The Effects Of Exercise On Neurological Function Recovery And Neuronal Cells Apotosis After Intracerebral Hmorrhage In The Rats

Stroke, including ischemic and hemorrhagic events, is a leading cause of death and disability worldwide. Hemorrhagic stroke is one of the least treatable types of stroke and represents~15% of all stroke cases in Western populations. Furthmore, up to 30% of ischemic strokes undergo hemorrhagic transformation. An intracerebral hemorrhage (ICH) occurs when a vessel ruptures and blood infiltrates surrounding brain tissue. It causes higt mortality (~50% by 1 month) and poor neurological recovery in survivors. Surgical removal of the hematoma and putative cytoprotectants aimed at treating the consequences of an ICH often fail to substantially improve outcome in humans. Whereas these failures may be partly attributed to study limitations, it is also likely that single cytoprotective agents will not sufficiently promote recovery after stroke. Instead, the use of multiple cytoprotective drugs or the combination of a cytoprotectant and rehabilitation (e.g, to enhance recovery of rescued tissue ) may be a more promising treatment stratege. Furthmore, severe insults will especially require a combination approach to achieve maximal recovery. It is still a disputable question that which is the suitable approach.At present, the rehabilitation methods for motor dysfunction are physical exercises, including active and passive physical exercises. Various rehabilitation interventions promote recovery after ischemic stroke in rats. Interestingly, even simple exercise (EX) treatments such as forced running promote recovery after ischemic stroke, and they also reduce cell death in some situations.There are few studys about hemorrhagic stroke. It is reported that stroke unit is beneficial for patients with ICH. Early rehabilitation can decrease mortality and improve outcome. The passive exercises began in 24 hours after ICH as soon as patient's consciousness is stable, or hematoma no breaking into brain ventricle. But the beginning time of active exercise is still a matter of controversy.Several studys show that rehabilitation also benefits rats suffering an ICH. One of the studys show that EX significantly reduced the lesion volume, decreased the apoptosis of neuronal cell in the striatum, and increases cell proliferation in the dentate gyrus of ICH rats with hyperglycemia. CIMT (Constraint-Induced Movement Therapy) is thought to promote functional improvement by overcoming learned nouse of the affected limb, promoting cortical and subcortical reorgnization,synaptogenesis, and angiogenesis. Unfortunately, most rehabilitation studies examine cortical lesions caused by ischemia or trauma, and there are little data on recovery after subcortical or hemorrhagic injury. There is not anyone study aboout this in our country.At prensent, there are four methods of ICH model.①ICH model was produced by injecting autologous blood into brain;②ICH model was produced by injecting bacterial collagenase into brain;③ICH model was produced by putting a little ball in brain;④ICH model spontaneously produce. In studies of stroke, the ICH model of injection of autologous blood into brain is usually used. But in rats, the size and location of hematola, and volune of blood were unstable. Rosenberg's ICH model by injecting of bacterial collagenase into brain was introduced from 1990, and it is simple and stable.The studies showed that hemorrhagic stroke not only suffer from hematoma directly damage on brain tissue, but also secondary brain injury, such as cell apoptosis. Therefor, our study tested whether the exercise would improve motor recovery, and supress neuronal apoptosis in rats after ICH. There are four parts in my article.The first part: we established the experimental ICH model by injecting bacterial collagenase into brain in rats, and observed the pathological changes of brain tissue around hematoma, and behavior changs of rats. The second part: the ICH rats received cage running exercise beginning 24 hours after ICH, then we investited behavioral change of the rats at 7d, 14d, 21d, 28d, after ICH. The third part: we observed pathological changes of brain tissue surrounding hemotoma by light microscopic, hippocampus tissue by electron microscope, and apoptosis of neuronal cells by TUNEL and FCA. The fourth part: we would survey protein expression of bcl-2, bax, caspase-3 by immunohistochemical, and western blotting techniques. RT-PCR method was used to measure the expression of bcl-2mRNA, baxmRNA and caspase-3mRNA.PartⅠThe establishment of experimental intracerebral hemorrhagic model and pathological and behavioral changes in ratsObjective: To make a stable and identical model of intracerebral hemorrhage (ICH) in rat and to study the changes in the behavior and tissue structure during absorption of the hematomaMethods: Seventy Sprague-Dawley rats ( weight, 270 to 300g) were randomly individed into two group, trial group (n=35) and control group(n=35). The rats in trial group were routinely anesthetized with an intramuscle injection of"quick sleepⅡ"( 0.8-1.0ml/kg). With the use of a sterile technique, a midline incision was made in the dorsal region of the scalp. A cranial burr hole 1mm in diameter was drilled near the cornal suture and 3.5mm to the right of the bregma, and the gauge needle was inserted stereotactically into the right caudate nucleus (coordinates: 1mm posterior, 6 mm ventral, and 3mm lateral to the bregma). Bacterial collagenaseⅦ(0.5U ) was infused by using a microinfusion pump into the right caudate nucleus of rats. After infusion, the needle was keeped in place for 10 minutes before being withdrawn. The control group (sham-operation) group received an equivalent dose of physiological saline with the same method. The behavior of rats with ICH was evaluated using 4 tests in each rat at 6h, 24h, 48h, 72h ,7d and 14d after operation. The specific 4 tests included (1) Bederson score; (2) beam-walking test; (3) bilateral forepaws grasp; and (4) and measurement of forelimb placing. Brain Water Content: All the rats were used for this experiment. At 6h, 24h, 48h, 72h,7d, and 14d after the motor-behavior tests were done, each rat was killed by beheading. The brain was quickly removed and placed on a cooled surface, and the frontal pole (approximately 4 mm thick), the cerebellum and brain stem were removed. The cerebrum was coronally cut into 2 parts: the first cut was through the needle entry site and the second through the midpoint of the posterior remnant. The first part (almost 2 mm thick) was used for brain edema measurement and was cut into two sections: ipsilateral and contralateral of ICH. Each section was wrapped in preweighed aluminum foil and weighed to obtain the wet weight (WW), then dried for 24h in an oven at 95～110℃and weighed again to obtain the dry weight (DW). Brain water content was calculated as the percentage change between wet weight and dry weight using the following formula: (WW-DW)/WW×100%. The second part were post-fixed, dehydrated by different concentrations of ethanol, embedded in paraffin, cut into 5μm slices, stained with hematoxylin-eosin and toluidine blue.Results: The results showed that the neurological deficit scores were the highest at 6h after operation in thefour tests because of the anesthesia and injuries during the operation. At 24h~72h, the scores of the ICH group leveled high until all the rats recovered and the scored fell to the lowest at 14d. At all time points, the scores of the Sham-operated rats were lower than that of the ICH rats and significant differences existed between the two groups. The line graph showed the dynamic tendency of brain water content in the ipsilateral, contralateral and sham groups at different time points after intracerebral infusion. At 6h and 14d, brain water contents of the ICH and sham groups had no significant differences, P>0.05. But at 24h,48h and 72h, the brain water content of ICH group was much higher than sham group. At 7d, the brain water content obviously revoery, and it was almost normal at 14d. The successful ratio of this model was 80%. There were obvious hematomas at 6h after hemorrhage and the size, shape and position of hematomas were stable. The diameter of hematoma was about 3.0-3.5mm. In the earlier period, the neuronal cells around the periphery of the hematoma were obviously swollen and necrotic, and the inflammatory cells were infiltrated. A lot of glial cells, endothelial cells of vessel and glial fiber appeared around the hematoma in the late period.Conclusions: The ICH model in rat by intracerebrally injection of collagenase was simple, reproducible, higt success rate, low mortality. The hematoma development is stable and identicial. The obvious behavioral change and histological changes were similar to clinical patient with ICH.PartⅡ: Exercise training effect on functional recovery after intrcerebral hemorrhage in ratsObjective: To determine whether exercise training would enhance the functional recovery after intracerebral hemorrhage(ICH) in rats.Methods: The 90 male SD rats were randomized into 3 groups, cage-running group, rats with ICH to running-cage (n=30); control group, rats with ICH to normal cages (n=30); sham operated rats to normal cages (n=30). Trial group rats recrived wheel-running exercise beginning 24 hours after ICH . The cage-running is a cylinder mading of steel wire( perimeter: 20cm, diameter: 50cm) with a support at the bottom, and a handle on the cage for controling running speed. The cage-running was divide into three sections for three rats running at same time. During 2-14days of ICH, rats were forced running at a speed of 4r/min, for 40min once a day, resting 2min every 5min. During 15-28days, running at a speed of 5r/min, and putting some sticks into cage for increasing jumping of rats. Animals of the control group left in the cages for the same duration of time, but were not made to run. Animals were weighed and given a bettery of tests that aimed to assess, posture-reflex, balance function, muscle strength and forelimb placing at 24h, 3d, 7d, 14d, 21d, 28d after the operation.Results: Rats with ICH to cage-running performed significantly better than rats in control group in balance, muscle strength and forelimb placing. Sham-operated rats had no obvious disfunction. In all experiments, body weight was similar among all groups at the beginning of the experiment. The rats in trial group and control group develop progressive loss of weight from 72h to 14d after ICH, and rising again of weight after ICH 21d. But the loss of rats weight in trial group was more obvious than that of control group, and there was a significant difference(p<0.01).Conclusion Exercise training (cage-running) can improve the functional recovery after ICH in rats.PartⅢ: The exercise effect on apoptosis of neuronal cells surrounding brain tissue and histological changes in ICH ratsObjective: To investigate the effects of cage-running exercise on apoptosis of neuronal cells surrounding the hematoma and histological changes in ICH rats.Methods: The 104 male SD rats(weight, 270 to 300g) were randomly individed into three groups, trial group ( ICH with EX n=48), control group( ICH with no EX n=48) and sham operated group ( no ICH, no EX n=8). The rats brains were removed at 24h, 7d,14d,21d,28d after ICH. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-bioti in situ nick end-labeling(TUNEL) was used to detect deoxyribonucleic acid( DNA) fragmentation. Flow cytometry assay(FCA) was quantified for DNA. Light microscope and electron microscope were used to observe histological changes of hippocampus and surrounding the hematoma.Results: (1) Under light mcroscope, tipical histological changes of ICH were seen after operation 24h in trial and control groups. After exercise, a lot of new capillary were seen in perimeter of hematoma and hippocampus of rats, in contrast, the control group and sham group had little. (2) Under electronic microscope, shrunken neuron and glial cell with pre-apoptotic signs of intensely stained cytoplasm and abnormally dense nucleus, swollen mitochondrials, blood vessel, Golgi apparatus, rough endplasmic reticulum were seen in control group rats. In trial group rats, the main alterations is dim premembrane and postmembrane of synapses structure. (3) TUNEL-positive cells appeared in the periphery of the hematoma and hippocampus. The number of TUNEL-positive cells was nearly zero in the sham-operation group, and this number was markedly increased in control group from 14d to 28d after ICH, but it was reduced in trial group at same time. Even though, the number of TUNEL-positive cells in the two groups were once decreased from 7d-14d, but the number of TUNEL-positive cells in trial group was less than that in control group. There was a significant difference in two groups(P<0.05). (4) There was no difference at 24h between trial and control group in Flow cytometry assay of PI. The DNA content of trial and control group droped from 7d to 14d after ICH, and rise again after 21d. The DNA content of trial group were less than that of control group. There was a significant difference in two groups(P<0.05. (5) The result Flow cytometry assay of AnnexinⅤ-PI was similar to PI, but the apoptotic rate of neuronal cells was more higher than that of PI.Conclusion Exercise training (cage-running) can supress the number of apoptotic cells, and increase regeneration of capillary after ICH in rats.PartⅣ: The exercise effects on expression of bcl-2, bax, caspase-3 of neuronal cells after ICH in ratsObjective: To investigate the effects of exercise on expression of bcl-2, bax, caspase-3 of neuronal cells after ICH in ratsMethods: The 90 male SD rats(weight, 270 to 300g) were individed into three groups, trial group ( ICH-induction and exercise group, n=40), control group(ICH-induction group, n=40) and sham operated group ( sham-operation, n=10). The rats brains were removed at 7d,14d,21d,28d after ICH. The activation of bcl-2, bax, caspase-3 was measured by Immunohistochemistery, Western blotting and RT-PCR.Results: (1) Bcl-2-positive, bax-positive and caspase-3-positive cells appeared in around the periphery of the hematoma and cortex. The number of bcl-2, bax, and caspase-3-positive cells was nearly zero in the sham-operation group. This number of bcl-2-positive cells was markedly increased, this of bax-positive cells decreased in trial group from 21d to 28d after ICH, and there was a significant difference compared with control group(P<0.05). Although the expression of caspase-3 had a down-regulation trend, but there was no diference between trial and control group. (2) Western blotting study showed that the protein expression of bcl-2 increased, and the protein expression of bax decreased in trial group than control group (p<0.05) , but the protein expression of caspase-3 had no difference in two groups. (3) RT-PCR for bcl-2mRNA showed higher expression, lower expression for baxmRNA in trial group than control group( p<0.05). RT-PCR for caspase-3mRNA had no difference in two groups.Conclusion The results suggested that exercise training (cage-running) can increase the expression of bcl-2 and bcl-2mRNA, decrease the expression of bax and baxmRNA. The was no markely supressing on caspase-3 and caspase-3mRNA .