Bilateral Common Carotid Artery Stenosis Research Articles

Identification of toll-like receptor 2 as a key regulator of neuronal apoptosis in vascular dementia by bioinformatics analysis and experimental validation

BackgroundVascular dementia (VaD), the second most prevalent type of dementia, lacks a well-defined cause and effective treatment. Our objective was to utilize bioinformatics analysis to discover the fundamental disease-causing genes and pathological mechanisms in individuals diagnosed with VaD. MethodsTo identify potential pathogenic genes associated with VaD, we conducted weighted gene co-expression network analysis (WGCNA), differential expression analysis, and protein–protein interaction (PPI) analysis. The exploration of potential biological mechanisms involved the utilization of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis. Moreover, a bilateral common carotid artery stenosis (BCAS) mouse model of VaD was established, and the expression of the hub gene, its relationship with cognitive function and its potential pathogenic mechanism were verified by cognitive behavior tests, cerebral blood flow measurement, Western blotting, and immunofluorescence experiments. ResultsThis study identified 293 DEGs from the brain cortex of VaD patients and healthy controls, among these genes, the Toll-like receptor 2 (TLR2) gene was identified as hub gene, and it was associated with the apoptosis-related pathway PI3K/AKT.The BCAS model demonstrated that the use of TLR2 inhibitors greatly enhanced the cognitive function of the mice (p < 0.05). Additionally, there was a notable decrease in the number of apoptotic cells in the brain cortex of the mice (p < 0.01). Moreover, significant alterations in the levels of proteins related to the PI3K/AKT pathway and cleaved-caspase3 proteins were detected (p < 0.05). ConclusionsTLR2 plays a role in the pathophysiology of VaD by enhancing the neuronal apoptotic pathway, suggesting it could be a promising therapeutic target.

Construction of a screening system for lipid-derived radical inhibitors and validation of hit compounds to target retinal and cerebrovascular diseases

Recent studies have highlighted the indispensable role of oxidized lipids in inflammatory responses, cell death, and disease pathogenesis. Consequently, inhibitors targeting oxidized lipids, particularly lipid-derived radicals critical in lipid peroxidation, which are known as radical-trapping antioxidants (RTAs), have been actively pursued. We focused our investigation on nitroxide compounds that have rapid second-order reaction rate constants for reaction with lipid-derived radicals. A novel screening system was developed by employing competitive reactions between library compounds and a newly developed profluorescence nitroxide probe with lipid-derived radicals to identify RTA compounds. A PubMed search of the top hit compounds revealed their wide application as repositioned drugs. Notably, the inhibitory efficacy of methyldopa, selected from these compounds, against retinal damage and bilateral common carotid artery stenosis was confirmed in animal models. These findings underscore the efficacy of our screening system and suggest that it is an effective approach for the discovery of RTA compounds.

Chuanzhitongluo capsule improves cognitive impairment in mice with chronic cerebral hypoperfusion via the cholinergic anti-inflammatory pathway

Vascular cognitive impairment (VCI) has become a common disease-causing cognitive deficit in humans, second only to Alzheimer's Disease (AD). Chuanzhitongluo capsule (CZTL) is a Traditional Chinese Medicine (TCM) preparation known for its effective protection against cerebral ischemia. However, its potential to ameliorate VCI remains unclear. This study aimed to investigate the cognitive improvement effects of CZTL in a mouse model of VCI. Chronic cerebral hypoperfusion (CCH) was induced in mice by bilateral common carotid artery stenosis (BCAS) to simulate the pathological changes associated with VCI. Spatial learning and memory abilities were assessed using the Morris Water Maze (MWM). RNA sequencing (RNA-Seq) was employed to identify differentially expressed genes (DEGs) in the hippocampus. Levels of inflammatory factors were measured through enzyme-linked immunosorbent assay (ELISA), while immunofluorescence (IF) determined the expression intensity of target proteins. Western Blot (WB) confirmed the final action pathway. Results indicated that CZTL significantly improved the spatial learning and memory abilities of CCH mice, along with alterations in gene expression profiles in the hippocampus. It also reduced neuroinflammation in the hippocampus and upregulated the choline acetyltransferase (ChAT) and α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR), which are in synaptic plasticity and neuronal development. Moreover, CZTL inhibited the NF-κB signaling pathway. In conclusion, CZTL may alleviate neuroinflammation induced by CCH and improve cognitive impairment in CCH mice by regulating the cholinergic anti-inflammatory pathway (CAIP) involving ChAT/α7nAChR/NF-κB.

Aqueous Extract of Pinellia ternata Breitenbach Tuber Ameliorates Bilateral Common Carotid Artery Stenosis-Induced Cognitive Deficit in Mice by Suppressing the Activation of Astrocytes

Background: The proportion of dementia patients in Asian countries is rapidly increasing, and because vascular dementia is more common than in the West, it is recognized as important as Alzheimer’s disease. Purpose: The aim of this study was to verify the benefits of Pinellia ternata Breitenbach tuber aqueous extract (PTex) in improving cognitive function and reducing inflammation in brain damage resulting from bilateral common carotid artery stenosis (BCAS). Materials and methods: Four groups of five C57BL/6 male mice each were randomly assigned (a sham group, a BCAS surgery group, and two experimental groups that received PTex concentrations of 25 and 50 mL/kg, respectively). Measuring behavioral learning and memory, the two concentrations of the extract were given orally three times a week for six weeks following BCAS surgery. Furthermore, the inflammatory response in the cerebral cortex was observed, and RNA sequencing analysis was carried out. Results: The novel object recognition test revealed that the PTex-50-treated group had improved cognitive function, and the anti-inflammatory effect was confirmed by immunofluorescence staining of brain tissue. Proteins such as Apob, Shh, Dscc1, Cdc45, and Cdc6 were identified through protein-protein interaction network analysis as having a beneficial effect on BCAS-induced cognitive deficit. Conclusion: Results show that PTex-50 was found to have a beneficial effect on BCAS-induced cognitive deficit.

MiR-30a-5p mediates ferroptosis of hippocampal neurons in chronic cerebral hypoperfusion-induced cognitive dysfunction by modulating the SIRT1/NRF2 pathway

ObjectiveChronic cerebral hypoperfusion (CCH) is a common cause of brain dysfunction. As a microRNA (also known as miRNAs or miRs), miR-30a-5p participates in neuronal damage and relates to ferroptosis. We explored the in vivo and in vitro effects and functional mechanism of miR-30a-5p in CCH-triggered cognitive impairment through the silent information regulator 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway. MethodsAfter 1 month of CCH modeling through bilateral common carotid artery stenosis, mice were injected with 2 μL antagomir (also known as anti-miRNAs) miR-30a-5p, with cognitive function evaluated by Morris water maze and novel object recognition tests. In vitro HT-22 cell oxygen glucose deprivation (OGD) model was established, followed by miR-30a-5p inhibitor and/or si-SIRT1 transfections, with Fe2+ concentration, malonaldehyde (MDA) and glutathione (GSH) contents, reactive oxygen species (ROS), miR-30a-5p and SIRT1 and glutathione peroxidase 4 (GPX4) protein levels, NRF2 nuclear translocation, and miR-30a-5p-SIRT1 targeting relationship assessed. ResultsCCH-induced mice showed obvious cognitive impairment, up-regulated miR-30a-5p, and down-regulated SIRT1. Ferroptosis occurred in hippocampal neurons, manifested by elevated Fe2+ concentration and ROS and MDA levels, mitochondrial atrophy, and diminished GSH content. Antagomir miR-30a-5p or miR-30a-5p inhibitor promoted SIRT1 expression and NRF2 nuclear translocation, increased GPX4, cell viability and GSH content, and reduced Fe2+ concentration and ROS and MDA levels. miR-30a-5p negatively regulated SIRT1. In vitro, miR-30a-5p knockout increased NRF2 nuclear translocation by up-regulating SIRT1, inhibiting OGD-induced ferroptosis in HT-22 cells. ConclusionmiR-30a-5p induces hippocampal neuronal ferroptosis and exacerbates post-CCH cognitive dysfunction by targeting SIRT1 and reducing NRF2 nuclear translocation.

Methanol extract of Ligusticum chuanxiong Hort. Rhizome ameliorates bilateral common carotid artery stenosis-induced cognitive deficit in mice by altering microglia and astrocyte activation.

In traditional Asian medicine, Ligusticum chuanxiong Hort also known as Conioselinum anthriscoides "Chuanxiong", is mainly used for improving blood circulation or for analgesic and anti-inflammatory purposes, but they also have a long history of use for pain disorders in the head and face, such as headache. Despite the possibility that the plant is effective for diseases such as cerebral infarction and vascular dementia (VaD), the mechanism of action is not well understood. To determine if the dried rhizomes of L. chuanxiong (Chuanxiong Rhizoma, CR) methanol extract (CRex) has activity in a VaD mice model. Through network analysis, we confirm that CR is effective in cerebrovascular diseases. In mice, we induce cognitive impairment, similar to VaD in humans, by chronically reducing the cerebral blood flow by performing bilateral common carotid artery stenosis (BCAS) and administering CRex for 6weeks. We measure behavioral changes due to cognitive function impairment and use immunofluorescence staining to confirm if CRex can inhibit the activation of astrocytes and microglia involved in the inflammatory response in the brain. We quantify proteins involved in the mechanism, such as mitogen-activated protein kinases (MAPK), in the hippocampus and surrounding white matter, and analyze gene expression and protein interaction networks through RNA sequencing to interpret the results of the study. CRex administration rescued cognitive impairment relating to a novel object and inhibited the activation of astrocytes and microglia. Western blotting analysis revealed that CRex regulated the changes in protein expression involved in MAPK signaling such as extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38). The administration of CRex suppressed approximately 44% of the pathological changes in gene expression caused by BCAS. CRex extract effectively inhibited cognitive impairment caused by BCAS, and the mechanism through which this occurred is inhibited activation of astrocytes and microglia.

Xinshubao tablet rescues cognitive dysfunction in a mouse model of vascular dementia: Involvement of neurogenesis and neuroinflammation

Vascular dementia (VaD) represents a severe cognitive dysfunction syndrome closed linked to cardiovascular function. In the present study, we assessed the potential of Xinshubao tablet (XSB), a traditional Chinese prescription widely used for cardiovascular diseases, to mitigate neuropathological damage in a mouse model of VaD and elucidated the underlying mechanisms. Our findings revealed that oral administration of XSB rescued the cardiac dysfunction resulting from bilateral common carotid artery stenosis (BCAS), improved the cerebral blood flow (CBF) and cognitive function, reduced white matter injury, inhibited excessive microglial and astrocytic activation, stimulated hippocampal neurogenesis, and reduced neural apoptosis in the brains of BCAS mice. Mechanistically, RNA-seq analysis indicated that XSB treatment was significantly associated with neuroinflammation, vasculature development, and synaptic transmission, which were further confirmed by q-PCR assays. Western blot results revealed that XSB treatment hindered the nuclear translocation of nuclear factor-κB (NF-κB), thereby suppressing the NF-κB signaling pathway. These results collectively demonstrated that XSB could ameliorate cognitive dysfunction caused by BCAS through regulating CBF, reducing white matter lesions, suppressing glial activation, promoting neurogenesis, and mitigating neuroinflammation. Notably, the NF-κB signaling pathway emerged as a pivotal player in this mechanism.

Ethoxyquin, a Lipid Peroxidation Inhibitor, Has Protective Effects against White Matter Lesions in a Mouse Model of Chronic Cerebral Hypoperfusion.

White matter lesions induced by chronic cerebral hypoperfusion can cause vascular dementia; however, no appropriate treatments are currently available for these diseases. In this study, we investigated lipid peroxidation, which has recently been pointed out to be associated with cerebrovascular disease and vascular dementia, as a therapeutic target for chronic cerebral hypoperfusion. We used ethoxyquin, a lipid-soluble antioxidant, in a neuronal cell line and mouse model of the disease. The cytoprotective effect of ethoxyquin on glutamate-stimulated HT-22 cells, a mouse hippocampal cell line, was comparable to that of a ferroptosis inhibitor. In addition, the administration of ethoxyquin to bilateral common carotid artery stenosis model mice suppressed white matter lesions, blood-brain barrier disruption, and glial cell activation. Taken together, we propose that the inhibition of lipid peroxidation may be a useful therapeutic approach for chronic cerebrovascular disease and the resulting white matter lesions.

Effects of Banhabaekchulcheonma-Tang on Brain Injury and Cognitive Function Impairment Caused by Bilateral Common Carotid Artery Stenosis in a Mouse Model.

Vascular dementia (VD) is the second most prevalent dementia type, with no drugs approved for its treatment. Here, the effects of Banhabaekchulcheonma-Tang (BBCT) on ischemic brain injury and cognitive function impairment were investigated in a bilateral carotid artery stenosis (BCAS) mouse model. Mice were divided into sham-operated, BCAS control, L-BBCT (40 ml/kg), and H-BBCT (80 ml/kg) groups. BBCT's effects were characterized using the Y-maze test, novel object recognition test (NORT), immunofluorescence staining, RNA sequencing, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The NORT revealed cognitive function improvement in the H-BBCT group, while the Y-maze test revealed no significant difference among the four groups. The CD68+ microglia and GFAP+ astrocyte numbers were reduced in the H-BBCT group. Furthermore, H-BBCT treatment restored the dysregulation of gene expression caused by BCAS. The major BBCT targets were predicted to be cell division cycle protein 20 (CDC20), Epidermal growth factor (EGF), and tumor necrosis factor receptor-associated factor 1 (TRAF1). BBCT regulates the neuroactive ligand-receptor interaction and neuropeptide signaling pathways, as predicted by KEGG and GO analyses, respectively. BBCT significantly improved cognitive impairment in a BCAS mouse model by inhibiting microglial and astrocyte activation and regulating the expression of CDC20, EGF, TRAF1, and key proteins in the neuroactive ligand-receptor interaction and neuropeptide signaling pathways.

I-C-F-6 attenuates chronic cerebral hypoperfusion-induced neurological injury in mice by modulating microglia polarization.

Chronic cerebral hypoperfusion (CCH) is the leading cause of chronic cerebral dysfunction syndrome with its complex pathological mechanisms involving cortical and hippocampal neuronal loss, white matter lesions, and neuroinflammation. I-C-F-6 is a septapeptide, which has anti-inflammatory and anti-fibrotic effects. This study aimed to evaluate the neuroprotective effect of I-C-F-6 in chronic cerebral hypoperfusion (CCH)-induced neurological injury. C57BL/6J mice were subjected to bilateral common carotid artery stenosis (BCAS), and BV2 microglia cells were induced with oxygen-glucose deprivation (OGD). In vivo, mice were divided randomly into four groups: Sham, BCAS, GBE (30mg/kg), and I-C-F-6 (0.5mg/kg). In vitro, microglia were divided randomly into four groups: control, OGD, I-C-F-6 (25μg/mL), and Shikonin (800nmol/L). Through LFB, TUNEL, and NeuN staining, we found that I-C-F-6 was able to mitigate myelin pathology and reduce the number of apoptotic neurons. Furthermore, immunofluorescence staining revealed that I-C-F-6 was able to reduce microglia clustering and downregulate NF-κB p65. We also observed a significant downregulation of M1 phenotype microglia signature genes, such as TNF-α, iNOS, and upregulation of anti-inflammatory cytokines, such as Arg-1 and IL-10, indicating that I-C-F-6 may mainly reduce polarization towards the M1 phenotype in microglia. Notably, I-C-F-6 downregulated the expression of NF-κB signaling pathway-related proteins IKK-β and NF-κB p65, as well as pro-inflammatory cytokines IL-1β and iNOS. In conclusion, I-C-F-6 can improve neurological damage, alleviate neuroinflammation, and inhibit microglia polarization to the M1 phenotype via the NF-κB signaling pathway.

Integrated Analysis of Chromatin and Transcriptomic Profiling Identifies PU.1 as a Core Regulatory Factor in Microglial Activation Induced by Chronic Cerebral Hypoperfusion

In addition to causing white matter lesions, chronic cerebral hypoperfusion (CCH) can also cause damage to gray matter, but the underlying molecular mechanisms remain largely unknown. In order to obtain a better understanding of the relationship between gene expression and transcriptional regulation alterations, novel upstream regulators could be identified using integration analysis of the transcriptome and epigenetic approaches. Here, a bilateral common carotid artery stenosis (BCAS) model was established for inducing CCH in mice. The spatial cognitive function of mice was evaluated, and changes in cortical microglia morphology were observed. RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on isolated mouse cortical brain tissue. Then, a systematic joint analysis of BCAS hypoperfusion-induced cortex-specific RNA-seq and ATAC-seq was conducted in order to assess the extent of the correlation between the two, and PU.1 was found to be greatly enriched through motif analysis and transcription factor annotation. Also, the core regulatory factor PU.1 induced by BCAS hypoperfusion was shown to be colocalized with microglia. Based on the above analysis, PU.1 plays a key regulatory role in microglial activation induced by CCH. And the transcriptome and epigenomic data presented in this study can help identify potential targets for future research exploring chronic hypoperfusion-induced brain injury.

Neddylation in the chronically hypoperfused corpus callosum: MLN4924 reduces blood-brain barrier injury via ERK5/KLF2 signaling

Blood-brain barrier (BBB) breakdown and cerebrovascular dysfunction may contribute to the pathology in white matter lesions and consequent cognitive decline caused by cerebral hypoperfusion. Neddylation is the process of attaching a ubiquitin-like molecule NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8) to specific targets. By modifying protein substrates, neddylation plays critical roles in various important biological processes. However, whether neddylation influences the pathogenesis of hypoperfused brain remains unclear. In the present study, cerebral hypoperfusion-induced white matter lesions were produced by bilateral common carotid artery stenosis in mice. The function of the neddylation pathway, BBB integrity, cerebrovascular dysfunction, myelin density in the corpus callosum and cognitive function were determined. We show that NEDD8 conjugation aberrantly amplified in microvascular endothelium in the corpus callosum following cerebral hypoperfusion. MLN4924, a small-molecule inhibitor of NEDD8-activating enzyme currently in clinical trials, preserved BBB integrity, attenuated glial activation and enhanced oligodendrocyte differentiation, and reduced hypoperfusion-induced white matter lesions in the corpus callosum and thus improved cognitive performance via inactivating cullin-RING E3 ligase (CRL). Administration of MLN4924 caused the accumulation of ERK5 and KLF2. The ERK5 inhibitor BIX 02189, down-regulated MLN4924-induced activation of KLF2 and reversed MLN4924-mediated increase in pericyte coverage and junctional proteins. Furthermore, BIX 02189 blocked MLN4924-afforded protection against BBB disruption and white matter lesions in the corpus callosum. Collectively, our results revealed that neddylation impairs vascular function and thus exacerbated the pathology of hypoperfused brain and that inhibition of neddylation with MLN4924 may offer novel therapeutic opportunities for cerebral hypoperfusion-associated cognitive impairment.

Neurotropin alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in mice with vascular dementia

Vascular dementia (VD) is the second most common cause of dementia after Alzheimer's disease. Neuroinflammation contributes to pathogenesis of VD. Neurotropin (NTP) is an analgesic that has been shown to suppress inflammation and neural repair. But its effects on VD are still unclear. Therefore, this study aimed to investigate the therapeutic effects and potential mechanisms of NTP in the VD model mice established by bilateral common carotid artery stenosis method. In VD mice, we found that NTP treatment increased cerebral blood flow by Laser speckle imaging, reduced neuron loss by Nissl, HE and immunochemistry staining, attenuated white matter damage by magnetic resonance imaging and ultrastructural damage by transmission electron microscope, improved cognitive functions by new object recognition test and three-chamber test, Y maze test and Morris water maze test, inhibited significantly glial activation by immunofluorescence methods, reduced the expression of TLR4, down-regulated expression of MyD88 and phosphorylation of NF-κB P65, decreased the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNFα. Further, we showed that administration of a TLR4 inhibitor TAK242 had a similar effect to NTP, while the TLR4 agonist CRX-527 attenuated the effect of NTP in the VD mice. Collectively, our study suggested that NTP alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in the VD mice. Thus, NTP may be a promising therapeutic approach and a potential TLR4 inhibitor for VD.

A novel needle mouse model of vascular cognitive impairment and dementia.

Bilateral common carotid artery (CCA) stenosis (BCAS) is a useful model to mimic vascular cognitive impairment and dementia (VCID). However, current BCAS models have the disadvantages of high cost and incompatibility with MRI scanning due to metal implantation. We have established a new low-cost VCID model that better mimics human VCID and is compatible with live-animal MRI. The right and the left CCAs were temporarily ligated to 32 and 34 gauge needles with three ligations, respectively. After needle removal, CCA blood flow, cerebral blood flow, white matter injury (WMI) and cognitive function were measured. In male mice, needle removal led to ∼49.8% and ∼28.2% blood flow recovery in the right and left CCA, respectively. This model caused persistent and long-term cerebral hypoperfusion in both hemispheres (more severe in the left hemisphere), and WMI and cognitive dysfunction in about 90% of mice, which is more reliable compared to other models. Importantly, these pathological changes and cognitive impairments lasted for up to 24 weeks after surgery. The survival rate over 24 weeks was 81.6%. Female mice showed similar cognitive dysfunction, but a higher survival rate (91.6%) and relatively milder white matter injury. A novel, low-cost VCID model compatible with live-animal MRI with long-term outcomes was established.Significance StatementBCAS is an animal model mimicking carotid artery stenosis to study VCID. However, current BCAS models cost high cost and is incompatibility with MRI scanning due to metal implantation. We established a new asymmetric BCAS model by ligating the common carotid artery to various needle gauges followed by an immediate needle removal. Needle removal led to moderate stenosis in the right CCA and severe stenosis in the left CCA. This needle model replicates the hallmarks of VCID well in about 90% of mice, which is more reliable compared to other models, has ultra-low cost, and is compatible with MRI scanning in live animals. It will provide a new valuable tool and offer new insights for VCID research.

The astrocytic TRPA1 channel mediates an intrinsic protective response to vascular cognitive impairment via LIF production.

Vascular cognitive impairment (VCI) refers to cognitive alterations caused by vascular disease, which is associated with various types of dementia. Because chronic cerebral hypoperfusion (CCH) induces VCI, we used bilateral common carotid artery stenosis (BCAS) mice as a CCH-induced VCI model. Transient receptor potential ankyrin 1 (TRPA1), the most redox-sensitive TRP channel, is functionally expressed in the brain. Here, we investigated the pathophysiological role of TRPA1 in CCH-induced VCI. During early-stage CCH, cognitive impairment and white matter injury were induced by BCAS in TRPA1-knockout but not wild-type mice. TRPA1 stimulation with cinnamaldehyde ameliorated BCAS-induced outcomes. RNA sequencing analysis revealed that BCAS increased leukemia inhibitory factor (LIF) in astrocytes. Moreover, hydrogen peroxide-treated TRPA1-stimulated primary astrocyte cultures expressed LIF, and culture medium derived from these cells promoted oligodendrocyte precursor cell myelination. Overall, TRPA1 in astrocytes prevents CCH-induced VCI through LIF production. Therefore, TRPA1 stimulation may be a promising therapeutic approach for VCI.

Transcriptome Profiling of Hippocampus After Cerebral Hypoperfusion in Mice.

Chronic cerebral hypoperfusion (CCH) is considered to be one of the major mechanism in the pathogenesis of vascular cognitive impairment (VCI). Increased inflammatory cells, particularly microglia, often parallel hypoperfusion-induced gray matter damage such as hippocampal lesions, but the exact mechanism remains largely unknown. To understand the pathological mechanisms, we analyzed hippocampus-specific transcriptome profiles after cerebral hypoperfusion. The mouse hypoperfusion model was induced by employing the 0.16/0.18mm bilateral common carotid artery stenosis (BCAS) procedure. Cerebral blood flow (CBF) was assessed after 3-week hypoperfusion. Pathological changes were evaluated via hematoxylin staining and immunofluorescence staining. RNA-sequencing (RNA-seq) was performed using RNA samples of sham- or BCAS-operated mice, followed by quantitative real-time PCR (qRT-PCR) validation. We found that the 0.16/0.18mm BCAS induced decreased CBF, hippocampal neuronal loss, and microglial activation. Furthermore, GSEA between sham and BCAS mice showed activation of interferon-beta signaling along with inflammatory immune responses. In addition, integrative analysis with published single-cell RNA-seq revealed that up-regulated differentially expressed genes (DEGs) were enriched in a distinct cell type of "microglia," and down-regulated DEGs were enriched in "CA1 pyramidal," not in "interneurons" or "S1 pyramidal." This database of transcriptomic profiles of BCAS-hypoperfusion will be useful for future studies to explore potential targets for vascular cognitive dysfunction.

3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage.

Chronic cerebral hypoperfusion is an important pathological factor in many neurodegenerative diseases, such as cerebral small vessel disease (CSVD). One of the most used animal models for chronic cerebral hypoperfusion is the bilateral common carotid artery stenosis (BCAS) mouse. For the therapy of CSVD and other diseases, it will be beneficial to understand the pathological alterations of the BCAS mouse, particularly vascular pathological changes. A mouse model of BCAS was used, and 8 weeks later, cognitive function of the mice was examined by using novel object recognition test and eight-arm radial maze test. 11.7 T magnetic resonance imaging (MRI) and luxol fast blue staining were used to evaluate the injury of the corpus callosum (CC), anterior commissure (AC), internal capsule (IC), and optic tract (Opt) in the cerebral white matter of mice. Three-dimensional vascular images of the whole brain of mice were acquired using fluorescence micro-optical sectioning tomography (fMOST) with a high resolution of 0.32 × 0.32 × 1.00 μm3. Then, the damaged white matter regions were further extracted to analyze the vessel length density, volume fraction, tortuosity, and the number of vessels of different internal diameters. The mouse cerebral caudal rhinal vein was also extracted and analyzed for its branch number and divergent angle in this study. BCAS modeling for 8 weeks resulted in impaired spatial working memory, reduced brain white matter integrity, and myelin degradation in mice, and CC showed the most severe white matter damage. 3D revascularization of the whole mouse brain showed that the number of large vessels was reduced and the number of small vessels was increased in BCAS mice. Further analysis revealed that the vessel length density and volume fraction in the damaged white matter region of BCAS mice were significantly reduced, and the vascular lesions were most noticeable in the CC. At the same time, the number of small vessels in the above white matter regions was significantly reduced, while the number of microvessels was significantly increased in BCAS mice, and the vascular tortuosity was also significantly increased. In addition, the analysis of caudal rhinal vein extraction revealed that the number of branches and the average divergent angle in BCAS mice were significantly reduced. The BCAS modeling for 8 weeks will lead to vascular lesions in whole brain of mice, and the caudal nasal vein was also damaged, while BCAS mice mainly mitigated the damages by increasing microvessels. What is more, the vascular lesions in white matter of mouse brain can cause white matter damage and spatial working memory deficit. These results provide evidence for the vascular pathological alterations caused by chronic hypoperfusion.

Tooth-Cutting-Induced Maxillary Malocclusion Exacerbates Cognitive Deficit in a Mouse Model of Vascular Dementia.

Treatments to restore the balance of the temporomandibular joint (TMJ) are performed in the field of complementary and alternative medicine; however, evidence supporting this approach remains weak. Therefore, this study attempted to establish such evidence. Bilateral common carotid artery stenosis (BCAS) operation, which is commonly used for the establishment of a mouse model of vascular dementia, was performed, followed by tooth cutting (TEX) for maxillary malocclusion to promote the imbalance of the TMJ. Behavioural changes, changes in nerve cells and changes in gene expression were assessed in these mice. The TEX-induced imbalance of the TMJ caused a more severe cognitive deficit in mice with BCAS, as indicated by behavioural changes in the Y-maze test and novel object recognition test. Moreover, inflammatory responses were induced via astrocyte activation in the hippocampal region of the brain, and the proteins involved in inflammatory responses were found to be involved in these changes. These results indirectly show that therapies that restore the balance of the TMJ can be effectively used for the management of cognitive-deficit-related brain diseases associated with inflammation.

Ebselen alleviates white matter lesions and improves cognitive deficits by attenuating oxidative stress via Keap1/Nrf2 pathway in chronic cerebral hypoperfusion mice

Oxidative stress is crucial in cerebral white matter lesions (WMLs) induced by chronic cerebral hypoperfusion. Therefore, ameliorating oxidative damage is considered to be a beneficial strategy for the treatment of WMLs. Ebselen (EbSe), a small lipid organoselenium compound, its lipid peroxidation activity is mediated through the glutathione peroxidase-mimetic properties. This study aimed to investigate the role of EbSe in WMLs after bilateral common carotid artery stenosis (BCAS). The BCAS model can moderately reduce cerebral blood flow, and mimics white matter damage caused by chronic cerebral hypoperfusion or small vessel disease. Laser Speckle Contrast Imaging (LSCI) was used to monitor the cerebral blood flow of mice. The spatial learning and memory were tested by using the eight-arm maze. LFB staining was used to detect demyelination. The expression of MBP, GFAP and Iba1 was assayed by immunofluorescence. The demyelination was assessed by Transmission Electron Microscope (TEM). The activities of MDA, SOD and GSH-Px were detected by assay kits. The mRNA levels of SOD, GSH-Px and HO-1 was detected by realtime PCR. The activation of the Nrf2/ARE pathway and the expression of SOD, GSH-Px and HO-1was assessed by Western blot. EbSe ameliorated cognitive deficits and white matter lesions induced by bilateral common carotid artery stenosis (BCAS). The expression of GFAP and Iba1 was decreased in the corpus callosum of BCAS mice after EbSe treatment. Moreover, EbSe alleviated the level of MDA by elevating the expression and mRNA of SOD, GSH-Px and HO-1 in BCAS mice. Furthermore, EbSe promoted the dissociation of the Keap1/Nrf2 complex, resulting in the accumulation of Nrf2 in the nucleus. This study demonstrates a favorable effect of EbSe on cognitive impairment in a chronic cerebral hypoperfusion model, and the improvement of EbSe's antioxidant property is mediated by Keap1/Nrf2 pathway.

Determination of the role of hippocampal astrocytes in the bilateral common carotid artery stenosis mouse model by RNA sequencing

IntroductionBilateral common carotid artery stenosis (BCAS) is used experimentally to model vascular dementia (VaD). Previous studies have primarily focused on the degradation of brain white matter after BCAS. However, hippocampal abnormalities are equally important, and hippocampal astrocytes are specifically involved in neural circuits that regulate learning and memory. Whether hippocampal astrocytes participate in the pathogenesis of BCAS-induced VaD has not been well studied. Therefore, in the present study, we attempted to explore the role of hippocampal astrocytes in BCAS. MethodsTwo months after BCAS, behavioral experiments were conducted to investigate changes in neurological function in sham and BCAS mice. A ribosome-tagging approach (RiboTag) profiling strategy was used to isolate mRNAs enriched in hippocampal astrocytes, and the RNA was sequenced and analyzed using transcriptomic methods. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was utilized to validate the results of RNA sequencing. Immunofluorescence analyses were conducted to evaluate the number and morphology of hippocampal astrocytes. ResultsWe observed significant short-term working memory impairment in BCAS mice. Moreover, the RNA obtained through RiboTag technology was specific to astrocytes. Transcriptomics approaches and subsequent validation studies revealed that the genes that showed expression changes in hippocampal astrocytes after BCAS were mainly involved in immune system processes, glial cell proliferation, substance transport and metabolism. Furthermore, the number and distribution of astrocytes in the CA1 region of the hippocampus tended to decrease after modeling. ConclusionIn this study, comparisons between sham and BCAS mice showed that the functions of hippocampal astrocytes were impaired in BCAS-induced chronic cerebral hypoperfusion-related VaD.