| Cerebral palsy(cerebral palsy,CP),hereinafter referred to as cerebral palsy is a group of persistent central movement and posture developmental disorders,activity restricted syndrome,this syndrome is due to the developing fetus or infant brain of progressive damage caused by spasm cerebral palsy mainly due to the cone system damage,is the most common type of cerebral palsy,accounting for 75%of the whole.Despite initial damage in the brain,but the symptoms and treatment in muscle levels(central nerve,muscle,muscle).Skeletal muscle abnormalities are common in children with spastic type cerebral palsy,its potential mechanism is not clear,in the treatment of muscle cramps would be a great challenge.With the deepening of research,preparation and further research in the future using animal models has been called one of the important means of brain spasm type.Therefore to establish a practical animal model of cerebral palsy in the experimental study of disease and is of great significance to the clinical work,a better understanding of the muscle between the clinical doctors and therapists,can make the treatment even novel treatment strategies.Such knowledge for us to go to the diagnosis and treatment from abnormal development and adjustment of the process capability is the most basic of muscle dysfunction.Therefore,it is necessary to explore the central nerve injury of skeletal muscle spasm after at cellular or molecular level exactly what are the specific effect on the skeletal muscle,further explain the mechanism transformation of molecules or cells,muscle fiber configuration for future gene therapy targets to provide new theoretical and experimental basis,for spastic muscle function provides scientific theoretical guidance.Objective Paralysis by copying spasm type model to discuss the long-term training cases spastic type paralysed rats gastrocnemius and soleus BDNF expression changes and the relationship of the level of methylation,the experiment by Western blotting and RT-PCR reaction observation BDNF protein levels and changes of BDNF m-RNA expression.Methods Choose healthy newborn 180 grade clean Wistar rats,7 days were divided into control group and the control group and experimental group 60,control group adopts improved HIE building method,experimental animals ligation of bilateral common carotid arteries,under 92%nitrogen,8%oxygen environment 1.5 h,control only bilateral neck skin incision,separation,common carotid arteries ligation not cut,and then suture the wound,not placed in the anoxic environment,after the building of experimental rats randomly selected 10 only do neuroethology test and pathology examination,identification of spastic type paralysis in animal models,the experimental group 4 weeks swimming,once a day,each time for 20 minutes until relatively exhaustion.First of all the animals for a 1 week preliminary experiments,rats swimming time determination using the timer,training time range,9:00am-12:00p.m.,swimming pool round transparent,wall smooth,45 cm in diameter,depth of 70 cm,the water temperature is 34-36 ?,ensure water quality fresh.Experimental exhaustion standard for water rat nasal tip,body sinking,to the surface again for more than five seconds,and three times in a row,the animal's movement during the course of the swimming ability and activity state observation records,choose good mental state and so on spastic paralysis type rats as the ultimate inclusion criteria.From the control group and the control group respectively for each 10 rats in HE staining,brain tissue pathological morphological changes under electron microscope observation,respectively from the control group and the control group,10 rats from each detected using immunohistochemical method respectively in two groups of 1,3,7,15 days of BDNF protein content in the soleus and gastrocnemius.4 weeks after the control group and experimental group 10 rats from each respectively by Western blotting and RT-PCR method for detecting the soleus and gastrocnemius and m-RNA expression of BDNF protein,in the control group and experimental group soleus total methylation level and the correlation of BDNF m-RNA expression.Results(1)general situation:after operation,the control group of rats appeared different degree of postoperative depression,limbs activity decreases,unresponsive,eat less,and the control group of rats in the recovery of 7 hours,spirit,limbs activity increased,responsive,eating.(2)HE brain pathology morphological changes:ischemia hypoxia caused by pathological changes mainly focused on the damage bilateral hippocampal CA3 area,control the rat hippocampal CA3 area and cortical neurons is rich,regular,dense,rich matrix,dyeing uniformity.Osteoporosis,control group rats had hippocampal CA3 region and cortex neuron density decreased,arranged loosely,gap increases,the matrix color becomes shallow,appear a large number of vacuoles and cell debris,some cells pyknosis hyperchromatic.(figure 1).(3)immunohistochemical results:brain were observed under optical microscope,reaction material is tan are observed in the cytoplasm of immunohistochemical staining positive cells.Control the hippocampus dentate gyrus,cortex BDNF protein positive closely packed,contour and the structure is clear.As shown in figure 2 A,B,and after building,BDNF protein positive cells in control group scarce,cell swelling,disordered arrangement of cells,inflammatory cells infiltration and proliferation of glial cells.(4)the neuroethology changes:suspension experiment,the control group rats off time was 1.70±1.30 points and the control group was 4.00±1.00 points and differences between the two groups have statistical significance(p<0.001);Slope experiment,the control group rats to head up time was 12.40±3.40 seconds,control group was 3.30 ±1.70 seconds,the differences between the two groups have statistical significance(p<0.001);Kuang field experiment,the control group was 3.80+/-1.20 points,the control group was 12.40±3.40 points,the differences between the two groups have statistical significance(p<0.001);Ere rejected reaction experiment,the control group on average score was 1.10±0.90 points,control group was 5.00±1.00 points,differences between the two groups have statistical significance(p<0.001);Muscle tension tests,the control group was 7.00±3.00 points,control group was10.00± points,the differences between the two groups have statistical significance(p<0.01);Involuntary movement test,the control group in average score was 8.80±1.20 points,control group was 10.00±10.00,and the differences between the two groups have statistical significance(p<0.01);In the inclined plate test,4 control group rats both positive,the other 6 appears only a positive,control without positive reaction;Compared with the control rats,the control group rats unresponsive,autonomous activity decreases,slow speed,flexibility and stability,reduce the abnormal muscle tone and etc.(5)muscle tissue immunohistochemical results:control group in 1,3 days soleus BDNF protein content increased significantly,but gradually decreases over time,7,15 days of BDNF levels were lower than control group,the differences between the two groups have statistical significance(p<0.05);1,3,7 and 15 days of the gastrocnemius muscle BDNF protein content and control all close,no statistically significant difference in the two groups(p>0.05);(6)the experimental group and the control group than the gastrocnemius BDNF m-RNA and protein expression changes is not statistically significant(p>0.05);Compared to the experimental group and control group in the soleus BDNF protein expression levels and m-RNA are rising,differences between the two groups have statistical significance(p<0.05);(7)the experimental results from the study measured the soleus DNA methylation level was significantly lower than control group(p<0.001),the experimental group the soleus m-RNA was significantly higher than that of control group(p<0.001).Control the soleus DNA methylation level and mRNA expression was very weak correlation(r=0.076,p<0.001),the experimental group the soleus DNA methylation level and mRNA expression quantity has very weak correlation(r=0.076,p<0.001).Conclusions(1)the bilateral carotid artery in the newborn rats of days of age can successfully construct the spasmodic paralyzed animal model.(2)hopoxic ischemic stress in rats can produce an endogenous self-protection mechanism.(3)rehabilitation training can increase the gene transcription of the soleus muscle,leading to an increase in BDNF expression.(4)rehabilitation training can improve the expression level of BDNF m-RNA in the soleus muscle and reduce the level of DNA methylation. |
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