| Cerebral ischemia is a disease aroused by reduced blood flow resulted from blood vessels blockage of the brain.It is one of the main diseases leading to disability in the world today.Until now,however,there are no other effective clinical drugs for the treatment of cerebral ischemia except the tissue plasminogen activator(tPA).To develop effective treatment strategies,we need to have a deeper understanding of cerebral ischemia.However,the knowledge about the pathophysiological process of cerebral ischemia and the related molecular mechanisms is poor at present.All the time we give priority to the therapies conducted in the acute phase of cerebral ischemia,but sometimes the effect of treatment in the acute phase is limited,or even have strong side effects.Thus,some ischemic strategies applied in the subacute phase showed up.Although the field is not substantial till now,it might be a very promising research direction in treatment of cerebral ischemia in the future.Histone deacetylase(HDAC)is a family consisting of some proteases.It exerts its effect mainly through modifying the acetylation of histones,regulating the structure of chromosome and thus changing the expression of genes.In addition,some of its subtypes also control the acetylation of other molecules in the cells leading to a corresponding biological effect.In the past,a lot of studies including the studies of our laboratory have proved that the HDAC family is closely related to neurogenesis,learning and memory.However the studies about the role of HDAC in the process of the cerebral ischemia were rare.Therefore,we designed two parts of this study:(1)Study that whether the delayed treatment of HDAC inhibitor suberoylanilide hydroxamic acid(SAHA,Vorinostat)after cerebral ischemia promotes functional recovery,and based on its function,study the change of the key subtype of HDAC after cerebral ischemia and the influence of intervention of the subtype on sensorimotor function.(2)Study the cellular and molecular mechanisms of HDAC2 which influences function recovery after cerebral ischemia.Chapter 1: Delayed intervention of HDAC2 affects functional recovery after cerebral ischemia.In order to study the effect of delayed intervention of HDAC on the functional recovery after cerebral ischemia,we applied the HDAC inhibitor SAHA.SAHA was administrated through microcannula at d 3-9 after photothrombotic stroke,and grid-walking task and cylinder task were conducted at d 10,17 and 24 to assess sensorimotor function.We found that administration of SAHA significantly reduced foot faults in the grid-walking task and asymmetry index in the cylinder task,ameliorated the ischemia-induced impairments of sensorimotor functions.SAHA can inhibit three subtypes of HDAC family(HDAC1,2 and 6),but which subtype is relayed on by the effect of delayed administration of SAHA? We first verified the effect of delayed inhibition of HDAC6.We gave animals Tubastatin A(TubA),a specific HDAC6 inhibitor,for seven consecutive days through microcannula.Then we test the activity of HDAC6 in the inject zone and found a decrease in HDAC6 activity,demonstrating that TubA could down-regulate HDAC6 activity.Then we used protein samples from the peri-infarct zone of the ischemic mice at d 1,3,7 and 14 respectively,detected the expression of HDAC6 and found no obvious change.Finally,TubA was administrated through microcannula at d 3-9 after photothrombotic stroke,and grid-walking task and cylinder task were conducted at d 10,17,24 and 31 to assess sensorimotor function.However,we found no change in sensorimotor function after TubA administration.Thus,delayed treatment of SAHA improves functional recovery after stroke,and the effect is not HDAC6 dependent.In order to study whether the HDAC2 is changed after cerebral ischemia,we conducted the following experiments.First,we prepared and maintained primary cultures of cortical neurons for 9 d,and used glutamate to induce excitotoxicity.Then we extracted protein from neurons at 1 h,2 h,3 h and 4 h respectively after induction and detected the expression of HDAC2.We found that the expression of HDAC2 decreased significantly at 3 h and 4 h after induction.Whether the decrease could turn over after a longer time? So we used oxygen and glucose deprivation(OGD)experiment,which is closer to real cerebral ischemia.We prepared and maintained primary cultures of cortical neurons for 8 d and exposed the neurons to OGD.Then we extracted protein from neurons at 4 h,8 h,24 h and 72 h respectively after OGD and detected the expression of HDAC2.We found that expression of HDAC2 rose at later time points,but was still lower than control.The above results showed that in the in vitro model of cerebral ischemia,the expression of HDAC2 decreased and after a short period of time gradually began to rise.Next,We investigated the effect of ischemia on the expression of HDAC2 on animals.We used protein samples from the peri-infarct zone and the infarct core of the ischemic mice at d 1,3,7 and 14 respectively and detected the expression of HDAC2.Our results showed that the expression of HDAC2 in the infarct core of the ischemic mice decreased,probably due to global cell death in this area.However,the expression of HDAC2 in the peri-infarct zone of the ischemic mice significantly increased at d 3,7 and 14 after cerebral ischemia,and such increase is a promising target for stroke therapy.Since the expression of HDAC2 increased in the peri-infarct zone,we wondered whether its activity would change after cerebral ischemia.W e used protein samples from the peri-infarct zone of the ischemic mice at d 1,3,7 and 14 respectively and detected the activity of HDAC2.As expected,HDAC2 activity increased after cerebral ischemia.Our data suggest that the expression and activity of HDAC2 increased after cerebral ischemia.We wondered whether the increase of HDAC2 expression and activity is the main target for the effect of SAHA when administrated in a delayed phase? So HDAC2 shRNA lentiviral particles(LV-HDAC2-shRNA-GFP)are recommended for the inhibition of HDAC2 expression.LV-HDAC2-shRNA-GFP could decrease the expression of HDAC2 both in vivo and in vitro.LV-HDAC2-shRNA-GFP was administrated immediately after photothrombotic stroke,and grid-walking task and cylinder task were conducted at d 7,14,28 and 42 to assess sensorimotor function.Our results showed that administration of LV-HDAC2-shRNA-GFP significantly ameliorated the ischemia-induced impairments of sensorimotor functions.Thus,inhibition of HDAC2 expression can improve functional recovery after stroke.Next,the recombinant adenovirus AD-HDAC2-Flag was constructed for the overexpression of HDAC2.AD-inactive-HDAC2-Flag was used as control.AD-HDAC2-Flag could increase the expression of HDAC2 whereas AD-inactive-HDAC2-Flag could not.AD-HDAC2-Flag and AD-inactiveHDAC2-Flag was administrated immediately after photothrombotic stroke,and grid-walking task and cylinder task were conducted at d 7,14 and 21 to assess sensorimotor function.Administration of LV-HDAC2-shRNA-GFP significantly exacerbated the ischemia-induced impairments of sensorimotor functions.Thus,overexpression of HDAC2 can exacerbate functional impairments after stroke.Chapter 2: How does delayed intervention of HDAC2 affect functional recovery after cerebral ische mia.We used protein samples from the peri-infarct zone of mice at d 1,3,7 and 14 respectively after photothrombotic stroke,detected the expression of synapsin and spinophilin and found significant decrease in both of them.Then,we administrated LV-HDAC2-shRNA-GFP or AD-HDAC2-Flag immediately after photothrombotic stroke and found that LV-HDAC2-shRNA-GFP could reverse the decrease of synapsin and spinophilin expression.These data indicated that intervention of HDAC2 expression can affect synaptogenesis after stroke.To confirm this,LV-HDAC2-shRNA-GFP was administrated immediately after photothrombotic stroke and Golgi-Cox staining was performed to show subtle morphological alterations in neuronal dendrites and dendritic spines at d 14 after ischemia.Treatment with LV-HDAC2-shRNA-GFP reversed the decrease in spine density,number of dendrites,length of dendrites and branching of dendrites.In addition,administration of LV-HDAC2-shRNA-GFP after OGD reduced the ischemia-induced cell death.Thus,delayed intervention of HDAC2 affects functional recovery after stroke probably through increasing the growth of dendrites and synaptogenesis and decreasing ischemia-induced cell death in the peri-infarct zone.Conclusion:(1)Delayed administration of SAHA promotes functional recovery after stroke probably through inhibition of HDAC2;(2)Delayed intervention of HDAC2 affects functional recovery might via increasing the growth of dendrites and synaptogenesis after cerebral ischemia. |
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