Chronic Cerebral Hypoperfusion In Rats Research Articles

Nicotinamide riboside alleviates brain dysfunction induced by chronic cerebral hypoperfusion via protecting mitochondria

Chronic cerebral hypoperfusion (CCH) is an enduring inadequate blood flow to the brain, resulting in vascular dementia (VaD). However, the effective treatment strategies are lacking. Supplementing with nicotinamide adenine dinucleotide (NAD+) has shown neuroprotective benefits in other neurodegenerative disorders. Nicotinamide riboside (NR), as a precursor of NAD+, is believed to hold promise in improving mitochondrial health, autophagy, and cognitive function. Meanwhile, NR has unique oral bioavailability, good tolerability, and minimal side effects, and it is the most promising for clinical translation. However, the effectiveness of NR in treating CCH-related VaD is still uncertain. The present study examined the neuroprotective effects of NR supplementation and its underlying mechanisms in a CCH rat model. The rats with CCH were given NR at a daily dosage of 400 mg/kg for 3 months. NR supplementation increased blood and brain NAD+ levels and improved brain function in CCH rats, including cognitive function and oxygenation capacity. It also reduced hippocampal neuronal loss and abnormalities and mitigated the decrease in dendritic spine density. The analysis of RNA sequencing in hippocampal tissue supports these findings. Electron microscopy and protein detection results suggest that NR may maintain mitochondrial structural integrity and exert a protective role by attenuating mitochondrial fission and impaired autophagy flux caused by CCH. In conclusion, these findings offer evidence for the neuroprotective potential of NR supplementation in ameliorating cognitive impairment induced by CCH.

Binary Nano-inhalant Formulation of Icariin Enhances Cognitive Function in Vascular Dementia via BDNF/TrkB Signaling and Anti-inflammatory Effects.

Vascular dementia (VaD) has a serious impact on the patients' quality of life. Icariin (Ica) possesses neuroprotective potential for treating VaD, yet its oral bioavailability and blood-brain barrier (BBB) permeability remain challenges. This research introduced a PEG-PLGA-loaded chitosan hydrogel-based binary formulation tailored for intranasal delivery, enhancing the intracerebral delivery efficacy of neuroprotective agents. The formulation underwent optimization to facilitate BBB crossing, with examinations conducted on its particle size, morphology, drug-loading capacity, in vitro release, and biodistribution. Using the bilateral common carotid artery occlusion (BCCAO) rat model, the therapeutic efficacy of this binary formulation was assessed against chitosan hydrogel and PEG-PLGA nanoparticles loaded with Ica. Post-intranasal administration, enhanced cognitive function was evident in chronic cerebral hypoperfusion (CCH) rats. Further mechanistic evaluations, utilizing immunohistochemistry (IHC), RT-PCR, and ELISA, revealed augmented transcription of synaptic plasticity-associated proteins like SYP and PSD-95, and a marked reduction in hippocampal inflammatory markers such as IL-1β and TNF-α, highlighting the formulation's promise in alleviating cognitive impairment. The brain-derived neurotrophic factor (BDNF)/tropomyosin related kinase B (TrkB) pathway was activated significantly in the binary formulation compared with the other two. Our study demonstrates that the intranasal application of chitosan hydrogel loaded with Ica-encapsulated PEG-PLGA could effectively deliver Ica into the brain and enhance its neuroprotective effect.

Inhibition of Endoplasmic Reticulum Stress Improves Chronic Ischemic Hippocampal Damage Associated with Suppression of IRE1α/TRAF2/ASK1/JNK-Dependent Apoptosis.

Chronic cerebral ischemia is a complex form of stress, of which the most common hemodynamic characteristic is chronic cerebral hypoperfusion (CCH). Lasting endoplasmic reticulum (ER) stress can drive neurological disorders. Targeting ER stress shows potential neuroprotective effects against stroke. However, the role of ER stress in CCH pathological processes and the effects of targeting ER stress on brain ischemia are unclear. Here, a CCH rat model was established by bilateral common carotid artery occlusion. Rats were treated with 4-PBA, URB597, or both for 4weeks. Neuronal morphological damage was detected using hematoxylin-eosin staining. The expression levels of the ER stress-ASK1 cascade-related proteins GRP78, IRE1α, TRAF2, CHOP, Caspase-12, ASK1, p-ASK1, JNK, and p-JNK were assessed by Western blot. The mRNA levels of TNF-α, IL-1β, and iNOS were assessed by RT-PCR. For oxygen-glucose deprivation experiments, mouse hippocampal HT22 neurons were used. Apoptosis of the hippocampus and HT22 cells was detected by TUNEL staining and Annexin V-FITC analysis, respectively. CCH evoked ER stress with increased expression of GRP78, IRE1α, TRAF2, CHOP, and Caspase-12. Co-immunoprecipitation experiments confirmed the interaction between TRAF2 and ASK1. ASK1/JNK signaling, inflammatory cytokines, and neuronal apoptosis were enhanced, accompanied by persistent ER stress; these were reversed by 4-PBA and URB597. Furthermore, the ASK1 inhibitor GS4997 and 4-PBA displayed synergistic anti-apoptotic effects in cells with oxygen-glucose deprivation. In summary, ER stress-induced apoptosis in CCH is associated with the IRE1α/TRAF2/ASK1/JNK signaling pathway. Targeting the ER stress-ASK1 cascade could be a novel therapeutic approach for ischemic cerebrovascular diseases.

Physical and social environmental enrichment alleviate ferroptosis and inflammation with inhibition of TLR4/MyD88/p38MAPK pathway in chronic cerebral hypoperfusion rats

A typical enriched environment (EE), which combines physical activity and social interaction, has been proven to mitigate cognitive impairment caused by chronic cerebral hypoperfusion (CCH). However, it remains unclear how the different components of EE promote cognitive recovery after CCH. This study stripped out the different components of EE into physical environmental enrichment (PE) and social environmental enrichment (SE), and compared the neuroprotective effects of PE, SE and typical EE (PSE) in CCH. The results of novel object recognition and Morris water maze tests showed that PE, SE, and PSE improved cognitive function in CCH rats. Additionally, Nissl and TUNEL staining revealed that three EEs reduced neuronal loss in the hippocampus. PSE exhibited superior neuroprotective and functional improvement effects compared to PE and SE, while there was no significant difference between PE and SE. Furthermore, three EEs reduced lipid peroxidation in the hippocampus with decreasing the levels of MDA and increasing the activities of SOD and GSH. The expression of SLC7A11 and GPX4 was increased, while the level of p53 was reduced in three EEs. This suggested that three EEs inhibited ferroptosis by maintaining the redox homeostasis in the hippocampus. Three EEs reduced the levels of IL-β, TNF-α, and IL-6, thereby inhibiting neuroinflammation. Additionally, Western blotting and immunofluorescence results indicated that three EEs also inhibited the TLR4/MyD88/p38MAPK signaling pathway. These findings collectively demonstrated that the three EEs alleviated hippocampal ferroptosis and neuroinflammation in CCH rats, thereby reducing neuronal loss, which might be associated with the inhibition of the TLR4/MyD88/p38MAPK signaling pathway. Moreover, the study results supported that it is only through the combination of physical exercise and social interaction that the optimal neuroprotective effects can be achieved. These findings provided valuable insights for the prevention and treatment of vascular cognitive impairment.

Hexahydrocurcumin attenuated demyelination and improved cognitive impairment in chronic cerebral hypoperfusion rats.

Age-related white matter lesions (WML) frequently present vascular problems by decreasing cerebral blood supply, resulting in the condition known as chronic cerebral hypoperfusion (CCH). This study aimed to investigate the effect of hexahydrocurcumin (HHC) on the processes of demyelination and remyelination induced by the model of the Bilateral Common Carotid Artery Occlusion (BCCAO) for 29days to mimic the CCH condition. The pathological appearance of myelin integrity was significantly altered by CCH, as evidenced by Transmission Electron Microscopy (TEM) and Luxol Fast Blue (LFB) staining. In addition, CCH activated A1-astrocytes and reactive-microglia by increasing the expression of Glial fibrillary acidic protein (GFAP), complement 3 (C3d) and pro-inflammatory cytokines. However, S100a10 expression, a marker of neuroprotective astrocytes, was suppressed, as were regenerative factors including (IGF-1) and Transglutaminase 2 (TGM2). Therefore, the maturation step was obstructed as shown by decreases in the levels of myelin basic protein (MBP) and the proteins related with lipid synthesis. Cognitive function was therefore impaired in the CCH model, as evidenced by the Morris water maze test. By contrast, HHC treatment significantly improved myelin integrity, and inhibited A1-astrocytes and reactive-microglial activity. Consequently, pro-inflammatory cytokines and A1-astrocytes were attenuated, and regenerative factors increased assisting myelin maturation and hence improving cognitive performance. In conclusion, HHC improves cognitive function and also the integrity of white matter in CCH rats by reducing demyelination, and pro-inflammatory cytokine production and promoting the process of remyelination.

Hexahydrocurcumin Attenuates Neuronal Injury and Modulates Synaptic Plasticity in Chronic Cerebral Hypoperfusion in Rats

Hexahydrocurcumin Attenuates Neuronal Injury and Modulates Synaptic Plasticity in Chronic Cerebral Hypoperfusion in Rats

ChemR23 activation attenuates cognitive impairment in chronic cerebral hypoperfusion by inhibiting NLRP3 inflammasome-induced neuronal pyroptosis

Neuroinflammation plays critical roles in vascular dementia (VaD), the second leading cause of dementia, which can be induced by chronic cerebral hypoperfusion (CCH). NLRP3 inflammasome-induced pyroptosis, the inflammatory programmed cell death, has been reported to contribute to the development of VaD. ChemR23 is a G protein-coupled receptor that has emerging roles in regulating inflammation. However, the role of ChemR23 signalling in NLRP3 inflammasome-induced pyroptosis in CCH remains elusive. In this study, a CCH rat model was established by permanent bilateral common carotid artery occlusion (BCCAO) surgery. Eight weeks after the surgery, the rats were intraperitoneally injected with the ChemR23 agonist Resolvin E1 (RvE1) or chemerin-9 (C-9). Additionally, primary rat hippocampal neurons and SH-SY5Y cells were adopted to mimic CCH injury in vitro. Our results showed that the levels of ChemR23 expression were decreased from the 8th week after BCCAO, accompanied by significant cognitive impairment. Further analysis revealed that CCH induced neuronal damage, synaptic injury and NLRP3-related pyroptosis activation in hippocampal neurons. However, pharmacologic activation of ChemR23 with RvE1 or C-9 counteracted these changes. In vitro experiments also showed that ChemR23 activation prevented primary neuron pyroptosis induced by chronic hypoxia. In addition, manipulating ChemR23 expression markedly regulated NLRP3 inflammasome-induced neuronal pyroptosis through PI3K/AKT/Nrf2 signalling in SH-SY5Y cells under hypoglycaemic and hypoxic conditions. Collectively, our data demonstrated that ChemR23 activation inhibits NLRP3 inflammasome-induced neuronal pyroptosis and improves cognitive function via the PI3K/AKT/Nrf2 signalling pathway in CCH models. ChemR23 may serve as a potential novel therapeutic target to treat CCH-induced cognitive impairment.

Combined use of hair follicle stem cells and CEPO (carbamylated erythropoietin)-Fc in a rat model of chronic cerebral hypoperfusion: A behavioral, electrophysiological, and molecular study

BackgroundIn dementia, synaptic dysfunction appears before neuronal loss. Stem cell therapy could potentially provide a promising strategy for the treatment of dementia models. The carbamylated erythropoietin fusion protein (CEPO-Fc) has shown synaptotrophic effects. This study aimed to determine the efficiency of the combined use of hair follicle stem cells (HFSC) and CEPO-Fc in the basal synaptic transmission (BST) and long-term plasticity (LTP) of chronic cerebral hypoperfusion (CCH) rats. MethodsWe divided 64 adult rats into control, sham, CCH+vehicle, CCH+CEPO, CCH+HFSC, and CCH+HFSC+CEPO groups. The CEPO-Fc was injected three times/week for 30 days. HFSC transplantation was done on days 4, 14, and 21 after surgery. The Morris water maze test and passive avoidance were used to assess memory. BST and LTP were assessed by a field-potential recording of the CA1 region. The hippocampal mRNA expression of IGF-1, TGF-β1, β1-Catenine, NR2B, PSD-95, and GSk-3β was evaluated by quantitative RT-PCR. ResultsFollowing combination therapy, spatial memory retention, and BST showed significant improvement relative to HFSC and CEPO-Fc groups. These effects were also confirmed by recovered mRNA expression of β1-catenin, TGF-β1, and NR2B. GSK-3β expression was downregulated in all treatment groups. The upregulated PSD-95 was identified in HFSC and combination groups compared to the vehicle group. ConclusionsThese findings indicate that the combined use of HFSC and CEPO-Fc may be more advantageous for treating memory disruption in the CCH model than CEPO-Fc or HFSC alone. This type of combination therapy may hopefully lead to a new approach to treatment for dementia.

Rosmarinic acid-rich ethanolic extract of Orthosiphon stamineus ameliorates cognitive and hippocampal long-term potentiation in chronic cerebral hypoperfusion rat model

Chronic cerebral hypoperfusion (CCH) is one of the main causes of vascular dementia caused by the reduced blood flow to the brain. Orthosiphon stamineus (OS) is a medicinal herb exhibiting pronounced neuroprotective, anti-oxidant and anti-inflammatory activities due to its high rosmarinic acid content. This study investigated the nootropic effect of OS ethanolic extract on cognitive functions in CCH rats. CCH was developed by permanent bilateral occlusion of the common carotid artery (PBOCCA). Passive avoidance task (PAT) and Morris water maze (MWM) test were conducted to evaluate cognitive functions followed by in vivo long-term potentiation (LTP) for assessing neuroplasticity. The rosmarinic acid content of OS ethanolic extract was quantified using a validated HPLC-PDA. Treatment with OS ethanolic extract significantly increased step-through latency in the PAT, decreased escape latency at a low dose of OS extract in the MWM and rescued the LTP impairment at the highest dose in CCH rats. These results strongly support the effectiveness of rosmarinic acid-rich OS extract (5.088 % w/w) in treating pathological vascular dementia caused by CCH.

The promoting effect of modified Dioscorea pills on vascular remodeling in chronic cerebral hypoperfusion via the Ang/Tie signaling pathway.

The objective of this study was to investigate the effect of modified Dioscorea pills (MDP) on microcirculatory remodeling in the hippocampus of rats with chronic cerebral hypoperfusion (CCH) through the angiopoietin (Ang)/tyrosine kinase receptor tyrosine kinase with immunoglobulin-like and EGF-like domains (Ang receptor) 2 (Tie-2) signaling pathways, which may underlie the cognitive improvement observed in CCH rats. Forty male Sprague-Dawley rats raised under specific pathogen-free conditions were randomly divided into three groups: control group (10 rats), model group (15 rats), and MDP group (15 rats). The rats in the model group and MDP group underwent bilateral common carotid artery occlusion using the 2-vessel occlusion (2-VO) method to induce CCH. Rats in the control group underwent the same surgical procedures as those in the model group, except for ligation and occlusion of the carotid arteries. After 1 week of 2-VO, rats in the MDP group were administered MDP condensed decoction intragastrically at a dose of 1 ml/100 g body weight (prepared by the Preparation Room of Hubei Provincial Hospital of Traditional Chinese Medicine) for 45 days, while rats in the other two groups received normal saline intragastrically with the same dose and duration as the MDP group. After the intervention, all rats were euthanized, and brain perfusion was performed to obtain the hippocampal tissue for analysis. Immunohistochemical staining for CD43 was performed to assess microvessel density (MVD); western blot and the reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze the expression of proteins and genes in angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), Tie-2, and vascular endothelial growth factor (VEGF) proteins and genes in the hippocampal tissue and compute the Ang-1/Ang-2 ratio. MDP treatment reduced neuronal loss and promoted restoration of the damaged hippocampal structure in CCH rats. The model group showed significantly higher MVD (14.93 ± 1.92) compared to the control group (5.78 ± 1.65) (P < 0.01), whereas MDP treatment further increased MVD (21.19 ± 2.62). Western blot and RT-PCR analysis revealed that CCH significantly increased the expression of Ang-1, Ang-2, Tie-2, and VEGF proteins and genes, while MDP treatment further significantly upregulated the expression of these proteins and genes. In addition, MDP significantly elevated the gene and protein expression of the Ang-1/Ang-2 ratio compared to the control group (P = 0.041, P = 0.029). CCH induces microvascular neogenesis in the hippocampus, and MDP promotes angiogenesis and microcirculation remodeling in CCH rats via the Ang/Tie signaling pathway, which may be an important mechanism for its restorative effects on hippocampal perfusion and improvement of cognitive function in CCH rats.

Bio-enhanced fraction from Clitoria ternatea root extract ameliorates cognitive functions and in vivo hippocampal neuroplasticity in chronic cerebral hypoperfusion rat model.

Research employing a bio-enhanced fraction of Clitoria ternatea (CT) to treat cognitive decline in the animal model has not yet been found. This study aimed to determine the neuroprotective effect of CT root bioactive fraction (CTRF) in chronic cerebral hypoperfusion (CCH) rat model. CTRF and its major compound, clitorienolactones A (CLA), were obtained using column chromatography. A validated HPLC-UV method was employed for the standardization of CTRF. CCH rats were given orally either vehicle or fraction (10, 20 and 40mg/kg). Behavioural and hippocampal neuroplasticity studies were conducted following 4 weeks post-surgery. The brain hippocampus was extracted for proteins and neurotransmitters analyses. HPLC analysis showed that CTRF contained 25% (w/w) of CLA. All tested doses of CTRF and CLA (10mg/kg) significantly restored cognitive deficits and reversed the inhibition of neuroplasticity by CCH. However, only CTRF (40mg/kg) and CLA (10mg/kg) significantly reversed the elevation of amyloid-beta plaque. Subsequently, treatment with CTRF (40mg/kg) and CLA (10mg/kg) alleviated the downregulation of molecular synaptic signalling proteins levels caused by CCH. The neurotransmitters level was restored following treatment of CTRF and CLA. Our finding suggested that CTRF improves memory and neuroplasticity in CCH rats which was mainly contributed by CLA.

Lipopolysaccharide-Induced Delirium-like Behaviour in a Rat Model of Chronic Cerebral Hypoperfusion Is Associated with Increased Indoleamine 2,3-Dioxygenase Expression and Endotoxin Tolerance.

Indoleamine 2,3-dioxygenase (IDO) and the tryptophan-kynurenine pathway (TRP-KP) are upregulated in ageing and could be implicated in the pathogenesis of delirium. This study evaluated the role of IDO/KP in lipopolysaccharide (LPS)-induced delirium in an animal model of chronic cerebral hypoperfusion (CCH), a proposed model for delirium. CCH was induced by a permanent bilateral common carotid artery ligation (BCCAL) in Sprague Dawley rats to trigger chronic neuroinflammation-induced neurodegeneration. Eight weeks after permanent BCCAL, the rats were treated with a single systemic LPS. The rats were divided into three groups: (1) post-BCCAL rats treated with intraperitoneal (i.p.) saline, (2) post-BCCAL rats treated with i.p. LPS 100 μg/kg, and (3) sham-operated rats treated with i.p. LPS 100 μg/kg. Each group consisted of 10 male rats. To elucidate the LPS-induced delirium-like behaviour, natural and learned behaviour changes were assessed by a buried food test (BFT), open field test (OFT), and Y-maze test at 0, 24-, 48-, and 72 h after LPS treatment. Serum was collected after each session of behavioural assessment. The rats were euthanised after the last serum collection, and the hippocampi and cerebral cortex were collected. The TRP-KP neuroactive metabolites were measured in both serum and brain tissues using ELISA. Our data show that LPS treatment in CCH rats was associated with acute, transient, and fluctuated deficits in natural and learned behaviour, consistent with features of delirium. These behaviour deficits were mild compared to the sham-operated rats, which exhibited robust behaviour impairments. Additionally, heightened hippocampal IDO expression in the LPS-treated CCH rats was associated with reduced serum KP activity together with a decrease in the hippocampal quinolinic acid (QA) expression compared to the sham-operated rats, suggested for the presence of endotoxin tolerance through the immunomodulatory activity of IDO in the brain. These data provide new insight into the underlying mechanisms of delirium, and future studies should further explore the role of IDO modulation and its therapeutic potential in delirium.

Untargeted metabolomics uncovering neuroprotective effect of Dl-3-n-butylphthalide on improving cognitive impairment induced by chronic cerebral hypoperfusion in rats

Chronic cerebral hypoperfusion (CCH) can cause cognitive impairments. Dl-3-n-butylphthalide (NBP) is widely used in neurological disorders; but, the role of NBP in CCH remains unclear. This study aimed to investigate the potential mechanism of NBP on CCH through untargeted metabolomics. Animals were divided into CCH, Sham, and NBP groups. A rat model of bilateral carotid artery ligation was used to simulate CCH. Cognitive function of the rats was assessed using the Morris water maze test. Additionally, we used LC-MS/MS to detect ionic intensities of metabolites between the three groups for off-target metabolism analysis and to screen for differential metabolites. The analysis showed an improvement in cognitive function in rats after NBP treatment. Moreover, metabolomic studies showed that the serum metabolic profiles of the Sham and CCH groups were significantly altered, and 33 metabolites were identified as potential biomarkers associated with the effects of NBP. These metabolites were enriched in 24 metabolic pathways.And the pathway of differential metabolite enrichment was further verified by immunofluorescence. Thus, the study provides a theoretical basis for the pathogenesis of CCH and the treatment of CCH by NBP, and supports a wider application of NBP drugs.

Protective effect of nanoparticle NLRP3 inhibitor (MCC950) on cranial nerves in rats with vascular cognitive impairment

Background: Chronic cerebral hypoperfusion (CCH) is a common cause of Vascular cognitive impairment (VCI). The neuroinflammation caused by ischemia play a key role in the progression of the ailment process. The over activation of NLRP3/Caspase-1/IL-1β signaling aggravates the inflammatory response. We investigated whether NLRP3 inhibitors have a protective effect on cranial nerves in rats with VCI. Methods: Male SD rats were selected arbitrarily and categorized into the sham operation group, CCH group and CCH+MCC950 group (10 mg/kg). The rat model of CCH was formed by bilateral common carotid artery occlusion (BCCAO). The changes in rat’s cerebral blood flow were observed by laser speckle imaging after operation. The sham operation and the CCH group were given intraperitoneal (i.p) injection of normal saline, and the therapeutic group was given i.p having dose of MCC950 (10 mg/kg). The behavior of the rats was analyzed through the Y maze and open field experiments, and the hematoxylin-eosin (HE) staining technique was employed to analyze the behavior of the rats. The histological changes of the rat hippocampus were observed under the following conditions, cell apoptosis was observed by Tune staining, and the protein expressions of NLRP3, IL (interleukin)-1β, and cleaved-Caspase-1 in the hippocampus were analyzed by Western blot. Results: MCC950 treatment enhanced the working memory of CCH rats, and the rats in each group showed no motor deficit. As shown by HE staining, inhibition of NLRP3 reduced neuronal damage caused by pyroptosis. In addition, MCC950 diminishes the appearance of NLRP3, IL-1β, and cleaved-Caspase-1 in the brain tissue of CCH rats by inhibiting inflammasome formation. Conclusions: inhibitors can improve the learning and cognitive ability of rats with vascular cognitive impairment, and the mechanism might be linked to inhibiting the NLRP3/Caspase-1/IL-1β signaling and decreasing apoptosis.

Honokiol prevents chronic cerebral hypoperfusion induced astrocyte A1 polarization to alleviate neurotoxicity by targeting SIRT3-STAT3 axis.

Alzheimer's Dementia (AD) and Vascular Dementia (VaD) are two main types of dementias for which no specific treatment is available. Chronic Cerebral Hypoperfusion (CCH) is a pathogenesis underlying AD and VaD that promotes neuroinflammatory responses and oxidative stress. Honokiol (HNK) is a natural compound isolated from magnolia leaves that can easily cross blood brain barrier and has anti-inflammatory and antioxidant effects. In the present study, the effects of HNK on astrocyte polarization and neurological damage in in vivo and in vitro models of chronic cerebral hypoperfusion were explored. We found that HNK was able to inhibit the phosphorylation and nuclear translocation of STAT3, A1 polarization, and reduce conditioned medium's neuronal toxicity of astrocyte under chronic hypoxia induced by cobalt chloride; STAT3 phosphorylation inhibitor C188-9 was able to mimic the above effects of HNK, suggesting that HNK may inhibit chronic hypoxia-induced A1 polarization in astrocytes via STAT3. SIRT3 inhibitor 3-TYP reversed, while Sirt3 overexpression mimicked the inhibitory effects of HNK on oxidative stress, STAT3 phosphorylation and nuclear translocation, A1 polarization and neuronal toxicity of astrocyte under chronic hypoxic conditions. For in vivo research, continuous intraperitoneal injection of HNK (1mg/kg) for 21 days ameliorated the decrease in SIRT3 activity and oxidative stress, inhibited astrocytic STAT3 nuclear translocation and A1 polarization, and prevented neuron and synaptic loss in the hippocampal of CCH rats. Besides, HNK application improved the spatial memory impairment of CCH rats, as assessed with Morris Water Maze. In conclusion, these results suggest that the phytochemical HNK can inhibit astrocyte A1 polarization via regulating SIRT3-STAT3 axis, thus improving CCH-induced neurological damage. These results highlight HNK as novel treatment for dementia with underlying vascular mechanisms.

Effects of Dl-3-n-butylphthalide on cognitive functions and blood–brain barrier in chronic cerebral hypoperfusion rats

Vascular cognitive impairment (VCI) has been one of the major types of cognitive impairment. Blood–brain barrier damage plays an essential part in the pathogenesis of VCI. At present, the treatment of VCI is mainly focused on prevention, with no drug clinically approved for the treatment of VCI. This study aimed to investigate the effects of DL-3-n-butylphthalide (NBP) on VCI rats. A modified bilateral common carotid artery occlusion (mBCCAO) model was applied to mimic VCI. The feasibility of the mBCCAO model was verified by laser Doppler, 13N-Ammonia-Positron Emission Computed Tomography (PET), and Morris Water Maze. Subsequently, the Morris water maze experiment, Evans blue staining, and western blot of tight junction protein were performed to evaluate the effect of different doses of NBP (40 mg/kg, 80 mg/kg) on the improvement of cognitive impairment and BBB disruption induced by mBCCAO. Immunofluorescence was employed to examine the changes in pericyte coverage in the mBCCAO model and the effect of NBP on pericyte coverage was preliminarily explored. mBCCAO surgery led to obvious cognitive impairment and the decrease of whole cerebral blood flow, among which the blood flow in the cortex, hippocampus and thalamus brain regions decreased more significantly. High-dose NBP (80 mg/kg) improved long-term cognitive function in mBCCAO rats, alleviated Evans blue leakage and reduced the loss of tight junction proteins (ZO-1, Claudin-5) in the early course of the disease, thereby exerting a protective effect on the blood–brain barrier. No significant changes in pericyte coverage were observed after mBCCAO. High-dose NBP improved cognitive function in mBCCAO rats. High-dose NBP protected the integrity of BBB by upregulating TJ protein expression, rather than regulating pericyte coverage ratio. NBP could be a potential drug for the treatment of VCI.

Enriched environment attenuates hippocampal theta and gamma rhythms dysfunction in chronic cerebral hypoperfusion via improving imbalanced neural afferent levels.

Chronic cerebral hypoperfusion (CCH) is increasingly recognized as a common cognitive impairment-causing mechanism. However, no clinically effective drugs to treat cognitive impairment due to CCH have been identified. An abnormal distribution of neural oscillations was found in the hippocampus of CCH rats. By releasing various neurotransmitters, distinct afferent fibers in the hippocampus influence neuronal oscillations in the hippocampus. Enriched environments (EE) are known to improve cognitive levels by modulating neurotransmitter homeostasis. Using EE as an intervention, we examined the levels of three classical neurotransmitters and the dynamics of neural oscillations in the hippocampus of the CCH rat model. The results showed that EE significantly improved the balance of three classical neurotransmitters (acetylcholine, glutamate, and GABA) in the hippocampus, enhanced the strength of theta and slow-gamma (SG) rhythms, and dramatically improved neural coupling across frequency bands in CCH rats. Furthermore, the expression of the three neurotransmitter vesicular transporters-vesicular acetylcholine transporters (VAChT) and vesicular GABA transporters (VGAT)-was significantly reduced in CCH rats, whereas the expression of vesicular glutamate transporter 1 (VGLUT1) was abnormally elevated. EE partially restored the expression of the three protein levels to maintain the balance of hippocampal afferent neurotransmitters. More importantly, causal mediation analysis showed EE increased the power of theta rhythm by increasing the level of VAChT and VGAT, which then enhanced the phase amplitude coupling of theta-SG and finally led to an improvement in the cognitive level of CCH. These findings shed light on the role of CCH in the disruption of hippocampal afferent neurotransmitter balance and neural oscillations. This study has implications for our knowledge of disease pathways.

Enhancement of oligodendrocyte autophagy alleviates white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in rats.

Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy-lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.

A comprehensive perspective on the disposition, metabolism, and pharmacokinetics of representative multi-components of Dengzhan Shengmai in rats with chronic cerebral hypoperfusion after oral administration.

Dengzhan Shengmai capsule (DZSM), an evidence-based Chinese medicine comprising Erigeron breviscapus (Vaniot) Hand. -Mazz., Panax ginseng C.A.Mey., Ophiopogon japonicus (Thunb.) Ker Gawl., and Schisandra chinensis (Turcz.) Baill., exhibits an excellent efficacy in treating cardio- and cerebrovascular diseases. It contains caffeoyl compounds, flavonoids, saponins, and lignans as primary active components. However, so far, the characteristics of disposition, metabolism, and pharmacokinetics of its active components remain mostly unclear. To elucidate disposition, metabolism, and pharmacokinetics of representative components of DZSM in rats with chronic cerebral hypoperfusion (CCH) by integrating ex vivo and in situ approaches. Exposure and distribution of absorbed prototypes and their metabolites were comprehensively investigated using sensitive LC-MS/MS and high-resolution LC-Q-TOF/MS. Pharmacokinetics of representative 16 components (12 prototypes and 4 metabolites) with different chemical categories, relatively high in vivo levels, wide tissue distribution, and reported neuroprotective activities were profiled. The ex vivo everted gut sac and in situ linked-rat models were adopted. Representative 12 prototypes including 6 caffeoyl compounds (CA, 5-CQA, 3-CQA, 4-CQA, 1,3-CQA, and 3,4-CQA), 1 flavonoid (Scu), 2 saponins (Rd and Rg2), and 3 lignans (SchA, SchB, and SolA) presented characteristic absorption, disposition, and pharmacokinetics profiles in CCH rats. The caffeoyl compounds and flavonoid were well absorbed, exhibited wide distribution, and underwent extensive intestinal metabolism, such as methylation, isomerization, and sulfoconjugation. For CA, 5-CQA, Scu, and 4 related metabolites, the enterohepatic circulation was observed and resulted in bimodal or multimodal pharmacokinetic profiles. Saponins showed relatively low systemic exposure and limited distribution. The PPD-type ginsenoside Rd exhibited longer elimination half-life and systemic circulation than the PPT-type ginsenoside Rg2. No enterohepatic circulation was observed regarding saponins, suggesting that the multimodal pharmacokinetic profile of Rd could be due to its multi-site intestinal absorption. Lignans presented a low in vivo exposure and broad distribution. They were mainly transformed into hydroxylated metabolites. Corresponding to its bimodal pharmacokinetic profile, one metabolite of lignans completed the enterohepatic cycle. The disposition, metabolism, and pharmacokinetic profiles of representative active components of DZSM were comprehensively characterized and elucidated.

Vesicular acetylcholine transporter in the basal forebrain improves cognitive impairment in chronic cerebral hypoperfusion rats by modulating theta oscillations in the hippocampus

The cholinergic transmission in the medial septum and ventral limb of the diagonal band of broca (MS/VDB)-hippocampal circuit and its associated theta oscillations play a crucial role in chronic cerebral hypoperfusion (CCH)-related cognitive impairment. However, the contribution and mechanism of the vesicular acetylcholine transporter (VAChT), a vital protein that regulates acetylcholine (ACh) release, in CCH-related cognitive impairment are not well understood. To investigate this, we established a rat model of CCH by performing 2-vessel occlusion (2-VO) and overexpressed VAChT in the MS/VDB via stereotaxic injection of adeno-associated virus (AAV). We evaluated the cognitive function of the rats using the Morris Water Maze (MWM) and Novel Object Recognition Test (NOR). We employed enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and immunohistochemistry (IHC) to assess hippocampal cholinergic levels. We also conducted in vivo local field potentials (LFPs) recording experiments to evaluate changes in hippocampal theta oscillations and synchrony. Our findings showed that VAChT overexpression shortened the escape latency in the hidden platform test, increased swimming time in the platform quadrant in probe trains, and increased the recognition index (RI) in NOR. Moreover, VAChT overexpression increased hippocampal cholinergic levels, improved theta oscillations, and improved the synchrony of theta oscillations between CA1 and CA3 in CCH rats. These results suggest that VAChT plays a protective role in CCH-induced cognitive deficits by regulating cholinergic transmission in the MS/VDB-hippocampal circuit and promoting hippocampal theta oscillations. Therefore, VAChT could be a promising therapeutic target for treating CCH-related cognitive impairments.