| Traumatic brain injury(TBI) is one of the commonest diseases which threaten the human life. The incidence rate and mortality rate of brain injury are just lower than those of cancer and cardiovascular diseasesAnd the mortality is highest in violence accident. Therefore, it's very important to study the mechanism for TBI treatment. The primary TBI then gives rise to secondary injuries, of which the apoptosis of neurocyte is among the most important .mechanism. The release of oxidant free radicals and the activation of caspase-3 play an important role of inducing the neuron apoptosis. This experiment aims to investigate how the caspase-3 inhibitor z-DEVD-fmk has effects on the neurocyte apoptosis and systematic level of free radicals with modified Feeney free dropping objective mode of rat, moreover, to provide the clues of developing new medicines.Materials and methods36 healthy, adult, male SD rats were tested,weighting 300 15g,provided byZhejiang University Medical Institute.All the rats were randomly divided into 3 groups:Group A(n=12) as sham operated group,group B(n=12) as NS treated group and group C(n=12) as z-DEVD-fmk treated group. There were no significant differences among the average values of body weight in the three groups by AVONA test(P =0.757).All the rats were anesthetized with Ketamine(40mg/kg) and placed in a stereotaxic apparatus. The scalp was steriled with iodine and 75% alcohol and incised on the midline. Then rats underwent a craniotomy, in which a circular region of skull (4.0 mm diameter, centered 2.0 mm caudal and 2.0 mm lateral to bregma) was removed over the right somatosensory cortex. A specific caspase-3 inhibitor z-DEVD-fmk(0.5 g/ul) was infused into the right lateral ventricle in a volume of l0l by a 25 l microsyringe with the speed of 21/min in the rats of group C.The puncture coordinate is at AP 1mm and LAT 1.5mm to the bregma and puncture depth is 4.5mm.Furthermore,needle of the microsyringe should be kept in lateral ventricle for 10 minnutes.30 minutes after infusion, a weight-drop device ,3 mm in diameter was placed stereotaxically over the dura and adjusted to stop an impact transducer (foot plate) at a depth of 3 mm below the dura. Then, a 50g weight was dropped from 16 cm above the dura, through a guide tube onto the foot plate to produce severe brain injury with intact dura.And 30 minutes after trauma,z-DEVD-fink was injected again with the same method as described above. Finally,close the bone window by dental base acrylic resin powder and suture the skin of scalp.The rats of group B were dealed with the same as group C except NS instead of z-DEVD-fmk.And the rats of group A were only dealt with craniotomy.Free radical detection:In 0 hour,6 hours, 12 hours, 24 hours,48 hours and 72 hours,Rat caudal venous blood samples(0.lml) were collected from all the subjects and heparin sodium was added as anticoagulant. The spectrophotometry of xanthine oxidase reactive substances was used to determine the erythrocytic SOD activity indicated as U/g.Hb. And the spectrophotometry of thiobarbituric acid reactive substances was applied to determine the erythrocytic MDA concentration expressed as nmol/mg.Hb. Histological methods for assessing cell damage and death: In 72 hours, all rats were anesthetized(Kitmine 40mg/kg) and decapitated.And brains were quickly removed from the cranial cavity on ice cubes, soaked in PBS(0), then were sliced in 2-mm-thickslabs in the coronal plane. For light microscopy, transverse serial sections, were made and stained with HE. Meanwhile, TUNEL method was used to observe apoptosis. Subsequently, ultrathin sections were made and fixed by 2.5% glutaraldehyde and then examined by electron microscopy.Medical Statistic Analysis: All data were statistically analyzed with SPSS/11.0 for Windows using a Intel Pentium IVYl.5G.They were expressed as mean plus or minus standard deviation (r s). Hypothesis testing methods used included one-way ANOVA test, LSD test and Dunnett's C test. In the statistical analysis of this study, the level of hy... |
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