Chronic Cerebral Hypoperfusion In Rats Research Articles

MicroRNA-153 impairs hippocampal synaptic vesicle trafficking via downregulation of synapsin I in rats following chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion (CCH) promotes the development of Alzheimer's pathology. However, whether and how CCH impairs the synaptic vesicle trafficking is still unclear. In the present study, we found that the hippocampal glutamatergic vesicle trafficking was impaired as indicated by a significant shortened delayed response enhancement (DRE) phase in CA3-CA1 circuit and decreased synapsin I in CCH rats suffering from bilateral common carotid artery occlusion (2VO). Further study showed an upregulated miR-153 in the hippocampus of 2VO rats. In vitro, overexpression of miR-153 downregulated synapsin I by binding the 3′UTRs of SYN1 mRNAs, which was prevented by its antisense AMO-153 and miRNA-masking antisense oligodeoxynucleotides (SYN1-ODN). In vivo, the upregulation of miR-153 elicited similar reduced DRE phase and synapsin I deficiency as CCH. Furthermore, miR-153 knockdown rescued the downregulated synapsin I and shortened DRE phase in 2VO rats. Our results demonstrate that CCH impairs hippocampal glutamatergic vesicle trafficking by upregulating miR-153, which suppresses the expression of synapsin I at the post-transcriptional level. These results will provide important references for drug research and treatment of vascular dementia.

Mfsd2a Reverses Spatial Learning and Memory Impairment Caused by Chronic Cerebral Hypoperfusion via Protection of the Blood-Brain Barrier.

Disruption of the blood–brain barrier (BBB) can lead to cognitive impairment. Major facilitator superfamily domain-containing protein 2a (Mfsd2a) is a newly discovered protein that is essential for maintaining BBB integrity. However, the role of Mfsd2a in vascular cognitive impairment has not been explored yet. In this study, a rat model of chronic cerebral hypoperfusion (CCH) was established by producing permanent bilateral common carotid artery occlusion (2VO) in rats. We found that after the 2VO procedure, the rats exhibited cognitive impairment, showed increased BBB leakage within the hippocampus, and had reduced expression of the Mfsd2a protein. The overexpression of Mfsd2a in the rat hippocampus reversed these changes. Further investigations using transmission electron microscopy revealed a significantly increased rate of vesicular transcytosis in the BBB of the hippocampus of the CCH rats; the rate reduced after overexpression of Mfsd2a. Moreover, Mfsd2a overexpression did not cause changes in the expression of tight junction-associated proteins and in the ultrastructures of the tight junctions. In conclusion, Mfsd2a attenuated BBB damage and ameliorated cognitive impairment in CCH rats, and its protective effect on the BBB was achieved via inhibition of vesicular transcytosis.

Dl-3-n-Butylphthalide Promotes Remyelination and Suppresses Inflammation by Regulating AMPK/SIRT1 and STAT3/NF-κB Signaling in Chronic Cerebral Hypoperfusion.

Demyelination in vascular dementia (VD) is partly attributable to inflammation induced by chronic cerebral hypoperfusion (CCH). Remyelination contributes to the recovery of cognitive impairment by inducing the proliferation and differentiation of oligodendrocyte progenitor cells. It was previously reported that Dl-3-n-butylphthalide (NBP) promotes cognitive improvement. However, whether NBP can stimulate remyelination and suppress inflammation after CCH remains unclear. To answer this question, the present study investigated the effects of NBP on remyelination in a rat model of CCH established by bilateral carotid artery occlusion. Functional recovery was evaluated with the Morris water maze (MWM) test, and myelin integrity, regeneration of mature oligodendrocytes, and inhibition of astrocyte proliferation were assessed by immunohistochemistry and histologic analysis. Additionally, activation of 5′ AMP-activated protein kinase (AMPK)/Sirtuin (SIRT)1 and Signal transducer and activator of transcription (STAT)3/nuclear factor (NF)-κB signaling pathways was evaluated by western blotting. The results showed that NBP treatment improved memory and learning performance in CCH rats, which was accompanied by increased myelin integrity and oligodendrocyte regeneration, and reduced astrocyte proliferation and inflammation. Additionally, NBP induced the activation of AMPK/SIRT1 signaling while inhibiting the STAT3/NF-κB pathway. These results indicate that NBP alleviates cognitive impairment following CCH by promoting remyelination and suppressing inflammation via modulation of AMPK/SIRT1 and STAT3/NF-κB signaling.

Effect of Methylene Blue and PI3K-Akt Pathway Inhibitors on the Neurovascular System after Chronic Cerebral Hypoperfusion in Rats.

Methylene blue (MB) has a protective effect on cognitive decline caused by chronic hypoperfusion, but the specific mechanism is not clear. This article aims to determine whether MB protects vascular neurons through PI3K/Akt and plays a role in improving cognitive impairment. Molecular biological methods, the hippocampal neuronal density test, the hippocampal vascular network density test, and dynamic enhanced magnetic resonance imaging (MRI) were used to detect the blood-brain barrier permeability and Evans blue leakage rate in the hippocampus. We also observed and evaluated the changes in the above results after administration of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway protein inhibitor LY294002. There were significant differences for cerebral blood flow (CBF) between the chronic cerebral hypoperfusion (CCH) + MB group (100ml/100g/min) and the CCH group (60ml/100g/min, P < 0.05). After using LY294002, the CBF of the CCH + MB + LY294002 group dropped to 82ml/100g/min. The vascular density in the CCH + MB group was 23%, which is significantly higher than that in the CCH group (15.1%) (P < 0.05). The vascular density (17.5%) in the CCH + MB + LY294002 group was significantly higher than that in the CCH group but lower than that in the CCH + MB group. Western blotting results showed that one week after intraperitoneal injection of MB, the expression of t-Akt and p-Akt in the CCH + MB group was increased after CCH, and LY294002 partially blocked this up-regulation effect (CCH + MB + LY294002 group). MB is a potential therapy for the relief of mild cognitive impairment associated with CCH, vascular dementia, and Alzheimer's disease.

Effects of Sodium Ferulate on Cognitive Function in Hippocampus of Chronic Cerebral Hypoperfusion Rats

Objective: To assess sodium ferulate’s effect on cognitive function and p38MAPK signaling in hippocampus of rats with chronic cerebral hypoperfusion (CCH). Methods: 45 male Sprague-Dawley (SD) rats were separated into sham-operation group, CCH model group and treatment group (intraperitoneal injection of 50 mg/kg sodium ferulate). The cognitive function was detected via open-field test, novel object recognition test and Morris water maze test. The activity of superoxide dismutase (SOD) in hippocampal tissues was detected using ELISA kit. Moreover, the neuronal damage in hippocampal tissues was assessed by immunohistochemical method, and apoptosis was assessed via TUNEL staining. Finally, p-p38MAPK signaling protein level in hippocampal tissues was detected via Western blotting. Results: No difference was found in rat autonomic activity among three groups (P &gt; 0.05). The novel object recognition ability of model rats was significantly impaired along with longer escape latency and shorter retention time in the target quadrant (P &lt; 0.01), which were all improved after sodium ferulate treatment (P &lt; 0.01). SOD activity in hippocampal tissues in model rats was significantly reduced and sodium ferulate could significantly increase SOD activity (P &lt; 0.01). There were a large number of damaged neurons and apoptotic cells in hippocampal tissues in model group and sodium ferulate could protect neurons (P &lt; 0.01). The levels of p-p38MAPK, GSP3β and P-tau protein in hippocampal tissues in model group were significantly increased, which were significantly reduced after treatment with sodium ferulate (P &lt; 0.01). Conclusion: Sodium ferulate can protect the CCH-induced cognitive impairment in rats and reduce the CCH-induced damage and apoptosis of hippocampal neurons, possibly through inhibition of p38MAPK signaling pathway.

Abstract TP446: Rosuvastatin Improves Cerebral Hemodynamics in Rats With Chronic Cerebral Hypoperfusion

Objective: Chronic cerebral hypoperfusion (CCH) is a common consequence of various cerebral vascular disorders, hemodynamics and blood composition changes, which can lead to neurodegenerative injury and cognitive dysfunction. Therefore, it is important to take measures to prevent or reverse its development. Methods: Eighty Sprague-Dawley rats were randomly divided into five groups: Sham group, Vehicle groups (2-week group and 4-week group), Rosuvastatin-treated groups (2-week group and 4-week group). Both vehicle and treated group rats were treated with bilateral common carotid artery occlusion (BCCAO). Then rats in the treated group were intravenously injection with rosuvastatin (0.2 mg/kg/day) daily for 14 days beginning 24 hours after BCCAO. The vehicle groups were given the same amount of saline. The MRI ASL technique was used to scan cerebral blood flow (CBF) at pre-occlusion, BCCAO, 1, 2, 3, and 4 weeks after operation. Morris water maze test detected spatial memory abilities of the treated and vehicle rats after BCCAO. Immunofluorescence staining was used to detect the expression of CD34, GFAP, NeuN and Caspase-3 positive cells in hippocampus. Results: CBF decreased significantly after BCCAO in both rosuvastatin-treated and vehicle groups and then increased gradually. The CBF of the treated group was basically recovered to baseline levels at 1 or 2 weeks after BCCAO. But it took the vehicle group 4 weeks to restore to baseline. The vertebral artery (VA) diameter of the treated group was greater than that of the vehicle group (p&lt;0.05), and the recovery of CBF and the dilation of VA were more evident during the administration period. Similarly, the escape latency of the treated group rats was also less than the vehicle group in Morris Water Maze test. In addition, the number of CD34 + and NeuN + cells in the hippocampus of the treated group was more than that in the vehicle group (p&lt;0.01), while the number of GFAP + and Caspase-3 + cells was less than in the vehicle group (p&lt;0.01). Conclusion: Rosuvastatin can accelerate the recovery of cerebral blood flow in CCH rats, which may be related to the dilation of VAs and angiogenesis; and its effects on reducing astrocyte activation and neuroprotection may help to ameliorate cognitive dysfunction in CCH rats.

Neuroprotective effects of andrographolide on chronic cerebral hypoperfusion-induced hippocampal neuronal damage in rats possibly via PTEN/AKT signaling pathway

To explore the potential effects of andrographolide on chronic cerebral hypoperfusion (CCH)-induced neuronal damage as well as the underlying mechanisms. Rat CCH model was established by 2-vessel occlusion (2VO). The CCH rats received andrographolide treatment for 4 weeks. The neuron loss was detected by using neuronal nuclei (NeuN) immunofluorescent staining. The expression levels of phospho-phosphatase and tensin homolog deleted on chromosome ten (p-PTEN), protein kinase B (AKT), p-AKT, and cysteinyl aspartate specific proteinase-3 (Caspase-3) proteins were accessed by Western blotting. Moreover, the neuronal apoptosis of hippocampus tissues was detected via terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) staining. CCH reduced the number of NeuN-positive cells, while the number was significant increased after andrographolide treatment. CCH increased the proteins expression level of p-PTEN, Caspase-3, and decreased the p-AKT, which were reversed by andrographolide treatment. Furthermore, andrographolide treatment also down-regulated CCH-induced TUNEL-apoptosis rate. Our results suggest that the PTEN/AKT pathway may be modulated by andrographolide and the damaging effects of CCH on hippocampus may be ameliorated by andrographolide treatment. Andrographolide may act as a potential therapeutic approach for chronic ischemic insults.

The Effects of P-Hydroxycinnamic Acid in Ameliorating Spatial Learning and Flexibility Deficits in Rats with Chronic Cerebral Hypoperfusion

Cerebral blood reduction resulting in oligemic energy failure and metabolic insufficiency initiates gradual neurodegeneration and cognitive impairments. We investigated the neuroprotective effects of p-hydroxycinnamic acid (pHCA) on neurodegeneration in association with cognitive impairments in rats with chronic cerebral hypoperfusion (CCH). Forty male Sprague-Dawley rats were randomly divided into 4 groups: Sham-veh, 2VO-veh, 2VO-pHCA50 and 2VO-pHCA100. We used modified 2-vessel occlusion (2VO) to induce CCH, and the 2 latter groups were given 50 mg/kg and 100 mg/kg of pHCA after the 2VO operation, respectively, which continued for 3 weeks. The behavioral tests consisted of anxiety-like behavior tested in an elevated plus maze (EPM) and hippocampal-dependent spatial learning and flexibility tested in a Morris water maze (MWM). Brain oxidative status, infarction, vulnerable hippocampus and corpus callosum (CC) white matter degeneration were evaluated. The results showed that modified 2VO induced reversible short-term anxiolytic-like behavior in the EPM (p 0.05) with significance infarction, vulnerable hippocampal CA1, CA3, DG and CC degeneration (p < 0.05). These were found along with significant spatial learning and flexibility deficits (p < 0.05). Additionally, 100 mg/kg of pHCA significantly ameliorated the spatial learning and flexibility deficits, which coincided with the significant decreases of infarction volume, CA1, CA3 and CC degeneration (p < 0.05). We conclude that pHCA’s improving effects on spatial learning and flexibility deficits are neuroprotective against oligemic energy failure-induced vulnerable neuronal and white matter degeneration in CCH rats.

Mumefural Ameliorates Cognitive Impairment in Chronic Cerebral Hypoperfusion via Regulating the Septohippocampal Cholinergic System and Neuroinflammation.

Chronic cerebral hypoperfusion (CCH) causes cognitive impairment and neurogenic inflammation by reducing blood flow. We previously showed that Fructus mume (F. mume) improves cognitive impairment and inhibits neuroinflammation in a CCH rat model. One of the components of F. mume, Mumefural (MF), is known to improve blood flow and inhibit platelet aggregation. Whether MF affects cerebral and cognitive function remains unclear. We investigated the effects of MF on cognitive impairment and neurological function-related protein expression in the rat CCH model, established by bilateral common carotid arterial occlusion (BCCAo). Three weeks after BCCAo, MF (20, 40, or 80 mg/kg) was orally administrated once a day for 42 days. Using Morris water maze assessment, MF treatment significantly improved cognitive impairment. MF treatment also inhibited cholinergic system dysfunction, attenuated choline acetyltransferase-positive cholinergic neuron loss, and regulated cholinergic system-related protein expressions in the basal forebrain and hippocampus. MF also inhibited myelin basic protein degradation and increased the hippocampal expression of synaptic markers and cognition-related proteins. Moreover, MF reduced neuroinflammation, inhibited gliosis, and attenuated the activation of P2X7 receptor, TLR4/MyD88, NLRP3, and NF-κB. This study indicates that MF ameliorates cognitive impairment in BCCAo rats by enhancing neurological function and inhibiting neuroinflammation.

Embelin Improves the Spatial Memory and Hippocampal Long-Term Potentiation in a Rat Model of Chronic Cerebral Hypoperfusion

Alzheimer’s disease (AD) is the second most occurring neurological disorder after stroke and is associated with cerebral hypoperfusion, possibly contributing to cognitive impairment. In the present study, neuroprotective and anti-AD effects of embelin were evaluated in chronic cerebral hypoperfusion (CCH) rat model using permanent bilateral common carotid artery occlusion (BCCAO) method. Rats were administered with embelin at doses of 0.3, 0.6 or 1.2 mg/kg (i.p) on day 14 post-surgery and tested in Morris water maze (MWM) followed by electrophysiological recordings to access cognitive abilities and synaptic plasticity. The hippocampal brain regions were extracted for gene expression and neurotransmitters analysis. Treatment with embelin at the doses of 0.3 and 0.6 mg/kg significantly reversed the spatial memory impairment induced by CCH in rats. Embelin treatment has significantly protected synaptic plasticity impairment as assessed by hippocampal long-term potentiation (LTP) test. The mechanism of this study demonstrated that embelin treatment alleviated the decreased expression of BDNF, CREB1, APP, Mapt, SOD1 and NFκB mRNA levels caused by CCH rats. Furthermore, treatment with embelin demonstrated neuromodulatory activity by its ability to restore hippocampal neurotransmitters. Overall these data suggest that embelin improve memory and synaptic plasticity impairment in CCH rats and can be a potential drug candidate for neurodegenerative disease-related cognitive disorders.

Honokiol Attenuates the Memory Impairments, Oxidative Stress, Neuroinflammation, and GSK-3β Activation in Vascular Dementia Rats.

Vascular dementia (VaD) is caused by chronic decreases in brain blood flow and accounts for 15-20% of dementia cases worldwide. In contrast to Alzheimer's disease (AD), no effective drug treatments are currently available for VaD. Previous studies have suggested that oxidative stress and neuroinflammation in the brain play important roles in the pathogenesis of VaD. Honokiol (HKL) is a well-known bioactive and nutraceutical compound that can act as an antioxidant and anti-inflammatory molecule. HKL can protect against memory impairments in AD mouse models. In this study, we explored whether the application of HKL was also protective against the insult of chronic cerebral hypoperfusion (CCH) in rats. We found that HKL supplementation prevented the memory impairments in the inhibitory avoidance step-down and Morris water maze tasks in CCH rats. HKL also suppressed the levels of oxidative stress and inflammation in CCH rats. Moreover, HKL prevented dendritic spines abnormalities in CCH rats. We also found that HKL inhibited the activity of GSK-3β, which may be critical for the neuroprotective activity of HKL. Thus, our study demonstrated the protective role of HKL in VaD.

Resveratrol Activates Autophagy via the AKT/mTOR Signaling Pathway to Improve Cognitive Dysfunction in Rats With Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a main cause of vascular dementia and is also an etiological factor of neurological diseases and mental disorders. However, few treatments are available for CCH, and new medications are needed. In the present study, we employed a rat model of CCH that was based on bilateral common carotid artery occlusion and investigated the therapeutic effects of resveratrol and its detailed mechanism of action. We evaluated neurological deficit scores and performed the Morris water maze test, hematoxylin and eosin staining, TUNEL staining, enzyme-linked immunosorbent assays, and Western blot. Resveratrol reduced neurological deficit scores in CCH rats and reduced pathological damage in the frontal cortex and hippocampus. Resveratrol activated autophagy and inhibited the expression of AKT/mechanistic target of rapamycin (mTOR) signaling pathway-related proteins. Treatment with a phosphoinositide-3 kinase inhibitor reversed the protective effect of resveratrol. These findings suggest that resveratrol improves cognitive function in a rat model of CCH and reduces oxidative stress-induced neuronal damage in the frontal cortex and hippocampus by activating autophagy and inhibiting neuronal apoptosis. These effects may be regulated by the AKT/mTOR signaling pathway.

Dl-3-n-Butylphthalide Reduces Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion Through GDNF/GFRα1/Ret Signaling Preventing Hippocampal Neuron Apoptosis.

Hippocampal neuron death is a key factor in vascular dementia (VD) induced by chronic cerebral hypoperfusion (CCH). Dl-3-n-butylphthalide (NBP) is a multiple-effects drug. Therefore, the potential molecular mechanisms underlying CCH and its feasible treatment should be investigated. This study had two main purposes: first, to identify a potential biomarker in a rat model of CCH induced VD using antibody microarrays; and second, to explore the neuroprotective role of NBP at targeting the potential biomarker. Glial cell line-derived neurotrophic factor (GDNF)/GDNF family receptor alpha-1 (GFRα1)/receptor tyrosine kinase (Ret) signaling is altered in the hippocampus of CCH rats; however, NBP treatment improved cognitive function, protected against hippocampal neuron apoptosis via regulation of GDNF/GFRα1/Ret, and activated the phosphorylation AKT (p-AKT) and ERK1/2 (p-ERK1/2) signaling. We also found that 1 h oxygen-glucose deprivation (OGD) followed by 48 h reperfusion (R) in cultured hippocampal neurons led to downregulation of GDNF/GFRα1/Ret. NBP upregulated the signaling and increased neuronal survival. Ret inhibitor (NVP-AST487) inhibits Ret and downstream effectors, including p-AKT and p-ERK1/2. Additionally, both GDNF and GFRα1 expression are markedly inhibited in hippocampal neurons by coincubation with NVP-AST487, particularly under conditions of OGD/R. GDNF/GFRα1/Ret signaling and neuronal viability can be maintained by NBP, which activates p-AKT and p-ERK1/2, increases expression of Bcl-2, and decreases expression of Bax and cleaved caspase-3. The current study showed that GDNF/GFRα1/Ret signaling plays an essential role in the CCH induced VD. NBP was protective against hippocampal neuron apoptosis, and this was associated with regulation of GDNF/GFRα1/Ret and AKT/ERK1/2 signaling pathways, thus reducing cognitive impairment.

Epigallocatechin-3-Gallate Attenuates Impaired Cognitive Function Induced by Chronic Cerebral Hypoperfusion via Activating HO-1 Through PI3K/Akt/Nrf2 Signaling Pathway

Heme oxygenase-1 (HO-1), the most important member of HO family, is sensitive to a wide range of pro-oxidant and stressors,and I may represent a target for treatment. Epigallocatechin-3-gallate (EGCG), has been widely reported to produce diverse anti-oxidative stress effects against chronic cerebral hypoperfusion, but the underlying mechanism has yet to be fully elucidated. The study was aimed to investigate whether EGCG can improve cognition by a neurorestorative effect in a chronic cerebral hypoperfusion (CCH) rat model, and to explore its potential mechanisms. Compared to the normal group and sham group, escape latency was significantly longer in the cerebral hypoperfusion model, but after EGCG treatment, the escape latency was found to be remarkably shorter, and it was in a dose-dependent manner (P<0.05). HE staining results showed EGCG reduced the histopathological changes induced by CCH. Moreover, the results showed that EGCG increased the concentration of superoxide dismutase (SOD) and the activity of HO-1, but decreased the malondialdehyde (MDA) contents. Furthermore, EGCG treatment induced the expression of PI3K, p-Akt, Nrf2 and HO-1 at protein levels, all of which were could be reversed by the PI3K inhibitor LY294002, siRNA-Nrf2 or HO-1 inhibitor protoporphyrin IX zinc(II) (ZnPP). Taken together, the findings suggest that the neuroprotective of EGCG on impaired cognition induced by CCH was by activating HO-1 via PI3K/Akt/Nrf2 intracellular signaling pathway, which might provide novel insights into understanding the mechanism for neuroprotective effects of EGCG. Funding: The work was supported by grants from the National Natural Science Foundation of China (11332003, 11572064), the National Key Technology R & D Program of China (2016YFC1102305), the Fundamental Research Funds for the Central Universities (CDJZRPY0012, CDJZRPY0021, DJPT230001) as well as the support from the Chongqing Engineering Laboratory in Vascular Implants and the Public Experiment Center of State Bioindustrial Base (Chongqing), China. Declaration of Interest: Author's declare none. Ethical Approval: All animal procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

DL-3-n-butylphthalide protects the blood-brain barrier against ischemia/hypoxia injury via upregulation of tight junction proteins.

Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia.Methods:A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0 μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3β (GSK-3β)/GSK-3β, and β-catenin/β-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy.Results:In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 μg/g vs. 12.3 ± 1.9 μg/g, P = 0.005; 6.7 ± 0.6 μg/g vs. 12.3 ± 1.9 μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 vs. 0.41 ± 0.06, P < 0.01; 0.97 ± 0.07 vs. 0.41 ± 0.06, P < 0.01), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05).Conclusion:NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3β/β-catenin signaling pathway.

Prostaglandin E1 Alleviates Cognitive Dysfunction in Chronic Cerebral Hypoperfusion Rats by Improving Hemodynamics.

Compensatory vascular mechanisms can restore cerebral blood flow (CBF) but fail to protect against chronic cerebral hypoperfusion (CCH)-mediated neuronal damage and cognitive impairment. Prostaglandin E1 (PGE1) is known as a vasodilator to protect against ischemic injury in animal models, but its protective role in CCH remains unclear. To determine the effect of PGE1 on cerebral hemodynamics and cognitive functions in CCH, bilateral common carotid artery occlusion (BCCAO) was used to mimic CCH in rats, which were subsequently intravenously injected with PGE1 daily for 2 weeks. Magnetic resonance imaging, immunofluorescence staining and Morris water maze (MWM) were used to evaluate CBF, angiogenesis, and cognitive functions, respectively. We found that PGE1 treatment significantly restored CBF by enhancing vertebral artery dilation. In addition, PGE1 treatment increased the number of microvascular endothelial cells and neuronal cells in the hippocampus, and decreased the numbers of astrocyte and apoptotic cells. In the MWM test, we further showed that the escape latency of CCH rats was significantly reduced after PGE1 treatment. Our results suggest that PGE1 ameliorates cognitive dysfunction in CCH rats by enhancing CBF recovery, sustaining angiogenesis, and reducing astrocyte activation and neuronal loss.

Huoluo Yinao decoction mitigates cognitive impairments after chronic cerebral hypoperfusion in rats

Ethnopharmacological relevanceHuoluo Yinao decoction (HLYND) has been used to ameliorate cognitive impairment induced by chronic cerebral hypoperfusion in clinical for years. However, the exact mechanisms remain unknown. Aim of the studyTo investigate the effects and mechanisms underlying HLYND-mediated improvement in cognitive deficits associated with chronic cerebral hypoperfusion. Materials and methodsThirty-six Sprague-Dawley rats were randomly allocated to three groups: sham, model, and HLYND. Daily administration of HLYND or volume-matched vehicle by gavage was initiated 1 day after bilateral carotid artery stenosis (BCAS) and continued for 42 days. The Morris water maze (MWM) test was used to assess cognitive functions from days 36–42. Via western blot and immunofluorescent staining, restoration of neuronal plasticity and remyelination of white matter were evaluated by analyzing the expression profiles of MAP-2, synaptophysin and MBP. In addition, macrophage/microglial activation was assessed by quantifying changes in Iba1, and macrophage/microglial polarization was assessed by changes in iNOS and CD16 (M1 markers), as well as Arg1 and CD206 (M2 markers). ResultsIn the MWM test, BCAS rats showed significantly extended escape latency and reduced platform crossing times, while those in the HLYND group had shortened escape latency and increased frequency of platform crossing. In addition, rats in the model group showed decreased levels and abnormal morphological changes of MAP-2, synaptophysin and MBP, whereas HLYND administration reversed these effects. As expected, Iba1 levels were elevated in both the model and HLYND groups but rats in the model group showed increased levels of the M1 markers, iNOS and CD16, and a correspondent decrease in the M2 marker, Arg1. In contrast, in the HLYND group, iNOS and CD16 levels were suppressed, while Arg1 levels were elevated. ConclusionsOur findings demonstrate that HLYND mitigates cognitive impairment after chronic cerebral hypoperfusion in rats through mechanisms involving increased neuronal plasticity and white matter remyelination, with a subtile modulation of macrophage/microglial polarization toward the M2 phenotype.

Dl-3-n-Butylphthalide regulates the Ang-1/Ang-2/Tie-2 signaling axis to promote neovascularization in chronic cerebral hypoperfusion

Previous findings have demonstrated, in a rat model, that chronic cerebral hypoperfusion (CCH) decreases cortical cerebral blood flow (CBF) while Dl-3-n-Butylphthalide (DNB) accelerates the timely recovery of CBF. However, potential biomarkers, therapeutic targets, and underlying mechanisms for these processes are unclear. In this study, a solid-phase antibody microarray for simultaneously detecting multiple proteins was used to search cortex biotargets in CCH compared to a sham control group, and these results were further examined by biological functional analysis. After DNB treatment, western blot and immunostaining were used to verify candidate protein expression. Importantly, we identified seven proteins that may serve as novel biotargets contributing to CCH. The levels of Tie-2, CNTFRα, IL-4, IL-10, ITGAM, MDC, and TROY were uniquely altered in the CCH. The Tie-2 level was significantly decreased and identified in CCH 2 week (W), CCH 4 W and CCH 8 W. In addition, Ang-1 level and Ang-1/Ang-2 ratio were significantly decreased in CCH 2 W and CCH 4 W while Ang-2 level was increased in the CCH, whereas DNB treatment created the inverse effect to some extent. Moreover, the expression of VEGF and CD34 in the earlier stage of CCH and the diameters of bilateral vertebral arteries (VAs), were significantly enlarged by DNB treatment. Together, we found that the Ang-1/Ang-2/Tie-2 signaling axis was altered in the CCH rat cortex, and DNB treatment could timely regulate this angiopoietin/Tie signaling axis to promote neovascularization in early stages.

Impaired functional recovery of endothelial colony-forming cells from moyamoya disease in a chronic cerebral hypoperfusion rat model.

OBJECTIVEEndothelial colony-forming cells (ECFCs) isolated from pediatric patients with moyamoya disease (MMD) have demonstrated decreased numbers and defective functioning in in vitro experiments. However, the function of ECFCs has not been evaluated using in vivo animal models. In this study, the authors compared normal and MMD ECFCs using a chronic cerebral hypoperfusion (CCH) rat model.METHODSA CCH rat model was made via ligation of the bilateral common carotid arteries (2-vessel occlusion [2-VO]). The rats were divided into three experimental groups: vehicle-treated (n = 8), normal ECFC-treated (n = 8), and MMD ECFC-treated (n = 8). ECFCs were injected into the cisterna magna. A laser Doppler flowmeter was used to evaluate cerebral blood flow, and a radial arm maze test was used to examine cognitive function. Neuropathological examinations of the hippocampus and agranular cortex were performed using hematoxylin and eosin and Luxol fast blue staining in addition to immunofluorescence with CD31, von Willebrand factor, NeuN, myelin basic protein, glial fibrillary acidic protein, and cleaved caspase-3 antibodies.RESULTSThe normal ECFC-treated group exhibited improvement in the restoration of cerebral perfusion and in behavior compared with the vehicle-treated and MMD ECFC-treated groups at the 12-week follow-up after the 2-VO surgery. The normal ECFC-treated group showed a greater amount of neovasculogenesis and neurogenesis, with less apoptosis, than the other groups.CONCLUSIONSThese results support the impaired functional recovery of MMD ECFCs compared with normal ECFCs in a CCH rat model. This in vivo study suggests the functional role of ECFCs in the pathogenesis of MMD.

Effect of berberine chloride on caspase-3 dependent apoptosis and antioxidant capacity in the hippocampus of the chronic cerebral hypoperfusion rat model.

Objective(s):The main goal of the current research was to examine the effects of Berberine (BBR) on apoptotic signaling and hippocampal oxidative stress induced by common carotid artery occlusion.Materials and Methods:Chronic cerebral hypoperfusion (CCH) model was created by occluding the two common carotid arteries (two-vessel occlusion [2VO]) permanently. BBR (50 and 100 mg/kg/daily) was intra-gastrically administered to ischemic rats. Neuronal survival was evaluated by Nissl staining. The levels of malondialdehyde (MDA) and antioxidant enzymes, including catalase (CAT) and superoxide dismutase (SOD), along with the activities of caspase 3 were estimated in the hippocampus 2 month after treating the rats with 2VO.Results:According to findings of the present research, the BBR therapy inhibited the neuro-degeneration of hippocampus. BBR also significantly decreased the amount of MDA and activity of caspase 3 in the hippocampus. Furthermore, the administration of BBR alleviated the lowered activities of SOD and CAT after 2VO surgery.Conclusion:The antioxidant and antiapoptotic properties of BBR might play important roles in improving functional outcomes and might have significant neuroprotective effects on the CCH damage.