Rat Model Of Cerebral Artery Occlusion Research Articles

Ozone reduces ischemic damage after a stroke by regulating the autophagy of astrocytes.

Stroke is a common and damaging disease of brain tissue, and has high morbidity, disability, and mortality rates. Ozone (O3) is an isomer of oxygen and can be applied to ozonate the isolated blood in specific containers outside the body and return it to the body. O3 can also alter the activity and function of multiple cellular components, thus affecting blood viscosity and altering hemodynamics. However, the question of whether O3 has clinical value in the treatment of stroke requires further investigation. This study sought to evaluate the protective effect and possible mechanism of O3 in brain injury after stroke. First, oxygen-glucose deprivation/reoxygenation (OGD/R)-induced human glioblastoma cell (A172) and middle cerebral artery occlusion (MCAO) rat models were established. Second, the levels of the associated ribonucleic acids and proteins were analyzed using reverse-transcription real-time-quantitative polymerase chain reaction, Western Blot, or immunofluorescence assays. Third, the concentration of glutamate and lactate dehydrogenase (LDH) were assessed using enzyme-linked immunoassays. The results showed that glial fibrillary acidic protein was upregulated in the OGD/R A172 cells. O3 decreased LDH and increased glutamate levels in the OGD/R A172 cells, which suggests that O3 reduced brain damage in the in vitro stroke model. We also showed that O3 attenuated brain infarction in the in-vivo stroke model. Further, we found that O3 alleviated stroke-induced brain damage by reducing the apoptosis of astrocytes. Further, the B-cell lymphoma 2 inhibitor propofol alleviated stroke-induced brain damage. Thus, O3 notably alleviated stroke-induced brain damage by inhibiting the apoptosis of astrocytes in the OGD/R-induced human glioblastoma cell and MACO rat models.

Neuroprotective effects of nobiletin on cerebral ischemia/reperfusion injury rats by inhibiting Rho/ROCK signaling pathway.

Nobiletin (NOB), an active natural flavonoid component of citrus, is used in Traditional Chinese Medicine for its anti-inflammatory activity, but its efficacy in cerebral ischemia/reperfusion (I/R) injury remains unclear. In a middle cerebral artery occlusion (MCAO) rat model, MCAO rats were administered (Sham group and MCAO model group treated with an equal volume of solvent, NOB group treated with 10 or 20 mg/kg NOB) once a day for 7 days before cerebral ischemia and again after reperfusion, 2,3,5-triphenyltetrazolium chloride (TTC) staining was applied to assess the infarct area. Neurological function was evaluated by the modified neurological severity score and Morris water maze. The levels of inflammatory factors, interleukin 6 (IL-6), interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α), were examined by enzyme-linked immunosorbent assay (ELISA). Histopathological staining evaluated neuron apoptosis in brain tissue. In an oxygen-glucose deprivation PC12 cell (OGD PC12) model, the proliferation, migration and apoptosis of OGD PC12 cells were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and cell migration assays and flow cytometry. The gene and protein expression levels of Ras homolog gene family, member A (Rho A), ras-related C3 botulinum toxin substrate 1 (Rac 1), Rho-associated kinase 1 (ROCK 1), ROCK 2 in the Rho/ROCK pathway were measured by Real-time PCR (RT-PCR), immunohistochemistry and western blot. In rats with cerebral I/R injury, NOB significantly decreased the infarcted area, neuron apoptosis in brain tissue and expressions of IL-6, IL-1β, and TNF-α. It also improved neurological deficits in brain tissue and enhanced learning and memory ability. Further, NOB had a protective effect on OGD PC12 cells, increasing proliferation and migration and decreasing apoptosis. The expressions of Rho A, Rac 1, ROCK 1 and ROCK 2 were high in cerebral I/R injury rats, but were downregulated by NOB in I/R injury rats' brain tissue and OGD PC12 cells. Nobiletin had a neuroprotective effect in rats with cerebral I/R injury, and its potential mechanism is decreasing neuron apoptosis by inhibiting the Rho/ROCK signaling pathway. These results suggest NOB is a promising neuroprotective agent for patients with cerebral ischemia.

Establishment of longitudinal transcranial stimulation motor evoked potentials monitoring of the forelimbs and hindlimbs in an ischemic stroke rat model

Evaluation of motor function ischemic stroke rat models includes qualitative assessments such as the modified neurological severity score (mNSS). However, mNSS cannot evaluate the function of forelimbs and hindlimbs separately. We quantitatively assessed motor function in a middle cerebral artery occlusion (MCAO) rat model of ischemic stroke. We recorded transcranial stimulation motor evoked potentials (tcMEPs) from MCAO rats and measured the changes in onset latency and amplitude at the forelimbs and hindlimbs up to 28 days after stroke. All MCAO subjects showed hemiparesis. The amplitudes of tcMEPs in both fore- and hindlimbs were inversely correlated with mNSS scores, but the amplitudes in the forelimbs improved later than those in the hindlimbs. The onset latency of tcMEPs in the forelimbs and hindlimbs remained almost unchanged during the follow-up period. Our results showed the differences in tcMEPs amplitude recovery times between the forelimbs and hindlimbs after MCAO, which emphasizes the importance of separately evaluating forelimbs and hindlimbs in post-ischemic stroke models. This minimally invasive and longitudinal quantitative method could be useful for further research on diseases and neurogenesis.

Protective Effect of Beta-Carotene against Myeloperoxidase- Mediated Oxidative Stress and Inflammation in Rat Ischemic Brain Injury.

Oxidative stress and inflammatory reaction play critical roles in ischemia/reperfusion (I/R) injury in the brain. β-carotene (βCAR) is a naturally occurring pigment present in fruits and vegetables that expresses antioxidant and anti-inflammatory activities. This study was conducted to investigate the involvement of Bcl2/Bax and NF-κB signaling pathways in the potential protective role of βCAR against brain injury in a middle cerebral artery occlusion (MCAO) rat model. A focal brain ischemia model was created for 2 h, followed by reperfusion. Rats were given 10 and 20 mg/kg of βCAR for 7 days orally before induction of ischemia, at the start of reperfusion, and 3 days after ischemia. Scores of neurological deficit were rated 24 h after induction of ischemia. Motor coordination and spontaneous coordinate activities were assessed using rotarod and activity cage, respectively. After 2 h of the last dose, the animals were killed and their brains were extracted for further examinations. The results of the study show that βCAR diminished the score of neurological deficits and ameliorated motor coordination, balance, and locomotor activity in the I/R control group. Further, βCAR resulted in diminution of malondialdehyde (MDA) and augmentation of reduced glutathione (GSH) contents, as well as the elevation of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) enzyme activities in the brain homogenates of I/R rats. βCAR treatment significantly reduced nuclear factor kappa B (NF-κB) brain content and myeloperoxidase (MPO) activity and ameliorated the histological alterations in the brain tissues. βCAR significantly suppressed Bcl-2-associated X protein (Bax) and caspase-3 expression, as well as upregulated B-cell lymphoma-2 (Bcl-2) expression, suggesting a neuroprotective potential via downregulating NF-kB and protecting the rat brain against the I/R-associated apoptotic injury.

Ligustrazine-Loaded Borneol Liposome Alleviates Cerebral Ischemia-Reperfusion Injury in Rats.

Our team's pharmacological and clinical trials proved that ligustrazine/borneol spray had a definite effect on ischemic stroke (IS). To solve the shortcomings of ligustrazine/borneol spray, such as low bioavailability, short half-life, and poor compatibility between borneol and ligustrazine, ligustrazine-loaded borneol liposomes (LIP@TMP) were successfully prepared by a thin-film ultrasonication method. The average particle size of LIP@TMP was 282.4 ± 3.6 nm, the drug loading rate was 14.5 ± 0.6%, and the entrapment efficiency was 42.7 ± 1.0%, which had excellent stability and sustained release ability. In addition, live/dead fluorescent staining and the CCK-8 test confirmed that LIP@TMP had good biocompatibility. Moreover, middle cerebral artery occlusion (MCAO) rat model experiments further demonstrated that LIP@TMP could significantly alleviate cerebral ischemia and reperfusion injury by improving neurological scores, reducing cerebral infarct volume, promoting neurogenesis, inhibiting inflammation, and reducing tissue damage. In addition, LIP@TMP enhanced neuronal marker doublecortin (DCX) and neuronal nuclei (NEUN), inhibited inflammatory factors (TNF-α and IL-1β), and reduced apoptosis signal molecules (TUNEL and caspase-3). The findings of this study suggested that the prepared LIP@TMP had tremendous potential for the treatment of cerebral ischemia.

Identifying potential therapeutic targets for ischemic stroke through immune infiltration analysis and construction of a programmed cell death‑related ceRNA network.

The present study aimed to uncover the underlying mechanisms and potential intervention targets of ischemic stroke (IS). An immune cell infiltration analysis using CIBERSORT was performed on two stroke-related datasets from the Gene Expression Omnibus database to generate a competitive endogenous RNA (ceRNA) network. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to predict potential biological functions of the network. Differentially expressed genes in the ceRNA network and programmed cell death-related genes were intersected to obtain common genes for a stroke ceRNA network related to programmed cell death. These genes were further verified through a middle cerebral artery occlusion rat model using reverse transcription-quantitative PCR. This built a ceRNA regulatory network based on bioinformatic analysis. In addition to the biological functions extracted from the GO and KEGG enrichment analyses, it was discovered that long non-coding (lnc)RNA-mediated ceRNA regulatory pathways were associated with programmed cell death. This included five for apoptosis (lncRNA deleted in lymphocytic leukemia 2 like (DLEU2L)/micro (miR)-4500/sulfite oxidase, lncRNA DLEU2L/miR-4500/transforming growth factor β receptor III, lncRNA DLEU2L/miR-4500/BTB and CNC homology 1 (BACH1), lncRNA DLEU2L/miR-4500/zinc finger and BTB domain containing 5 and lncRNA LINC00266-1/miR-363-3p/zinc finger protein 354B and two for ferroptosis (lncRNA DLEU2L/miR-4500/ribo-nucleotide reductase regulatory subunit M2 and lncRNA DLEU2L/miR-4500/BACH1). Based on the aforementioned results, the present study provided potential approaches for bridging programmed cell death, immune infiltration and ceRNA regulatory networks in IS. The present study may provide novel insights into the clinical diagnosis and treatment of IS, and may improve the knowledge of the regulation of pathophysiological processes for IS.

Molecular mechanisms for the prevention and promoting the recovery from ischemic stroke by nutraceutical laminarin: A comparative transcriptomic approach

Stroke is the second leading cause of death and a major cause of disability worldwide. Ischemic stroke caused by atherosclerosis accounts for approximately 87% of all stroke cases. Ischemic stroke is a preventable disease; therefore, a better understanding of the molecular mechanisms underlying its pathogenesis and recovery processes could provide therapeutic targets for drug development and reduce the associated mortality rate. Laminarin, a polysaccharide, is a nutraceutical that can be found in brown algae. Accumulating evidence suggests that laminarin could reduce the detrimental effects of neuroinflammation on brain damage after stroke. However, the molecular mechanism underlying its beneficial effects remains largely unknown. In the present study, we used a middle cerebral artery occlusion (MCAO) rat model and applied comparative transcriptomics to investigate the molecular targets and pathways involved in the beneficial effects of laminarin on ischemic stroke. Our results show the involvement of laminarin targets in biological processes related to blood circulation, oxygen supply, and anti-inflammatory responses in the normal brain. More importantly, laminarin treatment attenuated brain damage and neurodeficits caused by ischemic stroke. These beneficial effects are controlled by biological processes related to blood vessel development and brain cell death through the regulation of canonical pathways. Our study, for the first time, delineated the molecular mechanisms underlying the beneficial effects of laminarin on ischemic stroke prevention and recovery and provides novel therapeutic targets for drug development against ischemic stroke.

The Neuroprotective Effects of Exosomes Derived from TSG101-Overexpressing Human Neural Stem Cells in a Stroke Model.

Although tissue-type plasminogen activator was approved by the FDA for early reperfusion of occluded vessels, there is a need for an effective neuroprotective drug for stroke patients. In this study, we established tumor susceptibility gene (TSG)101-overexpressing human neural stem cells (F3.TSG) and investigated whether they showed enhanced secretion of exosomes and whether treatment with exosomes during reperfusion alleviated ischemia-reperfusion-mediated brain damage. F3.TSG cells secreted higher amounts of exosomes than the parental F3 cells. In N2A cells subjected to oxygen–glucose deprivation (OGD), treatment with exosomes or coculture with F3.TSG cells significantly attenuated lactate dehydrogenase release, the mRNA expression of proinflammatory factors, and the protein expression of DNA-damage-related proteins. In a middle cerebral artery occlusion (MCAO) rat model, treatment with exosomes, F3 cells, or F3.TSG cells after 2 h of occlusion followed by reperfusion reduced the infarction volume and suppressed inflammatory cytokines, DNA-damage-related proteins, and glial fibrillary acidic protein, and upregulated several neurotrophic factors. Thus, TSG101-overexpressing neural stem cells showed enhanced exosome secretion; exosome treatment protected against MCAO-induced brain damage via anti-inflammatory activities, DNA damage pathway inhibition, and growth/trophic factor induction. Therefore, exosomes and F3.TSG cells can affect neuroprotection and functional recovery in acute stroke patients.

Effect of Tetrahedral Framework Nucleic Acids on Neurological Recovery via Ameliorating Apoptosis and Regulating the Activation and Polarization of Astrocytes in Ischemic Stroke

Astrocytes, as the most plentiful subtypes of glial cells, play an essential biphasic function in ischemic stroke (IS). However, although having beneficial effects on stroke via promoting nerve restoration and limiting lesion extension, astrocytes can unavoidably cause exacerbated brain damage due to their participation in the inflammatory response. Therefore, seeking an effective and safe drug/strategy for protecting and regulating astrocytes in stroke is urgent. Here, we employ tetrahedral framework nucleic acid (tFNA) nanomaterials for astrocytes in stroke, considering their excellent biological properties and outstanding biosafety. In vitro, tFNA can inhibit calcium overload and ROS regeneration triggered by oxygen-glucose deprivation/reoxygenation (OGD/R), which provides a protective effect against astrocytic apoptosis. Furthermore, morphological changes such as hyperplasia and hypertrophy of reactive astrocytes are restrained, and the astrocytic polarization from the proinflammatory A1 phenotype to the neuroprotective A2 phenotype is facilitated by tFNA, which further alleviates cerebral infarct volume and facilitates the recovery of neurological function in transient middle cerebral artery occlusion (tMCAo) rat models. Moreover, the TLRs/NF-κB signaling pathway is downregulated by tFNA, which may be the potential mechanism of tFNA for protecting astrocytes in stroke. Collectively, we demonstrate that tFNA can effectively mediate astrocytic apoptosis, activation, and polarization to alleviate brain injury, which represents a potential intervention strategy for IS.

MiR-383-5p Regulated by the Transcription Factor CTCF Affects Neuronal Impairment in Cerebral Ischemia by Mediating Deacetylase HDAC9 Activity.

Stroke, the leading cause of long-term disability worldwide, is caused by the blockage or hemorage of cerebral arteries. The resultant cerebral ischemia causes local neuronal death and brain injury. Histone deacetylase 9 (HDAC9) has been reported to be elevated in ischemic brain injury, but its mechanism in stroke is still enigmatic. The present study aimed to unveil the manner of regulation of HDAC9 expression and the effect of HDAC9 activation on neuronal function in cerebral ischemia. MicroRNAs (miRNAs) targeting HDAC9 were predicted utilizing bioinformatics analysis. We then constructed the oxygen glucose deprivation (OGD) cell model and the middle cerebral artery occlusion (MCAO) rat model, and elucidated the expression of CCCTC binding factor (CTCF)/miR-383-5p/HDAC9. Targeting between miR-383-5p and HDAC9 was verified by dual-luciferase reporter assay and RNAi. After conducting an overexpression/knockdown assay, we assessed neuronal impairment and brain injury. We found that CTCF inhibited miR-383-5p expression via its enrichment in the promoter region of miR-383-5p, whereas the miR-383-5p targeted and inhibited HDAC9 expression. In the OGD model and the MCAO model, we confirmed that elevation of HDAC9 regulated by the CTCF/miR-383-5p/HDAC9 pathway mediated apoptosis induced by endoplasmic reticulum stress, while reduction of HDAC9 alleviated apoptosis and the symptoms of cerebral infarction in MCAO rats. Thus, the CTCF/miR-383-5p/HDAC9 pathway may present a target for drug development against ischemic brain injury.

Protective effects of fraxin on cerebral ischemia-reperfusion injury by mediating neuroinflammation and oxidative stress through PPAR-γ/NF-κB pathway

BackgroundInflammation and oxidative stress are associated with the pathogenesis of cerebral ischemia-reperfusion (I/R) injury. Fraxin, one of the primary active ingredients of Cortex Fraxini, may have potent anti-inflammatory activity. This study intended to investigate the function and mechanism of fraxin in a middle cerebral artery occlusion (MCAO) model. MethodsA middle cerebral artery occlusion (MCAO) rat model was engineered. Both in-vivo and in-vitro models were dealt with Fraxin. The profiles of inflammation-concerned cytokines, proteins and oxidative stress factors were determined by RT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA), and neuronal apoptosis and reactive oxygen species (ROS) levels were measured. The neurological functions of rats were evaluated by Morris water maze and modified neurological severity scores (mNSS). ResultsThe data revealed that fraxin abated the OGD/R-mediated release of inflammatory and oxidative stress mediators, enhanced “M2″-like BV2 microglia polarization, and mitigated HT22 cell apoptosis. Mechanistically, fraxin boosted PPAR-γ expression, activated the Nrf2/HO-1 pathway, and suppressed NF-κB, IKK-β，p38 MAPK, ERK1/2 and Keap1 in a dose-dependent manner. Furthermore, attenuating PPAR-γ through pharmacological treatment with GW9662 (a PPAR-γ antagonist) mainly weakened the neuroprotective and anti-inflammatory functions of fraxin. ConclusionFraxin could considerably ameliorate cerebral I/R damage by repressing oxidative stress, inflammatory response, and cell apoptosis through abrogating the PPARγ/ NF-κB pathway.

Effcacy-oriented compatibility for Tianma (), Yanlingcao () and Bingpian () on improving cerebral ischemia stroke by network pharmacology and serum pharmacological methods.

To evaluate the compatibility of Tianma (, TM), Yanlingcao (, YLC) and Bingpian (, BP), and their efficacy in the treatment of cerebral ischemic stroke. Network pharmacology was used to determine the compatibility of TM, YLC, and BP, and their potential mechanism. The middle cerebral artery occlusion (MCAO) rat model was used to evaluate the curative effect of the six combinations of TM, YLC, and BP (TZB1-TZB6) on cerebral ischemia, by using the weight matching method to form. The potential component changes of TM and YLC in the blood and brains of rats were analyzed using ultra performance liquid chromatography-mass spectrometry. Finally, molecular docking linked the results of animal experiments and network pharmacology, determining the potential component contributors of TM and YLC to treating ischemic stroke. TZB reduced the cerebral infarct volume and protected the nerve cells in MCAO rats. The components of TM and YLC were also identified in the blood and brain homogenate, and BP can facilitate the entry of the components of TM and YLC into the blood and brain. Diosgenin, pennogenin, and gastrodin induced effective binding activities with adenosine receptor a1. We investigate an approach that improves the means of folk prescription combined with multi technology that maybe promote the transformation of Chinese medicinal prescription into component-based Chinese medicine.

Downregulated XBP-1 Rescues Cerebral Ischemia/Reperfusion Injury-Induced Pyroptosis via the NLRP3/Caspase-1/GSDMD Axis.

Ischemic stroke is a major condition that remains extremely problematic to treat. A cerebral reperfusion injury becomes apparent after an ischemic accident when reoxygenation of the afflicted area produces pathological side effects that are different than those induced by the initial oxygen and nutrient deprivation insult. Pyroptosis is a form of lytic programmed cell death that is distinct from apoptosis, which is initiated by inflammasomes and depends on the activation of Caspase-1. Then, Caspase-1 mobilizes the N-domain of gasdermin D (GSDMD), resulting in the release of cytokines, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18). X-box binding protein l (XBP-1) is activated under endoplasmic reticulum (ER) stress to form an important transcription factor XBP-1 splicing (XBP-1s). The cerebral ischemia/reperfusion (CI/R) causes cytotoxicity, which correlates with the activation of splicing XBP-1 mRNA and NLRP3 (NOD-, LRR-, and pyrin domain-containing 3) inflammasomes, along with increases in the expression and secretion of proinflammatory cytokines and upregulation of pyroptosis-related genes in HT22 cells and in the middle cerebral artery occlusion (MCAO) rat model. However, whether XBP-1 plays a role in regulating pyroptosis involved in CI/R is still unknown. Our present study showed that behavior deficits, cerebral ischemic lesions, and neuronal death resulted from CI/R. CI/R increased the mRNA level of XBP-1s, NLRP3, IL-1β, and IL-18 and the expressions of XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, and IL-18. We further repeated this process in HT22 cells and C8-B4 cells and found that OGD/R decreased cell viability and increased LDH release, XBP-1s, NLRP3, Caspase-1, GSDMD-N, IL-1β, IL-18, and especially the ratio of pyroptosis, which were reversed by Z-YVAD-FMK and downregulated XBP-1. Our results suggest that downregulated XBP-1 inhibited pyroptosis through the classical NLRP3/Caspase-1/GSDMD pathway to protect the neurons.

Design, synthesis and biological evaluation of indoline derivatives as multifunctional agents for the treatment of ischemic stroke

In this work, a series of indoline derivatives as multifunctional neuroprotective agents for battling ischemic stroke were designed, synthesized, and biologically evaluated. In antioxidant assay, all compounds showed significant protective effects against H2O2-induced death of RAW 264.7 cells. In oxygen glucose deprivation/reperfusion (OGD/R)-induced neuronal damage, some compounds significantly elevated the cell survival rate. Among them, 7i, 7j and 7r exerted comparable neuroprotective effects to ifenprodil, and exhibited binding affinity to N-methyl-D-aspartic acid receptors 2B (NMDA-GluN2B). At the concentrations of 0.1, 1 and 10 μM, 7i, 7j and 7r dose-dependently lowered the LPS-induced secretion of inflammatory cytokines, including TNF-α, IL-6 and NO, by BV-2 cells. Importantly, 7i and 7j can dramatically reduce the cerebral infarction rate and improve neurological deficit scores in middle cerebral artery occlusion (MCAO) rat model. As demonstrated by the above results, 7i and 7j are potential neuroprotective agents for the treatment of ischemic stroke. Graphical abstract

A Reasonable Evaluation of Chuanxiong Rhizoma Processing with Wine through Comparative Pharmacokinetic Study of Bioactive Components: Dominant Effect on Middle Cerebral Artery Occlusion Model Rats.

According to the ancient documents and Chinese herbal medicine processing experience, Chuanxiong Rhizoma was usually processed with yellow rice wine to improve efficacy. However, the relevant mechanisms are still unclear so far. In this study, a validated ultrahigh-performance liquid chromatography tandem mass spectrometry method was used to compare the pharmacokinetics of four representative components in middle cerebral artery occlusion rats after oral administration of raw and wine-processed Chuanxiong Rhizoma. The neurobehavioral scores and 2,3,5-triphenyltetrazolium chloride staining were employed to evaluate the model. Biological samples were prepared by protein precipitation with methanol. All analytes were separated on an ACQUITY BEH C18 column through gradient elution using acetonitrile and 0.01% of formic acid as mobile phase, and the flow rate was 0.3 mL/min. The results showed that the maximum plasma concentrations, the area values under the concentration-time curves of senkyunolide A, and ferulic acid in wine-processed Chuanxiong Rhizoma were all higher than in raw Chuanxiong Rhizoma, which were completely opposite to our previous studies in normal rats. Compared with normal rats, the theory that wine processing could enhance the efficacy of Chuanxiong Rhizoma may be better reflected in model rats.

Macrophage membrane modified baicalin liposomes improve brain targeting for alleviating cerebral ischemia reperfusion injury

Baicalin (BA) has a good intervention effect on encephalopathy. In this study, macrophage membrane was modified on the surface of baicalin liposomes (BA-LP) by extrusion method. Macrophage membrane modified BA-LP (MM-BA-LP) was characterized by various analytical techniques, and evaluated for brain targeting. The results presented MM-BA-LP had better brain targeting compared with BA-LP. Pharmacokinetic experiments showed that MM-BA-LP improved pharmacokinetic parameters and increased the residence time of BA. Pharmacodynamic of middle cerebral artery occlusion (MCAO) rat model was studied to verify the therapeutic effect of MM-BA-LP on cerebral ischemia reperfusion injury (CIRI). The results showed that MM-BA-LP could significantly improve the neurological deficit, cerebral infarction volume and brain pathological state of MCAO rats compared with BA-LP. These results suggested that MM-BA-LP could significantly enhance the brain targeting and improve the circulation of BA in blood, and had a significantly better neuroprotective effect on MCAO rats than BA-LP.

Diosmetin alleviates cerebral ischemia-reperfusion injury through Keap1-mediated Nrf2/ARE signaling pathway activation and NLRP3 inflammasome inhibition.

Diosmetin was found to exert protective effect on renal and myocardial ischemia-reperfusion (IR) injury. This study aimed to investigate the role of diosmetin in cerebral IR (CIR) injury. PC12 neurons were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to establish CIR injury model in vitro and then incubated with diosmetin, and we found that diosmetin alleviated OGD/R-induced viability inhibition, LDH release, apoptosis, and oxidative stress in PC12 cells. Then our results showed that diosmetin downregulated kelch like ECH-associated protein 1 (Keap1) expression, and upregulated nuclear factor E2-related factor 2 (Nrf2) expression, antioxidant response element (ARE) activity and the mRNA and protein expression of heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1). Keap1 overexpression or Nrf2 silencing both attenuated the neuroprotective effect of diosmetin on PC12 cells. Moreover, diosmetin inhibited the levels of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) pyrin domain containing 3 (NLRP3) inflammasome pathway related proteins and inflammatory cytokines interleukin (IL)-1β and IL-18. Additionally, a middle cerebral artery occlusion (MCAO) rat model was established and diosmetin was injected for treatment. Diosmetin alleviated CIR-induced neurological deficits, cerebral infarction, brain edema and histopathological damage, and neuronal apoptosis and oxidative stress in MCAO rats. In conclusion, diosmetin attenuated OGD/R-induced PC12 cell viability inhibition, apoptosis, oxidative stress and inflammation through Keap1-mediated Nrf2/ARE signaling activation and NLRP3 inflammasome inhibition, and alleviated CIR-induced neurological injury in MCAO rat model. Our study may provide a novel therapeutic strategy for CIR injury.

Cranial Bone Transport Promotes Angiogenesis, Neurogenesis, and Modulates Meningeal Lymphatic Function in Middle Cerebral Artery Occlusion Rats.

Ischemic stroke is a leading cause of death and disability worldwide. However, the time window for quickly dissolving clots and restoring cerebral blood flow, using tissue-type plasminogen activator treatment is rather limited, resulting in many patients experiencing long-term functional impairments if not death. This study aims to determine the roles of cranial bone transport (CBT), a novel, effective, and simple surgical technique, in the recovery of ischemic stroke using middle cerebral artery occlusion (MCAO) rat model. CBT was performed by slowly sliding a bone segment in skull with a special frame and a speed of 0.25 mm/12 hours for 10 days following MCAO. Morris water maze, rotarod test, and catwalk gait analysis were used to study the neurological behaviors, and infarct area and cerebral flow were evaluated during CBT process. Immunofluorescence staining of CD31 and Nestin/Sox2 (sex determining region Y box 2) was performed to study the angiogenesis and neurogenesis. OVA-A647 (ovalbumin-Alexa Fluor 647) was intracisterna magna injected to evaluate the meningeal lymphatic drainage function. CBT treatment has significantly reduced the ischemic lesions areas and improved the neurological deficits in MCAO rats compared with the rats in the control groups. CBT treatment significantly promoted angiogenesis and neurogenesis in the brain of MCAO rats. The drainage function of meningeal lymphatic vessels in MCAO rats was significantly impaired compared with normal rats. Ablation of meningeal lymphatic drainage led to increased neuroinflammation and aggravated neurological deficits and ischemic injury in MCAO rats. CBT treatment significantly improved the meningeal lymphatic drainage function and alleviated T-cell infiltration in MCAO rats. This study provided evidence for the possible mechanisms on how CBT attenuates ischemic stroke injury and facilitates rapid neuronal function recovery, suggesting that CBT may be an alternative treatment strategy for managing ischemic stroke.

The Novel microRNA Rno-miR-686-3p Is Associated with the Ischaemic Penumbra

Introduction: We explored microRNA (miRNA) profiles correlated with the penumbra in three different phases of ischaemic stroke, using a permanent middle cerebral artery occlusion (p-MCAO) rat model. Materials and Methods: A 2-mm coronal section was cut from the optic chiasma in the caudal direction, and the penumbra was located in the area between a longitudinal line approximately 2 mm from the midline and a transverse diagonal line at the “2-o’clock” position. Total RNA was extracted from tissue specimens and peripheral blood samples, followed by deep sequencing analysis. Results: We identified nine novel miRNA candidates in tissues and evaluated their expression levels using real-time quantitative polymerase chain reaction. In situ hybridization was conducted to assess miRNA localization in the brain. Of these nine candidates, we identified and characterized a novel miRNA, rno-miR-686-3p, which was localized in cell nuclei of the cortex, and associated with the penumbra. rno-miR-686-3p was downregulated at 1 (p = 0.042), 3 (p = 0.032), and 4 h (p = 0.007) post-p-MCAO in the penumbra. A total of 297 potential target genes were predicted. Moreover, functional annotation clustering and pathway enrichment analysis predicted that rno-miR-686-3p participates in transcriptional regulation and the Wnt and cyclic adenosine monophosphate (cAMP) signalling pathways. Conclusion: rno-miR-686-3p is a novel miRNA associated with the ischaemic penumbra that is implicated in transcriptional regulation and modulation of the Wnt and cAMP signalling pathways. Furthermore, it may serve as a possible new biomarker with potential value for detecting the existence of the penumbra.

Remote ischemic preconditioning protects against cerebral ischemia injury in rats by upregulating miR-204-5p and activating the PINK1/Parkin signaling pathway.

Remote ischemic preconditioning (RiPC) is the process where preconditioning ischemia protects the organs against the subsequent index ischemia. RiPC is a protective method for brain damage. This study is to explore the effect and mechanism of RiPC in cerebral ischemia injury in rats through regulation of miR-204-5p/BRD4 expression. Middle cerebral artery occlusion (MCAO) rat model and glucose deprivation (OGD) neuron model were established. The effect of RiPC on neurological deficits, cerebral infarct size, autophagy marker, inflammatory cytokines and apoptosis was evaluated. miR-204-5p expression was analyzed using RT-qPCR, and then downregulated using miR-204-5p antagomir to estimate its effect on MCAO rats. The downstream mechanism of miR-204-5p was explored. RiPC promoted autophagy, reduced cerebral infarct volume and neurological deficit score, and alleviated apoptosis and cerebral ischemia injury in rats, with no significant effects on healthy rat brains. RiPC up-regulated miR-204-5p expression in MCAO rats. miR-204-5p knockdown partially reversed the effect of RiPC. RiPC promoted autophagy in OGD cells, and attenuated inflammation and apoptosis. miR-204-5p targeted BRD4, which partially reversed the effect of miR-204-5p on OGD cells. RiPC activated the PINK1/Parkin pathway via the miR-204-5p/BRD4 axis. In conclusion, RiPC activated the PINK1/Parkin pathway and prevented cerebral ischemia injury by up-regulating miR-204-5p and inhibiting BRD4.