and embedding in paraffin, the section of brain 3.3-4.3mm posterior to the bregma was sectioned serially coronoidly, 7μm in thickness. The tissue section followed by washing in xylen to dewax and rehydration through a graded series of ethanal and redist water. After that, HE staining and Nissl staining, immunohistochemical staining with nNOS, iNOS and caspase-3 were performed. TUNEL in situ was applied. The spatical learning and memory of rats following injury was measured at 11d, 12d, 13d, and 14d by means of Morris Water maze. nNOS, iNOS and caspase-3 immunohistochemical positive cell immunoreactivity was expressed with the OD values detected by color image analysis system. The count and morphological classification were compared in TUNEL positive cell. The obtained data were expressed as (x±s. SPSS 10.0 software was used for the one-way analysis of variance(ANOVA) and the student-neumom-keuls test was applied to define which group contributed to these differences. There was statistically significance if lateral p<0.05.Results: Following injury, decerebration and respirationsuppression occurred transiently. Hemorrhage or yellow-stain can be seen in subarachnoid of many rats. Scattered brain contusion can be noticed in the parietal lobe region and scattered hematomas in brain parenchyma. HE staining showed scattered degenerated and necrotic neuron 6h following injury, which peaked at 3d. The lesion at hippocampus was slight which improved to some extent at 7d and 14d after injury. Nissl staining showed the decreased Nissl in neuronal cytoplasmic of hippocampus at 1d following injury, which decreased further 3 days later and part of which declined. Nissl was less than in control group at 7 and 14d after injury. The above results were consistent with the manifestation described by Marmarou. nNOS immunoreactivity in trauma group increased 1h after injury and peaked at 6h which was obviously strengthened comparing with that in control group(p<0.01), and obviously declined at 24h; in contrast, immunoreactivity in AG group began to increase at 1h after TBI, and peaked at 6h and the peak continued till 12h, which was obviously strengthened comparing with that in trauma group(p<0.05), and began to decline at 24h. There was no difference between 7-NI treatment group and AG and 7-NI combined treatment group. iNOS immunoreactivity in trauma group began to increase at 6h following injury and peaked at 3d and declined at 7d but reacted positively at 14d. It was almost the same with iNOS in AG treatment group except that the peakwas slightly lower in AG treatment group, 7-NI treatment gro...
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