Middle Cerebral Artery Occlusion Stroke Research Articles

Increased sensitivity in detection of deficits following two commonly used animal models of stroke

Stroke is a leading cause of death and disability in the United States. Most strokes are ischemic, resulting in both cognitive and motor impairments. Animal models of ischemic stroke such as the distal middle cerebral artery occlusion (dMCAO) and photothrombotic stroke (PTS) procedures have become invaluable tools, with their own advantages and disadvantages. The dMCAO model is clinically relevant as it occludes the artery most affected in humans, but yields variability in the infarct location as well as the behavioral and cognitive phenotypes disrupted. The PTS model has the advantage of allowing for targeted location of infarct, but is less clinically relevant. The present study evaluates phenotype disruption over time in mice subjected to either dMCAO, PTS, or a sham surgery. Post-surgery, animals were tested over 28 days on standard motor tasks (grid walk, cylinder, tapered beam, and rotating beam), as well as a novel odor-based operant task; the 5:1 Odor Discrimination Task (ODT). Results demonstrate a significantly greater disturbance of motor control with PTS as compared with Sham and dMCAO. Disruption of the PTS group was detected up to 28 days post-stroke on the grid walk, and up to 7 days on the rotating and tapered beam tasks. PTS also led to significant short-term disruption of ODT performance (1-day post-surgery), exclusively in males, which appeared to be driven by motoric disruption of the lick response. Together, this data provides critical insights into the selection and optimization of animal models for ischemic stroke research. Notably, the PTS procedure was best suited for producing disruptions of motor behavior that can be detected with common behavioral assays and are relatively enduring, as is observed in human stroke.

Abstract WP14: Photobiomodulation Treatment Enhancesand Sustains Neurogenesis Following Acute Ischemic Stroke

Rationale: Previous work has indicated that photobiomodulation (PBM), may provide neuroprotection and improve neurological function after acute ischemic stroke. However, the underlying mechanism remains unclear. The present study was performed to examine whether PBM, a noninvasive neurostimulation technique, promotes neurogenesis in a middle cerebral artery occlusion (MCAO) stroke rat model. Methods: Beginning 6 h after MCAO, once-daily 2-min PBM treatments(808 nm) were applied to the infarcted hemisphere for 7 consecutive days. Samples were collected 7 days and 14 days after MCAO stroke. Fluorescent labeling, Western blotting, and behavioral tests were performed accordingly. Results: We found that PBM-treated animals displayed improved motor and sensory functions. PBM treatment significantly expanded the pool of neural progenitor cell (NPC) and newly generated immature neurons in the cortical peri-infarct region after acute ischemic stroke. Application of PBM strikingly diminished the MCAO-induced loss of NPCs and newly-formed neurons. In addition, the amount of newly generated mature neurons in the peri-infarct zone was significantly promoted by PBM treatment. Intriguingly, PBM reduced reactive gliogenesis significantly while promoting oligodendrogenesis and preserving myelination. Mechanistic studies uncovered that PBM enhances the expression of brain-derived neurotrophic factor (BDNF) and initiates the activation of the BDNF/TrkB signaling pathway. Conclusions: Our findings indicate that PBM promotes neurogenesis and functional recovery through stimulating NPC proliferation, reducing NPC and immature neuron cell death, and tamping excessive gliogenesis. These effects were due, in part, to the upregulated release of trophic factors. These findings support PBM as an effective neuroregenerative treatment for acute ischemic stroke. Keywords: Ischemic stroke, Photobiomodulation (PBM), Neurogenesis, Functional recovery, Neurotrophic factors

Abstract WMP12: Acid-Formulated Malonate Improves Neuroprotection Ischemia-Selective in a Mouse Model of Ischemic Stroke

Background: Malonate, a competitive inhibitor of succinate dehydrogenase, has shown much promise in ameliorating ischemia/reperfusion injury (IRI) in various diseases, including ischemic stroke. Its uptake via the monocarboxylate transporter 1 (MCT1) is enhanced by a decrease in local pH, enabling it to be preferentially taken up by ischemic tissue. We investigated whether an acid-formulated malonate could enhance uptake of malonate and, consequently, protection against stroke IRI. Methods: C57BL/6J mice were treated intravenously with either saline or malonate (640/320 mg/kg) at physiological (7.4) or low pH (6.0). Brain malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS. For the transient middle cerebral artery occlusion (tMCAO) stroke model, mice were subjected to 30 mins ischaemia followed by either 2 hour reperfusion. Brain infarct size was assessed by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining. Results: Malonate was acutely protective against stroke IRI with a reduction in brain infarct size from 21.98% to 6.57% after 2 hours of reperfusion. Malonate uptake into the brain in vivo dramatically increased when infused at pH 6 compared to neutral malonate. Furthermore, acid-formulation greatly enhanced the neuroprotective potency of malonate, enabling protection at a much lower dose compared to its non-acidic counterpart. Conclusions: Malonate is neuroprotective against stroke IRI. Its uptake via MCT1 and, ultimately its neuroprotective potency can be enhanced by acid formulation of the drug.

Neuroprotective action of hydroxybenzoic acid derivative with glycine (C40) in the middle cerebral artery occlusion in rats with chronic cerebral circulation insufficiency

The neuroprotective effect of a novel conjugate of hydroxybenzoic acid with glycine (C40) was investigated in a model of ischemic stroke in rats with chronic cerebral circulation insufficiency (CCCI). The study was conducted in two stages: first, CCCI was modeled by stenosing the common carotid arteries by 50 %, followed by a 60-minute middle cerebral artery occlusion (stroke) after 40 days. Compound C40 and the reference drug citicoline were administered for 10 days before stroke and for 3 days after. Further, neurological deficit (the Garcia and Combs D’Alecy scales), sensorimotor functions (Open Field test, Adhesive Removal test, and Rotarod test), cognitive impairments (Novel Object Recognition and Morris Water Maze tests), cerebral blood flow level, and endothelial function were evaluated. Animals receiving the test substances demonstrated a statistically significant reduction in neurological deficit, less pronounced motor coordination and sensorimotor function impairments, as well as fewer short-term and long-term memory deficits compared to the negative control group. The observed decrease in neurological deficit in the experimental groups was accompanied by improved endothelial function and increased cerebral blood flow. The neuroprotective effect of the novel hydroxybenzoic acid derivative with glycine (C40) was comparable to that of reference drug citicoline.

Comparative study of the efficacy of intra-arterial and intravenous transplantation of human induced pluripotent stem cells-derived neural progenitor cells in experimental stroke.

Cell therapy using neural progenitor cells (NPCs) is a promising approach for ischemic stroke treatment according to the results of multiple preclinical studies in animal stroke models. In the vast majority of conducted animal studies, the therapeutic efficacy of NPCs was estimated after intracerebral transplantation, while the information of the effectiveness of systemic administration is limited. Nowadays, several clinical trials aimed to estimate the safety and efficacy of NPCs transplantation in stroke patients were also conducted. In these studies, NPCs were transplanted intracerebrally in the subacute/chronic phase of stroke. The results of clinical trials confirmed the safety of the approach, however, the degree of functional improvement (the primary efficacy endpoint) was not sufficient in the majority of the studies. Therefore, more studies are needed in order to investigate the optimal transplantation parameters, especially the timing of cell transplantation after the stroke onset. This study aimed to evaluate the therapeutic effects of intra-arterial (IA) and intravenous (IV) administration of NPCs derived from induced pluripotent stem cells (iNPCs) in the acute phase of experimental stroke in rats. Induced pluripotent stem cells were chosen as the source of NPCs as this technology is perspective, has no ethical concerns and provides the access to personalized medicine. Human iNPCs were transplanted IA or IV into male Wistar rats 24 h after the middle cerebral artery occlusion stroke modeling. Therapeutic efficacy was monitored for 14 days and evaluated in comparison with the cell transplantation-free control group. Additionally, cell distribution in the brain was assessed. The obtained results show that both routes of systemic transplantation (IV and IA) significantly reduced the mortality and improved the neurological deficit of experimental animals compared to the control group. At the same time, according to the MRI data, only IA administration led to faster and prominent reduction of the stroke volume. After IA administration, iNPCs transiently trapped in the brain and were not detected on day 7 after the transplantation. In case of IV injection, transplanted cells were not visualized in the brain. The obtained data demonstrated that the systemic transplantation of human iNPCs in the acute phase of ischemic stroke can be a promising therapeutic strategy.

Growth arrest specific protein 6 alleviated white matter injury after experimental ischemic stroke

Ischemic white matter injury leads to long-term neurological deficits and lacks effective medication. Growth arrest specific protein 6 (Gas6) clears myelin debris, which is hypothesized to promote white matter integrity in experimental stroke models. By the middle cerebral artery occlusion (MCAO) stroke model, we observed that Gas6 reduced infarcted volume and behavior deficits 4 weeks after MCAO. Compared with control mice, Gas6-treatment mice represented higher FA values in the ipsilateral external capsules by MRI DTI scan. The SMI32/MBP ratio of the ipsilateral cortex and striatum was profoundly alleviated by Gas6 administration. Gas6-treatment group manifested thicker myelin sheaths than the control group by electron microscopy. We observed that Gas6 mainly promoted OPC maturation, which was closely related to microglia. Mechanically, Gas6 accelerated microglia-mediated myelin debris clearance and cholesterol transport protein expression (abca1, abcg1, apoc1, apoe) in vivo and in vitro, accordingly less myelin debris and lipid deposited in Gas6 treated stroke mice. HX531 (RXR inhibitor) administration mitigated the functions of Gas6 in speeding up debris clearance and cholesterol transport protein expression. Generally, we concluded that Gas6 cleared myelin debris and promoted cholesterol transportation protein expression through activating RXR, which could be one critical mechanism contributing to white matter repair after stroke.

Near-Infrared Afterglow ONOO--Triggered Nanoparticles for Real-Time Monitoring and Treatment of Early Ischemic Stroke.

Early detection and drug intervention with the appropriate timing and dosage are the main clinical challenges for ischemic stroke (IS) treatment. The conventional therapeutic agents relay fluorescent signals, which require real-time external light excitation, thereby leading to inevitable autofluorescence and poor tissue penetration. Herein, we report endogenous peroxynitrite (ONOO-)-activated BDP-4/Cur-CL NPs that release NIR afterglow signals (λmax 697 nm) for real-time monitoring of the progression of ischemia reperfusion (I/R) brain injury while releasing curcumin for the safe treatment of IS. The BDP-4/Cur-CL NPs exhibited bright NIR afterglow luminescence (maximum 732-fold increase), superb sensitivity (LOD = 82.67 nM), high energy-transfer efficiency (94.6%), deep tissue penetration (20 mm), outstanding antiapoptosis, and anti-inflammatory effects. The activated NIR afterglow signal obtained in mice with middle cerebral artery occlusion (MCAO) showed three functions: (i) the BDP-4/Cur-CL NPs are rapidly activated by endogenous ONOO-, instantly illuminating the lesion area, distinguishing I/R damage from normal areas, which can be successfully used for endogenous ONOO- detection in the early stage of IS; (ii) real-time reporting of in situ generation and dynamic fluctuations of endogenous ONOO- levels in the lesion area, which is of great value in monitoring the evolutionary mechanisms of IS; and (iii) dynamic monitoring of the release of curcumin drug for safe treatment. Indeed, the released curcumin effectively decreased apoptosis, enhanced survival, alleviated neuroinflammation, reduced brain tissue loss, and improved the cognition of MCAO stroke mice. This work is the first example of afterglow luminescence for early diagnosis, real-time reporting, drug tracing, and treatment for IS.

Association of intravascular enhancement sign on 3D-T1W TSE with collateral status in middle cerebral artery occlusion stroke

ObjectiveThe significance of the intravascular enhancement sign (IVES) on high-resolution magnetic resonance vascular wall imaging (HR-VWI) remains unclear. This study aimed to investigate the correlation between the IVES and collateral assessment derived from digital subtraction angiography (DSA). MethodA total of 75 patients with occlusion of the first segment of the middle cerebral artery (MCA) who underwent HR-VWI and DSA examinations at our research institution between November 2016 and February 2023 were included. The number of vessels with IVES, IVES-Alberta Stroke Program Early Computed Tomography Score (ASPECTS), American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology (ASITN/SIR) collateral grade, and DSA collateral blood flow grade were retrospectively evaluated. Correlations between these indicators were assessed using Spearman's correlation. ResultsInterrater agreement was good for the assessment of HR-VWI and DSA indicators. After adjustments for age, degree of wall enhancement, and hypertension, a multivariable ordinal logistic regression model identified both the number of IVES vessels (OR = 1.37; 95%CI [1.06–1.78]; P = 0.017) and IVES-ASPECTS (OR = 2.00; 95%CI [1.03–3.87]; P = 0.041) as independent predictors of ischemic stroke. In the patient group with acute ischemic stroke, we found weak correlations between the number of IVES vessels and the ASITN/SIR collateral grade (rho = −0.35; P = 0.002) and between the IVES-ASPECTS and ASITN/SIR collateral grade (rho = −0.27; P = 0.02). Moreover, there were strong correlations between the number of IVES vessels and the DSA collateral blood flow grade (rho = −0.74; P < 0.001) and between the IVES-ASPECTS and the DSA collateral blood flow grade (rho = −0.65; P < 0.001). The number of IVES vessels correlated strongly with the IVES-ASPECTS (rho = 0.92, P < 0.001). ConclusionWe find that the IVES is closely associated with sluggish collateral blood flow, which further confirms the hemodynamic mechanism underlying the IVES in MCA occlusion.

CD11c+ microglia promote white matter repair after ischemic stroke

Ischemic stroke leads to white matter damage and neurological deficits. However, the characteristics of white matter injury and repair after stroke are unclear. Additionally, the precise molecular communications between microglia and white matter repair during the stroke rehabilitation phase remain elusive. In this current study, MRI DTI scan and immunofluorescence staining were performed to trace white matter and microglia in the mouse transient middle cerebral artery occlusion (tMCAO) stroke model. We found that the most serious white matter damage was on Day 7 after the ischemic stroke, then it recovered gradually from Day 7 to Day 30. Parallel to white matter recovery, we observed that microglia centered around the damaged myelin sheath and swallowed myelin debris in the ischemic areas. Then, microglia of the ischemic hemisphere were sorted by flow cytometry for RNA sequencing and subpopulation analysis. We found that CD11c+ microglia increased from Day 7 to Day 30, demonstrating high phagocytotic capabilities, myelin-supportive genes, and lipid metabolism associated genes. CD11c+ microglia population was partly depleted by the stereotactic injecting of rAAV2/6M-taCasp3 (rAAV2/6M-CMV-DIO-taCasp3-TEVp) into CD11c-cre mice. Selective depletion of CD11c+ microglia disrupted white matter repair, oligodendrocyte maturation, and functional recovery after stroke by Rotarod test, Adhesive Removal test, and Morris Water Maze test. These findings suggest that spontaneous white matter repair occurs after ischemic stroke, while CD11c+ microglia play critical roles in this white matter restorative progress.

Ischemic Stroke Causes Disruptions in the Carnitine Shuttle System.

Gaining a deep understanding of the molecular mechanisms underlying ischemic stroke is necessary to develop treatment alternatives. Ischemic stroke is known to cause a cellular energy imbalance when glucose supply is deprived, enhancing the role for energy production via β-oxidation where acylcarnitines are essential for the transportation of fatty acids into the mitochondria. Although traditional bulk analysis methods enable sensitive detection of acylcarnitines, they do not provide information on their abundances in various tissue regions. However, with quantitative mass spectrometry imaging the detected concentrations and spatial distributions of endogenous molecules can be readily obtained in an unbiased way. Here, we use pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging (PA nano-DESI MSI) doped with internal standards to study the distributions of acylcarnitines in mouse brain affected by stroke. The internal standards enable quantitative imaging and annotation of endogenous acylcarnitines is achieved by studying fragmentation patterns. We report a significant accumulation of long-chain acylcarnitines due to ischemia in brain tissue of the middle cerebral artery occlusion (MCAO) stroke model. Further, we estimate activities of carnitine transporting enzymes and demonstrate disruptions in the carnitine shuttle system that affects the β-oxidation in the mitochondria. Our results show the importance for quantitative monitoring of metabolite distributions in distinct tissue regions to understand cell compensation mechanisms involved in handling damage caused by stroke.

DL-3-n-butylphthalide Attenuates Cerebral Ischemia-Reperfusion Injury by Inhibiting Mitochondrial Omi/HtrA2-Mediated Apoptosis.

Ischemic stroke is a major cause of death and disability worldwide and results from inadequate cerebrovascular blood supply; mitochondrial dysfunction plays an essential role in its pathogenesis. DL-3-n-butylphthalide (NBP) is an effective medicine for ischemic stroke that reduces cell apoptosis and improves long-term prognosis. Whether and how NBP regulates mitochondria-associated apoptosis in cerebral ischemia- reperfusion injury remains unclear. Male Sprague Dawley rats were subjected to a middle cerebral artery occlusion (MCAO) stroke and treated with low (20 mg/kg) or high (80 mg/kg) concentrations of NBP. The Omi/HtrA2 inhibitor UCF-101 was used as a positive control. Cerebral infarction, neuron injury and neuronal apoptosis were assessed to determine the efficacy of NBP compared to UCF-101. We assessed the expression of the Omi/HtrA2 signaling pathway by western blotting and tested the mRNA expression of mitochondrial metabolism-related genes by PCR. Compared to the MCAO group, both low and high concentrations of NBP substantially improved cerebral infarction, neuron injury, and neuronal apoptosis; high concentrations of NBP were more potent than low concentrations. The expression of proteins of the mitochondrial Omi/HtrA2 signaling pathway, including Omi/HtrA2, XIAP, PARL, OPA1, CHOP, and ClpP, was inhibited in the NBP group. Overall, early application of NBP attenuated cerebral ischemia-reperfusion injury by inhibiting mitochondrial Omi/HtrA2-mediated apoptosis in rats. Our study supports a novel neuroprotective mechanism of NBP, making it a promising therapeutic agent for ischemic stroke.

Astragaloside VI Ameliorates Post-Stroke Depression via Upregulating the NRG-1-Mediated MEK/ERK Pathway.

Post-stroke depression (PSD) has been identified as one of the most commonly occurring complications attributed to stroke. Astragaloside VI (AsVI), which is an active Radix Astragali (AR)-derived compound, has been reported to be a potential drug for post-stroke therapy, but its effects on PSD and the underlying mechanisms remain uncovered. In this study, healthy male SD rats underwent a middle cerebral artery occlusion (MCAO) stroke model. To create a PSD model, these rats were then kept in isolated houses and subjected to chronic unpredictable mild stress. The rats were examined every five days for a series of behavioral tests of depression. The antidepressant properties of AsVI were also investigated in vitro in a corticosterone (CORT)-induced major depression model using a CCK-8 assay. The release of neurotransmitters dopamine (DA)/5-hydroxytryptamine (5-HT) was measured using HPLC. The expression of the neurotrophic factor Neuregulin 1 (NRG-1) in rat brain tissues was detected by immunostaining. The protein expression of NRG-1, p-MEK1, and p-ERK1/2 was analyzed utilizing western blotting. AsVI treatment significantly reduced depression-like behaviors in PSD rats and attenuated the CORT-induced apoptotic cell death in neuronal PC-12 cells. Besides, AsVI treatment remarkably prevented the decrease of the levels of DA and 5-HT in the PSD rat brains and in CORT-induced PC-12 cells. Furthermore, AsVI treatment upregulated the NRG-1-mediated MEK/ERK pathway, which is associated with the improvement of PSD. These findings suggest that AsVI could improve PSD at least partially by upregulating NRG-1-mediated MEK/ERK pathway. AsVI could be a novel therapeutic option for treating PSD.

JAK2/STAT3 pathway mediates neuroprotective and pro-angiogenic treatment effects of adult human neural stem cells in middle cerebral artery occlusion stroke animal models.

Mismatches between pre-clinical and clinical results of stem cell therapeutics for ischemic stroke limit their clinical applicability. To overcome these discrepancies, precise planning of pre-clinical experiments that can be translated to clinical trials and the scientific elucidation of treatment mechanisms is important. In this study, adult human neural stem cells (ahNSCs) derived from temporal lobe surgical samples were used (to avoid ethical and safety issues), and their therapeutic effects on ischemic stroke were examined using middle cerebral artery occlusion animal models. 5 × 105 ahNSCs was directly injected into the lateral ventricle of contralateral brain hemispheres of immune suppressed rat stroke models at the subacute phase of stroke. Compared with the mock-treated group, ahNSCs reduced brain tissue atrophy and neurological sensorimotor and memory functional loss. Tissue analysis demonstrated that the significant therapeutic effects were mediated by the neuroprotective and pro-angiogenic activities of ahNSCs, which preserved neurons in ischemic brain areas and decreased reactive astrogliosis and microglial activation. The neuroprotective and pro-angiogenic effects of ahNSCs were validated in in vitro stroke models and were induced by paracrine factors excreted by ahNSCs. When the JAK2/STAT3 signaling pathway was inhibited by a specific inhibitor, AG490, the paracrine neuroprotective and pro-angiogenic effects of ahNSCs were reversed. This pre-clinical study that closely simulated clinical settings and provided treatment mechanisms of ahNSCs for ischemic stroke may aid the development of protocols for subsequent clinical trials of ahNSCs and the realization of clinically available stem cell therapeutics for ischemic stroke.

Combined Arterial Hypertension and Ischemic Stroke Exaggerate Anesthesia-Related Hypotension and Cerebral Oxygenation Deficits: A Preclinical Study.

Intraoperative arterial hypotension (IOH) is a common side effect of general anesthesia (GA), associated with poor outcomes in ischemic stroke. While IOH is more prevalent with hypertension, it is unknown whether IOH may differ when GA is induced during ischemic stroke, versus other clinical settings. This is important given that many stroke patients receive GA for endovascular thrombectomy. We evaluate the cardiovascular responses to volatile GA (isoflurane in 100% o2 ) before and during middle cerebral artery occlusion stroke in rats instrumented to record blood pressure (BP) and cerebral tissue oxygenation (p o2 ) in the projected penumbra, in clinically relevant cohorts of normotensive (Wistar rat, n = 10), treated hypertensive (spontaneously hypertensive [SH] + enalapril, n = 12), and untreated hypertensive (SH rat, n = 12). During baseline induction of GA, IOH was similar in normotensive, treated hypertensive, and untreated hypertensive rats during the induction phase (first 10 minutes) (-24 ± 15 vs -28 ± 22 vs -48 ± 24 mm Hg; P > .05) and across the procedure (-24 ± 13 vs -30 ± 35 vs -39 ± 27 mm Hg; P > .05). Despite the BP reduction, cerebral p o2 increased by ~50% in all groups during the procedure. When inducing GA after 2 hours, all stroke groups showed a greater magnitude IOH compared to baseline GA induction, with larger falls in treated (-79 ± 24 mm Hg; P = .0202) and untreated(-105 ± 43 mm Hg; P < .001) hypertensive rats versus normotensives (-49 ± 21 mm Hg). This was accompanied by smaller increases in cerebral p o2 in normotensive rats (19% ± 32%; P = .0144 versus no-stroke); but a decrease in cerebral p o2 in treated (-11% ± 19%; P = .0048) and untreated (-12% ± 15%; P = .0003) hypertensive rats. Sham animals (normotensive and hypertensive) showed similar magnitude and pattern of IOH when induced with GA before and after sham procedure. Our findings are the first demonstration that ischemic stroke per se increases the severity of IOH, particularly when combined with a prior history of hypertension; this combination appears to compromise penumbral perfusion.

A monoamine oxidase B inhibitor ethyl ferulate suppresses microglia-mediated neuroinflammation and alleviates ischemic brain injury.

Microglia are the resident macrophages in the brain, which play a critical role in post-stroke neuroinflammation. Accordingly, targeting neuroinflammation could be a promising strategy to improve ischemic stroke outcomes. Ethyl ferulate (EF) has been confirmed to possess anti-inflammatory properties in several disease models, including acute lung injury, retinal damage and diabetes-associated renal injury. However, the effects of EF on microglial activation and the resolution of post-stroke neuroinflammation remains unknown. Here, we found that EF suppressed pro-inflammatory response triggered by lipopolysaccharide (LPS) stimulation in primary microglia and BV2 cell lines, as well as post-stroke neuroinflammation in an in vivo transient middle cerebral artery occlusion (tMCAO) stroke model in C57BL/6 mice, consequently ameliorating ischemic brain injury. Furthermore, EF could directly bind and inhibit the activity of monoamine oxidase B (MAO-B) to reduce pro-inflammatory response. Taken together, our study identified a MAO-B inhibitor, Ethyl ferulate, as an active compound with promising potentials for suppressing post-stroke neuroinflammation.

Transient Intraluminal Filament Middle Cerebral Artery Occlusion Stroke Model in Rats: A Step-by-Step Guide and Technical Considerations

Stroke is a leading cause of disability and mortality worldwide. Related research, although already providing significant insights regarding the underlying pathophysiology and potential treatment strategies, has been far from conclusive. Stroke models have been proved of extreme significance for laboratories around the world. In the present report, we have described in detail the most popular to date focal stroke model, the transient intraluminal filament middle cerebral artery occlusion (tifMCAO) model in rats. This model reliably mimics stroke in humans and also approximates endovascular thrombectomy. The tifMCAO model was performed using Wistar rats weighing 300-400 g. We have described the surgical technique in a stepwise manner, with figures and/or high-definition video provided for each step. We have also introduced the use of complete arteriotomy of the external carotid artery stump during the procedure. We performed tifMCAO in 65 rats (male and female) involved in various experimental protocols. Although the initial mortality was 48%, practice reduced the rate to 10%. The mean procedural time was 53 minutes (range, 38-85 minutes). In a group of 8 rats ischemia was confirmed in 7 of them, with the stroke induction rate being 87.5%. The tifMCAO stroke model in rats is the most often used experimental model of focal ischemia because of its clinical relevance. We revisited the procedure and divided it, for instructional purposes, into 15 consecutive and distinct steps.

Apremilast exerts protective effects on stroke outcomes and blood-brain barrier (BBB) dysfunction through regulating Rho-associated protein kinase 2 expression.

AimsStroke is a devastating event and a huge public health concern worldwide. Apremilast (APR) is a selective inhibitor of phosphodiesterase‐4 involved in various neurological diseases, including stroke. However, the protective effects of APR on stroke have not been investigated. Here, we explored the effects of APR on stroke outcomes and blood–brain barrier (BBB) dysfunction using a middle cerebral artery occlusion (MCAO) stroke mice model.ResultsThe results show that APR attenuated neurological injury in MCAO mice with decreased neurological deficit scores and infarct size, as well as increased hanging grip time. The increased BBB permeability and decreased expression of the tight junction protein Claudin‐5 in MCAO mice were attenuated by APR treatment. APR treatment also mitigated neuroinflammation in MCAO mice, as shown by the decreased levels of inflammatory cytokines. In vitro assays also proved that APR ameliorated the oxygen/glucose deprivation/reoxygenation (OGD/R)‐induced increase in endothelial permeability and restored the expression of Claudin‐5 in bEnd.3 brain endothelial cells. Moreover, overexpression of ROCK2 in bEnd.3 cells abolished the protective effects of APR on endothelial permeability against OGD/R induction.ConclusionTaken together, our results demonstrate that APR showed significant efficacy on ischemic stroke outcomes by alleviating enhanced BBB permeability and neuroinflammation by inhibiting ROCK2. These findings suggest a novel therapeutic window for ischemic stroke.

NOX2-mediated reactive oxygen species are double-edged swords in focal cerebral ischemia in mice

BackgroundReactive oxygen species (ROS) often promote acute brain injury after stroke, but their roles in the recovery phase have not been well studied. We tested the hypothesis that ROS activity mediated by NADPH oxidase 2 (NOX2) contributes to acute brain injury but promotes functional recovery during the delayed phase, which is linked with neuroinflammation, autophagy, angiogenesis, and the PI3K/Akt signaling pathway.MethodsWe used the NOX2 inhibitor apocynin to study the role of NOX2 in brain injury and functional recovery in a middle cerebral artery occlusion (MCAO) stroke mouse model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7, 10 and 14 after reperfusion. In addition, dynamic NOX2-induced ROS levels were measured by dihydroethidium (DHE) staining. Autophagy, inflammasomes, and angiogenesis were measured by immunofluorescence staining and western blotting. RNA sequencing was performed, and bioinformatics technology was used to analyze differentially expressed genes (DEGs), as well as the enrichment of biological functions and signaling pathways in ischemia penumbra at 7 days after reperfusion. Then, Akt pathway-related proteins were further evaluated by western blotting.ResultsOur results showed that apocynin injection attenuated infarct size and mortality 3 days after stroke but promoted mortality and blocked functional recovery from 5 to 14 days after stroke. DHE staining showed that ROS levels were increased at 3 days after reperfusion and then gradually declined in WT mice, and these levels were significantly reduced by the NOX2 inhibitor apocynin. RNA-Seq analysis indicated that apocynin activated the immune response under hypoxic conditions. The immunofluorescence and western blot results demonstrated that apocynin inhibited the NLRP3 inflammasome and promoted angiogenesis at 3 days but promoted the NLRP3 inflammasome and inhibited angiogenesis at 7 and 14 days after stroke, which was mediated by regulating autophagy activation. Furthermore, RNA-Seq and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that apocynin injection resulted in PI3K–Akt signaling pathway enrichment after 7 days of MCAO. We then used an animal model to show that apocynin decreased the protein levels of phosphorylated PI3K and Akt and NF-κB p65, confirming that the PI3K–Akt–NF-κB pathway is involved in apocynin-mediated activation of inflammation and inhibition of angiogenesis.ConclusionsNOX2-induced ROS production is a double-edged sword that exacerbates brain injury in the acute phase but promotes functional recovery. This effect appears to be achieved by inhibiting NLRP3 inflammasome activation and promoting angiogenesis via autophagy activation.

Nt5e deficiency does not affect post-stroke inflammation and lesion size in a murine ischemia/reperfusion stroke model

SummaryExtracellular ATP released to the ischemic brain parenchyma is quickly metabolized by ectonucleotidases. Among them, the ecto-5′-nucleotidase CD73 encoded by Nt5e generates immunosuppressive adenosine. Genetic deletion of Nt5e led to increased infarct size in the murine photothrombotic stroke model. We aimed at validating this result using the transient middle cerebral artery occlusion (tMCAO) stroke model that represents pathophysiological aspects of penumbra and reperfusion. Three days after tMACO, we did not detect a difference in stroke size between CD73-deficient (CD73−/−) and control mice. Consistent with this finding, CD73−/− and control mice showed comparable numbers and composition of brain-infiltrating leukocytes measured by flow cytometry. Using NanoString technology, we further demonstrated that CD73−/− and control mice do not differ regarding glia cell gene expression profiles. Our findings highlight the potential impact of stroke models on study outcome and the need for cross-validation of originally promising immunomodulatory candidates.

Inhibition of PECAM-1 Significantly Delays Leukocyte Extravasation into the Subcortex Post-Stroke.

Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade. The transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post-stroke. Large ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function-blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM-1 Ab 62% ± 5.0%). High-resolution wide-field microscopy confirmed inhibition of TEM by PECAM-1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours. Our findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.