Effect Of Chronic Cerebral Hypoperfusion On Cognitive Impairment And Morphological Structure In Mice

Objective: To investigate the effect of chronic cerebral hypoperfusionon cognitive impairment and morphological features in mice brain, as wellas exploring the relationships and its mechanisms between Alzheimer’sdisease and chronic cerebral hypoperfusion. The outcome of the study willprovide laboratory evidence for the prevention and treatment of AD.Methods: Three months old C57mice were subjected to chroniccerebral hypoperfusion with bilateral common carotid artery permanentligation or sham operation. Anilin blue was irrigated to prove theeffectiveness of the model. Morris water maze and Elevated plus-maze testwas performed to test learning memory and anxious emotions. Thepathological changes such as neurons, neuronal apoptosis, astrocytes, β-siteAPP cleaving enzyme1(BACE1), γ-secretase presenilin-1(PS-1)andnicastrin(NCT), anterior pharynx defective-1(Aph-1), presenilin enhancer-2(Pen-2), β-amyloid (Amyloid β peptide, Aβ) deposition was examinedby immunohistochemical staining(IHC). Western blotting(WB) wasemployed to test the levels of apoptosis-related proteins, GAP43, PSD95, GFAP and BACE1in brain. The co-expression of AQP4and GFAP wereobserved by immunofluorescent staining. ELISA analysis was used todetect the Aβ40and Aβ42level.Results:1.The result of anilin blue irrigation: After the anilin blueirrigation the model group cerebrum was not dyed, while the control groupwas dyed dark blue.2.Behavioral test:1）Elevated plus-maze test: Compared with the shamgroup, open arm entries and open arm time in the model group weresignificantly decreased(P<0.05).2) Morris water maze test: The shamgroup and the model group had similar escape latency(P>0.05) and pathlength(P>0.05) during the first day of visible platform tests. In the hiddenplatform experiments, the escape latency and path length in sham groupand1-m model group were decreased with swimming training, while theywere not significant improvement in4-m and6-m group. The4-m and6-mmodel group had a significantly longer escape latency and path length toget the platform(P<0.01) in relative to sham group. However, the escapelatency increased significantly (P<0.05), whereas we did not find anystatistical difference in the1-m model group about path length（P>0.05). Inaddition, the platform-passing times in the model group was significantlylower than the sham group(P<0.01).3.Morphological test:1) Immunohistochemical staining: The numberof neurons in the hippocampus (P<0.01)and cortex (P<0.01)were significantly decreased, and the longer ischemia last, the fewer neurons was.Immunohistochemical staining of MAP2and Tublin III showed that theprocesses in the hippocampus and cortex was long and arranged neatly,regularly in the sham group, while they were shorter, apparently segmental,disorganized, irregular and deeply stained. Astrocytes were proliferatedafter chronic cerebral hypoperfusion and reflect the changes of the cellquantity, cellular hypertrophy and processes. We found that some of themwere overlapped and the processes became crossover, there was glial scarformation. In addition, senile plaques were selectively appeared in thecerebral cortex and hippocampus from4month. By contrast, there was nopositive plaques in the sham group. The longer ischemia keeped, the largerof the sp espressed. Moreover, the structure was more deep-stained andcompact. The6month group appeared intracellular Aβ deposition in thelateral geniculate body. Compared with the sham group, analysis showedthat the IOD of PS-1, NCT and Aph-1increased significantly from1monthgroup (P<0.05). But the difference in PEN-2was not statisticallysignificant(P>0.05). The increase was also observed in the BACE1expression and was statistically significant(P<0.01).3) TEM analysisrevealed that the ultra-structure of neurons and astrocytes in model groupwere changed, such as karyolysis, swollen of cytoplasm, and mild oedemaaround cells while it was not displayed in sham group. The vascular wasalso edema.4)Immunofluorescent staining: The co-expression of AQP4 and GFAP was very weak in the sham group. And the expression of AQP4and GFAP were significantly increased in model group（P<0.01), comparedwith the sham group.4.Western blot: The expression of Bax(P<0.01), Caspase-3(P<0.01),GFAP(P<0.05) and BACE1（P<0.05) were significantly increased, whilethe Bcl-2protein and PSD95were decreased dramatically. Meanwhile,chronic cerebral ischemia had no significant effect on GAP43（P>0.05).5.ELISA assay: Compared with the sham group, the Aβ40and Aβ42level were gradually increased in model group, and the difference wasstatistically significant (P <0.01).Conclusion:1.Chronic cerebral ischemia potentiates the memorydeficit and anxiety. The initial memory deficit emerge from1-m modelgroup, and it exists time-dependent manner.2.Chronic cerebral hypoperfusion leads to loss of neurons by affectingthe apoptosis-related proteins in mice.3.Chronic cerebral ischemia damages the normal neuronalmorphology and the processes, and the expression of PSD95reducedsignificantly that affects synaptic plasticity and synaptic reorganization.These pathological changes appeared from1-m, which may have aimpotant relationship with initial cognitive dysfunction.4.Chronic cerebral ischemia can activate astrocytes and increase theco-expression of GFAP and AQP4, it also can destroy the BBB. 5. Chronic cerebral ischemia can lead to Intracellular Aβdeposited,which may reduce the expresstion of PSD95significantly that affectssynaptic plasticity and integrity.6. Chronic cerebral ischemia facilitates Aβ deposition byup-regulating γ-secretase and β-secretase activity and increases cognitiveimpairment.