Cognitive Deficits In Rats Induced By Chronic Cerebral Hypoperfusion And Its Correlative Factor

Objective：Chronic hypoperfusion is a risk factor for age-related cognitive decline, vasculardementia（VaD）and Alzheimer’s disease（AD）as well. Studies has shown that chronichypoperfusion may lead to cognitive impairment, but the potential neurologicalmechanism is poorly understood. Permanent bilateral common carotid artery occlusion(BCCAO) can be used to establish the animal model of chronic cerebral hypoperfusionmodel. In our study, a rat model of BCCAO has been used to investigate the alterationsof neuronal damage, glial activation, oxidative stress and central cholinergic dysfunctionand try to know their relationship with cognitive which is caused by chronic cerebralhypoperfusion. This may provide a further insight into the neurobiological mechanism ofthe development of cognitive deficits under the condition of chronic cerebralhypoperfusion.Methods：Adult male Sprague-Dawley rats which were at the age6months. 1. Use the hypoperfusion rats mode which were established by BCCAO.2. Use Morris water maze test to investigate all experiment animal’s ability ofacquired spatial learning and spatial memory. Use T-maze test to assess workingmemory.3. Immunohistochemical and histological analysis is used to etimate neuronaldamage, activated astrocytes and activated microglia.4. By using commercially available assay kits to measure the levels ofmalondialdehyde (MDA) and protein carbonyls as well as the activities of superoxidedismutase (SOD), and glutathione peroxidase (GPX)in order to assess the oxidant andantioxidant status of rat brain which is under hypoperfusion status. Use commercial assaykits to measure central cholinergic system function.5. Using correlation analysis method to evaluate the relationship between thecognitive deficits and the measures of neuronal damage, activated glial cells, cholinergicand oxidative markers in BCCAO rats.Results：1.The spatial learning and memory functions of all rats were assessed by Morriswater maze. The results of the time required to find the hidden platform (escape latency)for the two groups of rats during the water maze acquisition training are shownsignificant difference. The spatial memory of all rats were assessed by the probe trialshown in BCCAO rats spent less time in the target quadrant than sham-operated rats. Theworking memory were assessed by T maze test shown in BCCAO rats markedlydecreased compared to sham-operated rats under30-s-delay condition.2. Neuronal loss in the cerebral cortex and hippocampal CA1region in BCCAO rats:Compared to sham-operated rats, BCCAO rats showed marked damaged neurons,reduced cell densities and a remarkable reduction of intact neurons both in the cerebralcortex and hippocampus CA1region. Glial activation in BCCAO rats: compared to sham-operated control rats, BCCAO rats revealed that a significant increase inGFAP-immuopositive astrocytes and Iba1-immuopositive microgliawas in the cerebralcortex and hippocampal CA1region. Oxidative stress is increased in BCCAO rats:Compared to sham-operated rats, the activities of endogenous antioxidant enzymes weremarked decreased in BCCAO rats, and the measure of lipid oxidation and proteinoxidation were significantly increased in BCCAO rats. The contents of Ach and ChATwere significantly reduced in BCCAO rats, however, the content of AchE wassignificantly elevated in BCCAO rats.3. Correlation of cognitive impairment with neuronal damage, activated glial cells,cholinergic and oxidative markers in BCCAO rats:(1) The spatial learning performancerevealed a significant positive correlation with MDA level, protein carbonyl level, andAchE level and a significant inverse correlation with Ach level, ChAT level, SOD levelGPX level, as well as the number of intact neurons in the cerebral cortex andhippocampus;(2) The performance of spatial memory showed a significant inversecorrelation with MDA level, protein carbonyl leve, and AchE level. It is also strikinglycorrelated to Ach level, ChAT level, SOD level, GPX level, and the number of intactneurons in the cerebral cortex and hippocampus;(3) the performance of working memoryshowed a significant inverse correlation with MDA level, protein carbonyl level, andAChE level as well as a significant positive correlation with ACh level, ChAT level, SODlevel, GPX level, and the number of intact neurons. The correlations also remainedsignificant after Bonferroni correction for multiple comparisons (at a significantthreshold of P <0.025). These findings indicate that neuronal damage, increasedoxidative stress and decreased cholinergic function may be involved in the developmentof cognitive impairments in BCCAO rats.4. Correlations between cholinergic and oxidative measures in BCCAO rats:(1) Theresult is that Ach level is correlated with ChAT level, SOD level and GPX level. It alsohas a marked inverse correlation with MDA level, protein carbonyl level, and AchE level; (2) ChAT is strikingly correlated with SOD level, GPX level and it also has a inversecorrelation with MDA level and protein carbonyl level;(3) AchE level showed asignificant inverse correlation with SOD level, GPX level and it also is positivecorrelated with MDA level, protein carbonyl level. Correlation of morphologicalmeasures with cholinergic and oxidative markers in BCCAO rats:(1) the numbers ofintact neurons showed a significant positive correlation with ChAT, AchE, Ach, SOD,GPX and it also is significant inverse correlated with MDA and protein carbonyls in thecerebral cortex of the brain and hippocampus;(2) the numbers of activated astrocytes andmicroglia showed a significant positive correlation with MDA and protein carbonyls inthe cerebral cortex and hippocampus.Conclusion：In summary, the results of the time required to find the hidden platform (escapelatency) for the two groups of rats during the water maze acquisition training are shownsignificant difference. The spatial memory of all rats were assessed by the probe trialshown in BCCAO rats spent less time in the target quadrant than sham-operated rats.Compared to sham-operated rats, BCCAO rats showed marked damaged neurons, asignificant increase in GFAP-immuopositive astrocytes and Iba1-immuopositivemicrogliawas in the cerebral cortex and hippocampal CA1region. Compared tosham-operated rats, the activities of endogenous antioxidant enzymes were markeddecreased in BCCAO rats, and the measure of lipid oxidation and protein oxidation weresignificantly increased in BCCAO rats. The contents of Ach and ChAT were significantlyreduced in BCCAO rats, however, the content of AchE was significantly elevated inBCCAO rats. This provides further evidence that long-term chronic cerebralhypoperfusion may cause cognitive deficits, neuronal damage, glial activation, decreasedcholinergic function and increased oxidative damage in the hypoperfused brain.Importantly, the result of the correlation of cognitive impairment with neuronal damage, activated glial cells, cholinergic and oxidative markers and the result of the correlationsbetween cholinergic and oxidative measures in BCCAO rats, suggest that cognitivedeficits induced by chronic cerebral hypoperfusion might be due to neuronal damage,central cholinergic dysfunction and increased oxidative damage in the cerebral cortex andhippocampus, which strongly supports the emerging view that chronic cerebralhypoperfusion plays an important role in mediating the initial pathogenic processes ofdementia such as VaD and AD.