Fasudil Hydrochloride Improve Learning/Memory Abilities Of AD Rats, And Its Mechanism

BackgroundAlzheimer disease (AD), most common and important neuro degenerative disease in the eldly people is clinically characterized by cognitive impairment and behavioral disorders, especially spatial learning and memory deficit. This terrible disease has been proved to be the fourth leading cause preceded by heart disease, cancer and stroke as the world population becoming old and life expectancy increasing. Mounting evidence shows that several pathologic features are tightly connected with AD including amyloid plaques, neurofibrillary tangles (NFT) and neuronal death mainly at frontal lobe and hippocampus. However, the etiological factors and pathogenesis of AD are still largely unknown, and there is no effective therapy method, therefore more deeply studies on it are still eagerly needed. Now studies have showed that the progressive impairment of learning/memory that typifies AD is thought to result from synaptic dysfunction and synaptic structure and function is considered the best pathological correlate of cognitive impairment in AD patients. So synaptic dysfunction can be considered an important target for treatments aimed at improving cognitive deficits and preventing the development of AD. Fasudil hydrochloride (FH), an active metabolite of fasudil is a typical Rho kinase (ROCK) inhibitor. It been reported to improve cognitive deficits in aged rats and in rats with cerebral ischemia. However, whether FH exerts anti-dementia effects in AD models remains unclear, and few studies have focused on the mechanism(s) of how it exerts anti-dementia properties. In this study, an Intracerebroventricularly (ICV) injected streptozotocin (STZ) in vivo model was used to observe the effect of FH on learning/memory, as well as its mechanism(s).ObjectiveThis study discusses the connections between the spatial learning and memory deficit and synaptic changes in hippocampus in AD rats, as well as the molecular mechanisms about FH's anti-dementia effects through testing the learning and memory abilities, the ultramicrostructure of CA1region in hippocampus, the gene and protein expression of synaptophysin (SYP) and the phosphorylation level of LIMK2and cofilin in hippocampus of ICV STZ rats and FH's effects to these factors. The current study provides a valuable foundation for further study of how FH can be used for the treatment of AD.MethodsMale Sprague-Dawley rats (weights250-300g) were obtained from the Experimental Animal Centre of Central South University (Hunan, China). We randomly divided the rats into4groups (n=12in each group):(1) sham-operated group (control);(2) sham-operated followed by FH administration group (sham+FH);(3) STZ-treated group (STZ); and (4) STZ treatment followed by FH administration group (STZ+FH). Rats in the STZ and STZ+FH groups received2divided doses of STZ (1.5mg/kg) intracerebroventricularly on days1and3, whereas control and sham+FH group rats were given citric acid/sodium citrate buffer. Rats in the sham+FH and STZ+FH groups were then treated intraperitoneally with FH (10mg/kg) for4weeks, and rats in the STZ and control groups were treated with saline. Learning and memory were measured using the Morris water maze The neuronal morphous in CA1region of the hippocampus were observed by using HE staining and Nissl's staining. The synaptic ultrastructure in the CA1region of the hippocampus was observed using electronic microscopy and the texture parameters of Gray type I synaptic interface in CA1region of the hippocampus was analysised by using transmission electron microscope and image analyzer. The expression of synaptophysin (SYP) was measured using real-time polymerase chain reaction and western blot analyses; the expression of p-LIMK2and p-cofilin were also detected using western blot analysis. Statistical analyses were conducted using SPSS version18.0software. Data are expressed as mean±(SD). Escape latency and swimming speed were analyzed using repeated measures2-way analysis of variance followed by the least significant difference test. Times of crossing were analyzed using the Wilcoxon signed-rank sum test, and the remaining variables were analyzed by performing separate measures of1-way analysis of variance followed by the least significant difference test. Values of p<0.05were considered significant.Results1. Compared with control rats, the escape latency of STZ rats on day2to day5was significantly higher, indicating poorer learning in the STZ rats; and the time they spent in the target quadrant and the number of times that they crossed the former location of the platform significantly decreased, reflecting impairment in memory. These disrupted performance was significantly improved by treatment with FH in the STZ+FH group. Sham+FH rats exhibited performance similar to that of control rats in the MWM test, indicating that FH had no effect on learning and memory in the control group per se.2. Compared with control rats, the neurons in hippocampal CA1region of STZ rats were decreased and distributed irregularly; and the degeneration of the ultrastructure of neuronic organelles in CA1region were more significant. After FH administration, In FH+STZ group, showed increased neurons in the hippocampal CA1region, which is also distributed more regularly; and the damages of the ultrastructure of neuronic organelles in CA1region are ameliorated.3. Compared with control rats, the SYP expression in the STZ group was significantly decreased at gene and protein levels, and STZ+FH group rats had a higher level of SYP mRNA and protein expression than the STZ rats.4. In STZ rats (compared with those in the control group), the number of synapses decreased along with the proportion of negatively curved and perforated synapses. In addition, the width of the synaptic cleft increased, the thickness of the PSD decreased, the length of synaptic appositions shortened, and the curvature of the synaptic cleft diminished. FH reversed these changes in the STZ+FH group.5. Compared with control rats, STZ induced the upregulation of phosphorylation levels of LIMK2and cofilin. However, the administration of FH decreased the phosphorylation levels of both LIMK2and cofilin in the STZ+FH group.Conclusion1. We injected STZ into the rat's lateral ventricles in order to found the animal model of AD. This rat model of AD had a good reproducibility and stabilization, which is an ideal model of further studies on AD.2. STZ induced structural impairment of neurons and synapses in hippocampus may be responsible for the cognitive impairment in STZ rats. FH could improve learning and memory abilities in STZ+FH rats by ameliorating structural and function of neurons and synapses.3. FH might protect synaptic structure and function by the molecule mechanism that it induced dephosphorylation (inactivation) of LIMK2and subsequent dephosphorylation (activation) of cofilin.