The Changes Of Cytoskeletons In The CA1 Subfield Of Rat Hippocampus After Transient Global Ischemia

Objective:Cerebral ischemic injury is a multifactorial patholgical process and often leads to cognitive or memory disturbances,but there is still no effective therapy.Transient global ischemia is thought to trigger delayed neuronal death and the abnormalities of cytoskeleton in the CA1 region.However,the underlying mechanisms are not completely understood.Microtubules and F-actin are two important cytoskeletons that play important roles in the growth and development of neurons.In this study,we adopted a transient 4-VO cerebral ischemia model to detect the morphological changes of microtubules and F-actin at different time points after ischemia of reperfusin.At the same time,we detected the expression of F-actin and microtubules associated proteins by western blot.Methods:In this study,we adopted a transient global ischemia model occluding for 13 min.Immunostaining against Neun and histofluorescence labeling of F-JB was used to examine the extent of neuronal cell loss;Immunostaining of MAP2 and β-Tubulin to investigate the change of microtubules;F-actin was revealed by phalloidin staining.The change of β-Tubulin、acetylated α Tubulin、cofilin and p-cofilin were detected by Western blot analysis.The morphological changes of microtubules and F-actin were detected directly by in-vitro experiments.Results:1.The delayed death of CA1 pyramidal cells has been confirmed by immunostaining against Neun and histofluorescence labeling of F-JB.The loss became remarkable at day 2 ~ 3,and reached a maximal level at day 7 after ischemia/reperfusion.2.Phalloidin staining revealed that the damage to F-actin in the CA1 subfields became apparent at day 2,and became gradually severe from 3 to 7d.The typical puncta of F-actin were rarely observed in the CA1 stratum radiatum at 2 ~ 3 d post-ischemia/reperfusion.3.Western blot revealed that the total level of cofilin was increased at day 3 and 7 after ischemia/reperfusion,and the phosphorylation of cofilin(p-cofilin)was easily detected in sham group,but rarely observed after ischemia/reperfusion.4.A moderate reduction of β-Tubulin was noted at day 1 after ischemia/reperfusion,which was followed by a sharp loss at day 2.From the 3rd day,the β-Tubulin in the CA1 subfield became progressively decreased in the dendritic processes.5.Western blot analysis of the whole hippocampus show little difference of β-Tubulin between sham and ischemia/reperfusion groups.However,as compared to the sham group,acetylated α Tubulin showed a slight decrease at day 3,at day 7,the decrease of acetylated α Tubulin became more serious.6.The staining of F-actin and MAP2 were decreased after treatment with cytochalasin D,and the fragmented F-actin and MAP2 became more serious with longer time;nocodazale resulted in similar damage to F-actin and MAP2.Conclusions:1.The F-actin and microtubules in the hippocampus became progressively damaged after transient global ischemia,which is higly consistent with the delayed neuronal death in time course and spatial distribution.2.The activation of cofilin may trigger depolymerization of F-actin after teansient global ischemia.3.The microtubule acetylation decreased,may related to microtubule destabilization.