The Crosstalk Between ERK1/2and ROCKpathways On The Regulation Of The Neurovascular Unit After Cerebral Infarction

ObjectiveTo establish an animal model for permanent cerebral infarction and on basis of that,firstly, to observe the changes in the expression of the signal transduction molecules ofERK1/2and ROCK; secondly, to investigate the crosstalk of these two molecules inmodulating the survival of the neurovascular unit by activating their common downstreameffector molecule of PARP-1.Methods1.60healthy male SPF SD rats weighing250to300g provided by laboratory animalcenter in Shandong University of traditional Chinese Medicine were used for theexperiment. These rats were randomly divided into the sham operation group(S) group andMCAO (M) group. After preoperative overnight-fasting and free access to water, rats inthe latter group underwent the operation of middle cerebral artery occlusion (MCAO),while the sham group rats were disposed to the same procedure except that the middlecerebral arteries were not ligated. Both of the two groups were then divided into sixsubgroups, as1hour,3hours,12hours,24hours,3days,7days groups after the operation,with5rats in each. At their corresponding time point, rats in each group were assessed forneurological function scale and western blotting was employed to detect the level ofERK1/2, p-ERK1/2, ROCK protein.2. Another40healthy SPF male SD rats were randomly divided into five groups, thesham (S) group, the MCAO group (M), the U0126(U) group, the Fasudil(F) group and theU0126+Fasudil group. Rats in each group were given DMSO, U0126, Fasudil and U0126plus Fasudil correspondingly at the time of30minutes before and12hours after operationby injection into the caudal vein.24hours after the operations, neurological function wereassessed. After that the brains were harvested and the cerebral cortex of the infarctedhemispheres were used for tests of infarction area and the level of ERK1/2, p-ERK1/2, ROCK, PARP-1protein.Results1. Analysis of western blotting showed that, among the sham subgroups, the totalERK1/2at each time point remained unchanged, while phosphorylated ERK1/2(p-ERK1/2)protein increased with time, and peaked at24hours, declining gradually after that.Compared to the corresponding sham operation subgroup, p-ERK1/2protein tested in theMCAO subgroups were lower at time1h and3h (P<0.05), but apparently higher at12h and24h (P<0.05). It then slowly declined with time, but still be higher than correspondingsham subgroups at3d and7d (P<0.05). ROCK protein showed no difference betweensham subgroups, but exhibited an increasing with time, peaked at12h subgroup. TheROCK protein in MCAO3h,12h and24h subgroups indicated statisticaldifferences(P<0.05), but not in3d and7d subgroups (P>0.05). There were no limbdysfunctions to the sham subgroups rats. For the MCAO subgroups rats, neurological testsgot a highest score of2.8±0.45at24h, and declined to1.2±0.45at7d.2. For the second part, compared to the sham groups, p-ERK1/2, ROCK and PARP-1protein got an obvious increase for the MCAO group (P<0.05). Making a comparison tothe MCAO group, p-ERK1/2and ROCK protein increased in the Fasudil group, whiledeclined in the U0126group. While ROCK protein remained unchanged in both groups.For PARP-1protein, there were visible declining in Fasudil, U0126and Fasudil+U0126groups compared to the MCAO group, especially in the Fasudil+U0126group. For theneurological function tests, rats in MCAO group exhibit obvious symptoms, but none forthe rats in sham group. Rats in the MCAO group got a score of2.8±0.45, and the meaninfarction area was26.8±3%, both of statistical significances compared to the sham group.U0126+Fasudil group rats demonstrated the best improvement in neurological function test,compared to MCAO, U0126and Fasudil group, with a mean score of1.3±0.5(P<0.05),and infarction area of15.8±2(P<0.05).Conclusions1. ERK1/2and ROCK are two important cell signal transduction molecules and playcrucial roles in cell proliferation, growth and development. Previous studies showed thatthese two pathways mediate the survival and death of neurons. Our research showed that24hours after cerebral infarction, neurological function test got the worst, with a highestp-ERK1/2protein at the same time point, which indicated a relation between theneurological function and the activation of ERK1/2. Meanwhile, there was an increasingexpression in ROCK, so we inferred that there is a crosstalk between ERK1/2and ROCK after cerebral infarction to modulate the damages and repairmen of neurons.2. ROCK inhibitors could inhibit the activation of ROCK ERK1/2in this research, butERK1/2inhibitor didn’t inhibit the protein expression of ROCK, in view of which weinferred ERK1/2might be a downstream molecule for ROCK. Besides, we deduced thatERK1/2and ROCK might together modulate the protein expression of PARP-1to regulatethe survival and death of neurovascular unit after cerebral infarction in consideration thatboth inhibitors for ERK1/2and ROCK could affect the protein expression of PARP.