| BackgroundTraumatic brain injury (TBI), the majority of patients are young adults, is caused by external force severely impaired brain disease with high incidence rate, high mortality and high disability rate. Because most of the survivors leave over varying degrees of neurological and psychological dysfunction, bring heavy mental and economic burden to family and society. Until today the treatment of TBI is limited, therefore, a thorough study on the mechanism of TBI physiological and pathological change and to find a newly and effectively therapeutic target has been the hotspot and difficulty. TBI leading to further brain damage called secondary brain injury(SBI), secondary brain injury occurred from a few minutes to several days or even longer period after TBI, based on the gradual development of pathological changes and is main reasons of death, so the key to improve the prognosis of patients with TBI is the treatment of secondary brain injury. Recent studies have shown that the damage of neurons caused by TBI is the superoxide anion. After the primary injury, the oxygen free radicals such as lipid peroxide (LPO), H2O2, O2-, OH, etc. can be generated due to ischemia, hypoxia, ischemia reperfusion and bleeding. The free radical is lively and can easily react with other substances to form new free radicals or active oxygen oxides. The oxidative stress can cause protein oxidation, lipid oxidation, cell nuclear dissolution, respiratory chain disruption, apoptosis, etc. Therefore, it is a correct way to reduce the free radical damage and find the special medicine which can selectively neutralize oxygen free radicals and inhibit lipid peroxidation. At present, the study on the oxidation of hydrogen has become the focus of the domestic and foreign scholars, hydrogen is a colorless and tasteless double atom gas with low reduction. In recent years, the related research shown that hydrogen is not the physiological inert gas but an ideal antioxidant substance. Hydrogen selective antioxidation specifically and intracellular harmful free radicals protect DNA protein from damage to keep normal mitochondrial function and prevent apoptosis. In order to study effects of hydrogen on brain injury, this study using self-made rich hydrogen saline intervention in rats’ model of traumatic brain injury, after seven consecutive days of intraperitoneal injection, to study the effect and mechanism of protective effect of brain damage by observed the behavior, histopathological and molecular biology aspects, provided theoretical basis for further study of neuroprotection of hydrogen rich saline after cerebral injury.Part I Effects of hydrogen rich saline on the behavior of rats with traumatic brain injuryObjective:To observe the effect of hydrogen rich saline on motor function and learning and memory in rats with brain damage model.Methods:After randomization using modified Feeney method to establish the model of rat brain injury, rats were randomly divided into sham operation group (sham group), group of brain injury (TBI), a brain damaged rich hydrogen saline group (HS group), HS group given daily hydrogen rich saline by intraperitoneal injection (10ml/kg), continuous 7d, postoperative 1,3,7,14,21 and 28 days were used MNSs nerve function score of neural function defect assessment of symptoms and balance the balance beam and walking experiments to detect rat balance and movement coordination ability, in postoperative 10-14,24-28d by water maze test in rats changes of learning and memory ability.The general behavior observed in TBI group and HS rats eating in the early damage, reduce the amount of drinking water and late recovery with the sham group, the same, repeated measurement analysis showed no significant difference (F[food intake]=3.256, F[water intake]=10.256, P> 0.05); TBI rats in the postoperative weight lower than HS group and sham group, HS rats weight lower than sham group, repeated measure analysis of variance showed that the difference was statistically significant (F=l2.680, P< 0.05). MNSs score showed that the score of 28 days of sham group 1.80+0.45, TBI group score for 8.96+1.25, HS group, the score is 4.56 +2.00, rank sum test showed differences have statistical significance (x2 2=24.65, P < 0.05). Repeated measures analysis of variance analysis found that TBI rats balance balance beam time was shorter than that of sham group and HS group (F=23.86, P< 0.05), HS group and TBI groups were no statistical significance (F=3.42 (P> 0.05); TBI rats balance beam walk time is longer than that of the sham group and HS group (F= 10.55, P< 0.05), HS group walking time was longer than that of the sham group (F=5.72, P< 0.05). Morris water maze test were found with the extension of training time, incubation period in the three groups of rats of two stages gradually shortened, but compared with the sham group and HS group, TBI group time delays (F=9.80, P < 0.05), HS group and sham group compared the difference was not statistically significant (F=2.27. (P> 0.05). On day 14 in sham group target quadrant time is 42.3+4.55% and TBI group for 35.6+8.34%, HS group was 36.7+7.65%. Statistical analysis showed differences had no statistical significance (27 F=2., P> 0.05); 28d exploring test in sham group target quadrant time for 65.6+6.80% and TBI group was 45.7+5.27%, HS group was 56.1+7.00%. Statistical analysis showed that there was a statistically significant difference (F=13.57, P< 0.05); TBI rats through the platform number was significantly less than that of HS group and sham group, sham group and HS group compared no significant (F=7.05 (P> 0.05)Conclusion:Hydrogen rich saline can promote the recovery of body weight after injury, improve the neurological function, motor function,learning and memory of TBI’s rats.PartⅡ Hydrogen rich saline on the improvement of pathological changes in rats with traumatic brain injuryObjectiveTo observe effects of the damage area and pathological changes of rats with hydrogen rich saline,including cerebral edema and blood-brain barrier, and the survival of neurons.Method:after randomization using modified Feeney method to establish the model of rat brain injury, rats were randomly divided into sham group, TBI group, HS group, HS group given daily hydrogen rich saline by intraperitoneal injection (10ml/kg), continuous 7d, randomly selected from each group of 5 rats, after 3 d,7 d,14 d. HE staining was used to observe the pathological damage,28d Nissl staining observed in the dentate gyrus of hippocampus nerve cell survival, and to observe the change of the area of brain injury; 3 h after operation,12h,24h,3d,7d by wet and dry weight measurement of cerebral edema content and EB content were used to observe the changes of the ipsilateral cerebral permeability.Results:HE staining showed that TBI injury group of presents a large number of early neuronal necrosis, edema, inflammatory cell infiltration, advanced see gliosis; HS group injury presents early neuronal loss reduce and alleviate edema, late proliferation of glial cells reduced. The damage area of the TBI group was 31.2+ 4.35mm3, the damage area of the HS group was 18.90+3.58mm3 (t=12.03, P< 0.05). The neuronal cells in the TBI area around the damage zone and the neurons in the hippocampus of the DG area of the hippocampus were significantly less than that in the DG group (F=21.35, P<0.05). The contents of brain water content and 3h in TBI group and HS group increased gradually after operation, and the water content of 24h reached the peak at 3d and 7d HS after operation, and the water content of group was lower than that of TBI group (F=11.82, P< 0.05). EB content determination of reaction of blood brain barrier permeability changes and postoperative 24hTBI group and HS group EB content higher than sham group about 9 times (F=14.86, P< 0.05), 3 days after HS group EB content lower than TBI group, the difference was statistically significant (F=9.81, P< 0.05).Conclusion:Hydrogen rich saline can delay damage and pathological changes, reduce the damage area, promote the survival of neurons, decrease the permeability of cerebral edema and blood-brain barrier.Part Ⅲ Molecular mechanism of the protective effect of the hydrogen rich saline in rats with traumatic brain injuryObjective:To observe the effect of rich hydrogen saline on expression of inflammation cytokines IL-1, IL-6, TNF-a, analyse the content of malondialdehyde (MDA),the change of superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH PX), the apoptosis of nerve cell and apoptosis related protein expression changes in rats with traumatic brain injury.Methods:After randomization using modified Feeney method to establish the model of rats’traumatic brain injury, rats were randomly divided into sham group, TBI group, HS group, HS group given daily hydrogen rich saline by intraperitoneal injection (10ml/kg), continuous 7d, randomly selected from each group of 5 rats. Postoperative 3h,12h,24h,3d,7d by ELISA determination of differences in the expression of inflammatory cytokines IL-1, IL-6, TNF-a; brain tissue made homogenate according to kit instructions for the determination of activity of superoxide dismutase (SOD), content of malondialdehyde (MDA), activity of glutathione peroxidase (GSH PX). The expression of apoptosis and GFAP protein was observed by TUNEL after 7d, and the expression of Bax, Bcl-2 and caspase-3 was detected by Western blot.Results:The expression of inflammatory cytokines IL-1 and IL-6 in the TBI group and HS group which began to rise after 3h of operation by ELISA.They reached the peak on 24h. The expression of the same trend, but the difference was not statistically significant between two group (F[IL-l]=1.85,P>0.05; F[IL-6]=2.64,P>0.05).The expression level was significantly higher than that in sham group (F[IL-1]=41.07,P< 0.05; F[IL-6]=22.65,P<0.05). That in HS group was lower than that of the TBI group after 3d and 7d.The difference was statistically significant (F[IL-1]=25.15,P<0.05; F[IL-6]=14.30,p<0.05). The expression of TNF-a in TBI group and HS group sustained increase and declined after 3d.The difference which the two groups compared was not significant (F=2.04,P>0.05), but higher than those in sham group(F=15.39,P<0.05). The content of SOD in the TBI group and HS group was significantly lower than that in sham group (F=316.5, P< 0.05), HS group higher than TBI group, the difference was statistically significant (F=124.2, P<0.05). The content of MDA in the TBI group and HS group was significantly higher than that of sham group (F=8.103, P<0.05), but in HS group was significantly lower than that of the TBI group, the difference was statistically significant (F=165.5, P<0.05). The expression of GSH-px in TBI group and HS group was significantly lower than that in sham group (F=11.61, P<0.05), but in HS group was higher than that of the TBI group, the difference is statistically significant (F=6.098, P<0.05). Apoptotic cells of TBI group in 7d after operation were more than that of GFAP group (P< 0.05).The expression of Caspase-3 was higher than that of Bax group (P<0.05), and the expression of Bcl-2 protein was lower than HS group (P<0.05).Conclusion:Hydrogen rich saline can inhibit the expression of IL-1, IL-6, antioxidant damage, and increase the level of expression of Bax protein, and decrease the level of apoptosis of Bcl-2, and play a protective role in brain. |
PDF Full Text Request