Study Of Cerebral Protection Of Trichostatin A Preconditioning On MCAO Rats And Its Merchanism

Part?:Study on the betterment of suture method to make MCAO model in ratsBackground and objectiveIt was reported that the middle cerebral artery occlusion (MCAO) accounts for the largest proportion of clinic ischemic cerebrovascular diseases and it often has automatic recanalization, which is similar to the pathological symptom of middle cerebral artery occlusion in rats model. Only is the animal experiment applicable for drug pre-conditioning when the selected animal model satisfies clinic need, therefore, this study used MCAO model to learn the functions to the brain tissues when Trichostatin A preconditioned.The uncertainty of cerebral infarction volume in MCAO rats even if the equal time of middle cerebral artery occlusion is worst defects of this model. To make stable MACO model is the good foundation for later researches. This study used modified suture method to make stable MACO, thus building the base of sequent experiment.Material and methodsExperimental animals:Healthy clean SD male rats in sham operation group and cerebral ischemia-reperfusion group,6 rats per group.Animal model:The suture method was used to block the middle cerebral artery for 90 mins, and then a reperfusion for 24 hours was performed in rats. For rats in sham operation group, the suture was merely put in external carotid artery.Measurement indicators:neurological function disfunction score, cerebral infarction volume, pathological issue change in ischemic rat brainTesting methods:The hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; Zea-Longa method was used to assessment the neurological disfunction status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume of rats.Statistical analysis:Excel 2007 was used to build dataset and for data entering. Median and Inter-quartile were used to describe neurological function scores and two-independent sample t test was used to compare the difference in neurological function score between sham group and cerebral ischemia-reperfusion group.'P<0.05'was selected as statistical test level. All of statistical analysis was implemented using the SPSS 17.0.Results(1)The result of HE staining between sham group and cerebral ischemia-reperfusion group was different. The damage of the cerebral ischemia-reperfusion group was great and the sham group had no.(2) Substantial differences in neurological function score between sham group and cerebral ischemia-reperfusion group at I/R6,12 and 24 hours (P<0.05).(3) TTC staining showed that rats in cerebral ischemia-reperfusion group had clear infarction when compare with the lack of brain infarction in sham control.ConclusionThe modified suture and other methods are reliable in making MACO model in rats.Part?:Study of TSA preconditioning on MCAO rats and the relevance to IL-1?and bcl-2Background and objectiveCerebral ischemia-reperfusion injury is a common clinically pathological symptom. Ischemic preconditioning (IPC) is the most powerful intrinsic nerve protective phenomenon that has been reported currently. Because ischemic preconditioning is an injurious preconditioning, it is necessary to seek for a drug-based preconditioning as a substitute. As a new generation HDACIs, whether does TSA has an cerebral protection effect on cerebral ischemia-reperfusion injury and its mechanism remain unreported. The primary objective of this study aims to examine the cerebral protection impact of TSA preconditioning on MCAO rats and to examine the relationship between cerebral protection of TSA and IL-1?and bcl-2.Material and methodsExperimental animals:Healthy SD male rats. Four groups were determined as followed:control group,1%DMSO preconditioning group (DMSO group), TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.Measurement indicators:neurological function defect score, cerebral infarction volume, cerebrospinal fluid and blood IL-1?, bcl-2 positive cell count.Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1?in cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the level of bcl-2 protein.Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1?, and bcl-2 count. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'?=0.05'was selected as statistical test level. All of statistical analysis were implemented using the SPSS 17.0.Results(1) The results of analysis of variance showed that there was no significant difference in neurological function scores between four groups (control group, DMSO group, TSA 1 group and TSA 2 group) at all time points (P>0.05) except for that TSA 1 group had lower score than the control group at 6 hours (P<0.05).(2) Results showed that the cerebral infarction volume ratio of rats in TSA 1 group was less than that of the other three groups (P<0.05); the rats in TSA 2 group and DMSO group had less cerebral infarction volume than those in control group (P<0.05), but did not differ from eAc-H other (P>0.05).(3) Results displayed that the blood and the cerebrospinal fluid IL-1?of rats in TSA 1 group was lower than that in control and DMSO groups (P<0.05), but show no statistical difference from that in rats of TSA 2 group; The blood IL-1?of rats had no significant difference between TSA 2 group and DMSO group (P>0.05), but both were lower than those for control group (P<0.05).(4) Pearson correlation showed the cerebral infarction volume highly correlated with blood and cerebrospinal fluid IL-1?(P<0.05), having a correlation coefficient of 0.841 and 0.618, respectively.(5) Results showed statistical significance between four groups at eAc-H time point. The multi-comparisons revealed that at the 6h, the bcl-2 positive cell count of rats in control group was lower than the other three groups, the rats in DMSO group and TSA 2 group had less bcl-2 positive cell number than those in TSA 1 group (P<0.05); at the 12h, the first three groups had no statistical difference between themselves in bcl-2 positive cell count (P>0.05), while all of them had smaller bcl-2 positive cell count than TSA 1 group (P<0.05); at the 24h, the bcl-2 count of control group was lower than the rest three groups, and DMSO and TSA 2 groups were also less than that of TSA 1 group; and at the 48h, a statistical difference existed between each pair (P<0.05).Conclusion(1) TSA preconditioning can reduce cerebral infarction and have a dose-depended effect to lower level of IL-1?in blood and cerebrospinal fluid, and higher sum of anti-apoptosis protein bcl-2 cells.(2) The protection effect of TSA cerebral ischemia-reperfusion injury is related to the level of IL-1?and anti-apoptosis protein bcl-2, suggesting a cerebral protection of TSA preconditioning to local cerebral ischemia-reperfusion injury in rats.Part?:Study of TSA preconditioning on MCAO rats and the relevance to HDAC?Ac-H3 and Ac-H4Backgroud and objectiveCerebral drug pre-conditioning is based on ischemic pre-conditioning and uses drug to stimulate the body to produce endogenous protective secretion, so as to protect brain. Histone deacetylases inhibitors (HDACIs) increase the plasticity of neuron after ischemia and can promote the rehabilitation of function. The previous study reported that TSA preconditioning can decrease the level of IL-1?level in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection is unknown. This study was aimed to examine the relationship between TSA pre-conditioning and HDAC, Ac-H3 and Ac-H4 in rats receiving focal cerebral ischemia-reperfusion injury and explore the association between related cerebral protective factors.Material and methodsExperimental animals:Healthy SD male rats. Four groups were determined as followed:control group, TSA 0.1mg/kg preconditioning group (TSA 1 group), and TSA 0.03mg/kg preconditioning group (TSA 2 group) and 1%DMSO preconditioning group (DMSO group). Each group included four sub-groups for reperfusion for 6,12,24 and 48 hours, respectively,6 rats per sub-group.Animal model:The modified suture method was used to block the middle cerebral artery for 90min, then did a reperfusion for 6,12,24 and 48 hours, respectively, thus creating the cerebral ischemia-reperfusion animal model. Four groups were determined as followed: control group, TSA 1 group, TSA 2 group and DMSO group. TSA and DMSO were injected through caudal vein 20 minutes before ischemia implementation. The same volume of normal saline was injected into rats in the control group.Measurement indicators:HDAC level in infracted brain issue, Ac-H3 and Ac-H4 level.Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of Ac-H3 and Ac-H4 in the infracted brain issue; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1?in cerebrospinal fluid and blood; and the immunohisto-chemistry method was used to detect the count of bcl-2 protein positive cell.Statistical analysis:Excel 2007 was used to build dataset and for data entering. Two-way analysis of variance was used to test the main effects of treatment factor, time factor and their interaction on HDAC level, Ac-H3 and Ac-H4. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. Spearman correlation was used to examine the relationship between related cerebral protective factors. The'?=0.05'was selected as statistical test level. Statistical analysis was implemented using the SPSS 17.0.Results(1) After receiving a cerebral ischemia injury, the HDAC level grew over the time of reperfusion in a linear way, with the linear equation HDAC=0.116+0.060×time (P<0.05). Two-way analysis of variance showed that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Multiple comparisons between four groups revealed:TSA 1 group had lower HDAC level than control group and DMSO group (P<0.05); the HDAC level in TSA 2 group and DMSO group were lower than control group (P<0.05); and there no statistical difference between other groups (P>0.05).(2) For Ac-H3 and Ac-H4, two-way analysis of variance showed that that the main effects of treatment factor and time factor were statistically significant (P<0.05), but the effect of interaction of treatment and time was insignificant (P>0.05). Both Ac-H3 and Ac-H4 increased from 6 hours and reAc-Hed the peak at 12 hours, then decreased gradually at all of four groups.Multiple comparisons for Ac-H3 showed that the level of Ac-H3 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group and DMSO group had higher Ac-H3 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).(3)Multiple comparisons for Ac-H4 showed that the level of Ac-H4 in TSA 1 group was statistically higher than the other three groups (P<0.05), and TSA 2 group had higher Ac-H4 level than control group (P<0.05). There no statistical difference between other groups (P>0.05).(4)The bcl-2 positive cell count significantly correlated with HDAC, Ac-H4, with the Spearman rank correlation coefficient of -0.775 and 0.346 (P<0.01). The correlation coefficient between Ac-H3 and Ac-H4 was 0.800 (P<0.01). The rest of correlation were not statistically significant (P>0.05).(5) Ac-H3 had a higher correlation with cerebrospinal fluid IL-1?and blood IL-1?(rs=1.0 and 0.8, P<0.01); the cerebrospinal fluid IL-1?highly correlated with Ac-H4 (rs=0.8, P<0.01); and the correlation coefficient between cerebrospinal fluid IL-1?and blood IL-1?was 0.8 (P<0.01). The rest of correlation were not statistically significant (F>0.05).Conclusion(1) The HDAC level rise over the time of reperfusion after the rats receive cerebral ischemia injury. TSA can reduce the expression of HDAC.(2) TSA pre-conditioning can raise the level of histone Ac-H3 and Ac-H4 in cerebral issue. Histone Ac-H3 positively associate with cerebrospinal fluid IL-1?and blood IL-1?; and cerebrospinal fluid IL-1?has a high and positive correlation with Ac-H4.Part?:Study of Association between age and TSA preconditioning and related factors (IL-1?, bcl-2)Backgroud and objectiveAs China step into the aging society, ischemic cerebral vasnacular diseases have become more and more frequent than before. The implementation of family planning policy has attracted more and more attention to the health of singe child for eAc-H family. The cerebral function injury from neurological system diseases is one of important and unresolved pediatric research problem. Few studies involve the epigenetics of ischemic cerebral vanascular diseases although there are lots of researches exploring the pathological characteristics and treatment. It has been reported that TSA preconditioning can decrease the level of IL-1?level in the cerebrospinal fluid and raise the expression of apoptosis protein bcl-2, thus yielding protection of cerebral ischemia-reperfusion injury. However, the mechanism of its cerebral protection at different ages of rats keeps unreported. To deepen the mechanism of cerebral ischemia-reperfusion injury at rats of different ages, this study aimed to examine the relationship between age and cerebral protection of TSA preconditioning and related factors (IL-1?, bcl-2).Material and methodsExperimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group.Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.Measurement indicators:neurological function score, cerebral infarction volume, cerebrospinal fluid and blood IL-1?, bcl-2 positive cell count.Testing methods:Zea-Longa method was used to assessment the neurological function status; triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volume ratio of rats; the hematoxylin-eosin (HE) staining was used to observe pathological changes of ischemic brain issues; the enzyme-linked immunosorbent assay (ELISA) was used to test the level of IL-1?in cerebrospinal fluid and blood; and the immunohistochemistry method was used to detect the count of bcl-2 protein positive cell.Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in neurological function score, cerebral infarction volume ratio, cerebrospinal fluid and blood IL-1?, and bcl-2 positive cell count between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The '?=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.Results(1) One-way analysis of variance revealed that there was no statistical difference in Zea-Longa neurological function score between three age groups at 6h,12h and 24h (P>0.05).(2)No significant difference in modified neurological function score were observed between three age groups at 6h and at 12h (P>0.05); but there the differences at 24h were statistically significant between three age groups. The mult-comparion between three age group at 24h showed, the scores of old group were different from those of young group (P<0.05), and there no significant difference between other groups (P>0.05).(3)The total balance scores of young group were obviously lower than those of the other age groups (P<0.05); the total sport scores of young group differed from those of old group (P<0.05); The total reflection scores of young group and adult group were less than those of old group (P<0.05); the total sensation scores of three age groups were not significantly different from each other (P>0.05).(4) One-way analysis of variance showed that no statistical difference was found in cerebral infarction volume between three age groups (P>0.05).(5) One-way analysis of variance showed that rats of three age group showed no significant difference in cerebrospinal fluid and blood IL-1?level (P>0.05).(6) One-way analysis of variance showed that the bcl-2 count differed between three age groups (P<0.05). Multi-comparisons between three groups showed that the old group had higher bcl-2 count than the other two groups (P<0.05), but no significant difference was detected between young group and adult group (P>0.05). ConclusionAge is related to the cerebral protection of TSA pre-conditioning. The different function of the level of cerebrospinal fluid IL-1?and the bcl-2 count of the three groups changed with TSA preconditioning.Part?:Study of Association between age and TSA preconditioning and related factors (HDAC, Ac-H3 and Ac-H4)Background and objectivePrevious study reported that the occurrence of ischemic resistance is the adoptive chain of reaction, including genetic dependent reaction and non-genetic dependent reaction, the existed decoration of protein translation. The pre-conditioning is the re-programming process of organs to ischemia reaction at genetic level. Histone acetylation and deacetylation is the the most explored decoration mechanism after protein translation. Prior studies suggested that TSA pre-conditioning can produce protection to cerebral ischemia-reperfusion injury. To deepen the mechanism of cerebral ischemia-reperfusion injury in rats of different age, this study aimed to explore the relationship between age and HDAC, Ac-H3 and Ac-H4 in rats those receive TSA preconditioning, so as to provide base for the exploration of new treatment method.Material and methodsExperimental animals:18 healthy and clean SD male rats that received TSA preconditioning (0.1mg/kg) were divided into three groups based on their age:young group, adult group and old group. Animal model:The modified suture method was used to block the middle cerebral artery for 1.5 hours, and then a 24-hour reperfusion was done, thus creating the cerebral ischemia-reperfusion animal model. TSA was injected through caudal vein 20 minutes before ischemia implementation.Measurement indicators:HDAC level in infracted brain issue, acetylaced H3 and H4 level.Testing methods:The colorimetry method was used to test the level of HDAC in the infracted brain issue; and the Western blotting was used to test the level of acetylaced H3 and H4 in the infracted brain issue.Statistical analysis:Excel 2007 was used to build dataset and for data entering. One-way analysis of variance was used to compare the difference in HDAC, Ac-H3 and Ac-H4 between three age groups. Non-parametric test or robust test was used when the assumptions of analysis of variance were violated. The'?=0.05'was selected as statistical test level. Statistical analysis was performed using the SPSS 17.0.Results(1) The HDAC level showed significant differences between three age groups (P<0.05). The old group had higher HDAC level than young and adult groups (P<0.05); there was no significant difference in HDAC level between the young group and the adult group (P<0.05).(2) Rats of three groups showed statistical difference in Ac-H3 level (P<0.05). The old group had lower Ac-H3 level than the other two groups (P<0.05), but no significant difference was detected between the young group and the adult group (P>0.05). There was significant differences in Ac-H4 level between three age groups (P>0.05).(3) Rats of three groups showed no statistical difference in Ac-H4 level (P<0.05). The old group had higher H4 level than the other two groups, but no significant difference was detected of the three groups (P>0.05).Conclusion (1) Age has an effect on the expression of HDAC in rats that receive TSA pre-conditioning.(2) Age affects the expression of Ac-H3 in rats receiving TSA pre-conditioning but has no effect on the expression of Ac-H4.