Intraluminal Middle Cerebral Artery Occlusion Research Articles

Neural precursor cell delivery induces acute post-ischemic cerebroprotection, but fails to promote long-term stroke recovery in hyperlipidemic mice due to mechanisms that include pro-inflammatory responses associated with brain hemorrhages

BackgroundThe intravenous delivery of adult neural precursor cells (NPC) has shown promising results in enabling cerebroprotection, brain tissue remodeling, and neurological recovery in young, healthy stroke mice. However, the translation of cell-based therapies to clinical settings has encountered challenges. It remained unclear if adult NPCs could induce brain tissue remodeling and recovery in mice with hyperlipidemia, a prevalent vascular risk factor in stroke patients.MethodsMale mice on a normal (regular) diet or on cholesterol-rich Western diet were exposed to 30 min intraluminal middle cerebral artery occlusion (MCAO). Vehicle or 106 NPCs were intravenously administered immediately after reperfusion, at 3 day and 7 day post-MCAO. Neurological recovery was evaluated using the Clark score, Rotarod and tight rope tests over up to 56 days. Histochemistry and light sheet microscopy were used to examine ischemic injury and brain tissue remodeling. Immunological responses in peripheral blood and brain were analyzed through flow cytometry.ResultsNPC administration reduced infarct volume, blood–brain barrier permeability and the brain infiltration of neutrophils, monocytes, T cells and NK cells in the acute stroke phase in both normolipidemic and hyperlipidemic mice, but increased brain hemorrhage formation and neutrophil, monocyte and CD4+ and CD8+ T cell counts and activation in the blood of hyperlipidemic mice. While neurological deficits in hyperlipidemic mice were reduced by NPCs at 3 day post-MCAO, NPCs did not improve neurological deficits at later timepoints. Besides, NPCs did not influence microglia/macrophage abundance and activation (assessed by morphology analysis), astroglial scar formation, microvascular length or branching point density (evaluated using light sheet microscopy), long-term neuronal survival or brain atrophy in hyperlipidemic mice.ConclusionsIntravenously administered NPCs did not have persistent effects on post-ischemic neurological recovery and brain remodeling in hyperlipidemic mice. These findings highlight the necessity of rigorous investigations in vascular risk factor models to fully assess the long-term restorative effects of cell-based therapies. Without comprehensive studies in such models, the clinical potential of cell-based therapies cannot be definitely determined.

Distinctions between the Koizumi and Zea Longa methods for middle cerebral artery occlusion (MCAO) model: a systematic review and meta-analysis of rodent data

Ischemic stroke in rodents is usually induced by intraluminal middle cerebral artery occlusion (MCAO) via the common carotid artery plugging filament invented by Koizumi et al. (MCAO-KM), or the external carotid artery plugging filament created by Zea Longa et al. (MCAO-LG). A systematic review of the distinctions between them is currently lacking. Here, we performed a meta-analysis in terms of model establishment, cerebral blood flow (CBF), and cerebral ischemia–reperfusion injury (CIRI) between them, Weighted Mean Differences and Standardized Mean Difference were used to analyze the combined effects, Cochrane's Q test and the I2 statistic were applied to determine heterogeneity, sensitivity analysis and subgroup analysis were performed to explore the source of heterogeneity. Literature mining suggests that MCAO-KM brings shorter operation time (p = 0.007), higher probability of plugging filament (p < 0.001) and molding establishment (p = 0.006), lower possibility of subarachnoid hemorrhage (p = 0.02), larger infarct volume (p = 0.003), severer brain edema (p = 0.002), and neurological deficits (p = 0.03). Nevertheless, MCAO-LG shows a more adequate CBF after ischemia–reperfusion (p < 0.001), a higher model survival rate (p = 0.02), and a greater infarct rate (p = 0.007). In conclusion, the MCAO-KM method is simple to operate with a high modeling success rate, and is suitable for the study of brain edema under long-term hypoperfusion, while the MCAO-LG method is highly challenging for novices, and is suitable for the study of CIRI caused by complete ischemia–reperfusion. These findings are expected to benefit the selection of intraluminal filament MCAO models before undertaking ischemic stroke preclinical effectiveness trials.

An Assessment of Physical and N6-Cyclohexyladenosine-Induced Hypothermia in Rodent Distal Focal Ischemic Stroke.

Therapeutic hypothermia (TH) mitigates damage in ischemic stroke models. However, safer and easier TH methods (e.g., pharmacological) are needed to circumvent physical cooling complications. This study evaluated systemic and pharmacologically induced TH using the adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA), with control groups in male Sprague-Dawley rats. CHA was administered intraperitoneally 10 minutes following a 2-hour intraluminal middle cerebral artery occlusion. We used a 1.5 mg/kg induction dose, followed by three 1.0 mg/kg doses every 6 hours for a total of 4 doses, causing 20-24 hours of hypothermia. Animals assigned to physical hypothermia and CHA-hypothermia had similar induction rates and nadir temperatures, but forced cooling lasted ∼6 hours longer compared with CHA-treated animals. The divergence is likely attributable to individual differences in CHA metabolism, which led to varied durations at nadir, whereas physical hypothermia was better regulated. Physical hypothermia significantly reduced infarction (primary endpoint) on day 7 (mean reduction of 36.8 mm3 or 39% reduction; p = 0.021 vs. normothermic animals; Cohen's d = 0.75), whereas CHA-induced hypothermia did not (p = 0.33). Similarly, physical cooling improved neurological function (physical hypothermia median = 0, physical normothermia median = 2; p = 0.008) and CHA-induced cooling did not (p > 0.99). Our findings demonstrate that forced cooling was neuroprotective compared with controls, but prolonged CHA-induced cooling was not neuroprotective.

Age-Associated Resilience Against Ischemic Injury in Mice Exposed to Transient Middle Cerebral Artery Occlusion.

Ischemic stroke is the leading cause of death and disability. Although stroke mainly affects aged individuals, animal research is mostly one on young rodents. Here, we examined the development of ischemic injury in young (9-12-week-old) and adult (72-week-old) C57BL/6 and BALB/c mice exposed to 30 min of intraluminal middle cerebral artery occlusion (MCAo). Post-ischemic reperfusion did not differ between young and adult mice. Ischemic injury assessed by infarct area and blood-brain barrier (BBB) integrity assessed by IgG extravasation analysis was smaller in adult compared with young mice. Microvascular viability and neuronal survival assessed by CD31 and NeuN immunohistochemistry were higher in adult than young mice. Tissue protection was associated with stronger activation of cell survival pathways in adult than young mice. Microglial/macrophage accumulation and activation assessed by F4/80 immunohistochemistry were more restricted in adult than young mice, and pro- and anti-inflammatory cytokine and chemokine responses were reduced by aging. By means of liquid chromatography-mass spectrometry, we identified a hitherto unknown proteome profile comprising the upregulation of glycogen degradation-related pathways and the downregulation of mitochondrial dysfunction-related pathways, which distinguished post-ischemic responses of the aged compared with the young brain. Our study suggests that aging increases the brain's resilience against ischemic injury.

MicroRNA-210 Downregulates TET2 (Ten-Eleven Translocation Methylcytosine Dioxygenase 2) and Contributes to Neuroinflammation in Ischemic Stroke of Adult Mice.

Stroke is a leading cause of morbidity and mortality worldwide. Neuroinflammation plays a key role in acute brain injury of ischemic stroke. MicroRNA-210 (miR210) is the master hypoxamir and regulates microglial activation and inflammation in a variety of diseases. In this study, we uncovered the mechanism of miR210 in orchestrating ischemic stroke-induced neuroinflammation through repression of TET2 (ten-eleven translocation methylcytosine dioxygenase 2) in the adult mouse brain. Ischemic stroke was induced in adult WT (wild type) or miR210 KO (miR210 deficient) mice by transient intraluminal middle cerebral artery occlusion. Injection of TET2 silencing RNA or miR210 complementary locked nucleic acid oligonucleotides, or miR210 KO mice were used to validate miR210-TET2 axis and its role in ischemic brain injury. Furthermore, the effect of TET2 overexpression on miR210-stimulated proinflammatory cytokines was examined in BV2 microglia. Post assays included magnetic resonance imaging scan for brain infarct size; neurobehavioral tests, reverse transcription-quantitative polymerase chain reaction, and Western blot for miR210; and TET2 levels, flow cytometry, and ELISA for neuroinflammation in the brain after stroke or microglia in vitro. miR210 injection significantly reduced TET2 protein abundance in the brain, while miR210 complementary locked nucleic acid oligonucleotides or miR210 KO preserved TET2 regardless of ischemic brain injury. TET2 knockdown reversed the protective effects of miR210 inhibition or miR210 KO on ischemic stroke-induced brain infarct size and neurobehavioral deficits. Moreover, flow cytometry and ELISA assays showed that TET2 knockdown also significantly dampened the anti-inflammatory effect of miR210 inhibition on microglial activation and IL (interleukin)-6 release after stroke. In addition, overexpression of TET2 in BV2 microglia counteracted miR210-induced increase in cytokines. miR210 inhibition reduced ischemic stroke-induced neuroinflammatory response via repression of TET2 in the adult mouse brain, suggesting that miR210 is a potential treatment target for acute brain injury after ischemic stroke.

Influence of sex, age and diabetes on brain transcriptome and proteome modifications following cerebral ischemia

Ischemic stroke is a major cause of death and disability worldwide. Translation into the clinical setting of neuroprotective agents showing promising results in pre-clinical studies has systematically failed. One possible explanation is that the animal models used to test neuroprotectants do not properly represent the population affected by stroke, as most of the pre-clinical studies are performed in healthy young male mice. Therefore, we aimed to determine if the response to cerebral ischemia differed depending on age, sex and the presence of comorbidities. Thus, we explored proteomic and transcriptomic changes triggered during the hyperacute phase of cerebral ischemia (by transient intraluminal middle cerebral artery occlusion) in the brain of: (1) young male mice, (2) young female mice, (3) aged male mice and (4) diabetic young male mice. Moreover, we compared each group's proteomic and transcriptomic changes using an integrative enrichment pathways analysis to disclose key common and exclusive altered proteins, genes and pathways in the first stages of the disease. We found 61 differentially expressed genes (DEG) in male mice, 77 in females, 699 in diabetics and 24 in aged mice. Of these, only 14 were commonly dysregulated in all groups. The enrichment pathways analysis revealed that the inflammatory response was the biological process with more DEG in all groups, followed by hemopoiesis. Our findings indicate that the response to cerebral ischemia regarding proteomic and transcriptomic changes differs depending on sex, age and comorbidities, highlighting the importance of incorporating animals with different phenotypes in future stroke research studies.

Near infrared spectroscopy detection of hemispheric cerebral ischemia following middle cerebral artery occlusion in rats

Timely and sensitive in vivo estimation of ischemic stroke-induced brain infarction are necessary to guide diagnosis and evaluation of treatments’ efficacy. The gold standard for estimation of the cerebral infarction volume is magnetic resonance imaging (MRI), which is expensive and not readily accessible. Measuring regional cerebral blood flow (rCBF) with Laser Doppler flowmetry (LDF) is the status quo for confirming reduced blood flow in experimental ischemic stroke models. However, rCBF reduction following cerebral artery occlusion often does not correlate with subsequent infarct volume. In the present study, we employed the continuous-wave near infrared spectroscopy (NIRS) technique to monitor cerebral oxygenation during 90 min of the intraluminal middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats (n = 8, male). The NIRS device consisted of a controller module and an optical sensor with two LED light sources and two photodiodes making up two parallel channels for monitoring left and right cerebral hemispheres. Optical intensity measurements were converted to deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) changes relative to a 2-min window prior to MCAO. Area under the curve (auc) for Hb and HbO2 was calculated for the 90-min occlusion period for each hemisphere (ipsilateral and contralateral). To obtain a measure of total ischemia, auc of the contralateral side was subtracted from the ipsilateral side resulting in ΔHb and ΔHbO2 parameters. Infarct volume (IV) was calculated by triphenyl tetrazolium chloride (TTC) staining at 24h reperfusion. Results showed a significant negative correlation (r = −0.81, p = 0.03) between ΔHb and infarct volume. In conclusion, our results show feasibility of using a noninvasive optical imaging instrument, namely NIRS, in monitoring cerebral ischemia in a rodent stroke model. This cost-effective, non-invasive technique may improve the rigor of experimental models of ischemic stroke by enabling in vivo longitudinal assessment of cerebral oxygenation and ischemic injury.

High-resolution micro-CT for 3D infarct characterization and segmentation in mice stroke models

Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.

Phosphodiesterase 10A deactivation induces long-term neurological recovery, Peri-infarct remodeling and pyramidal tract plasticity after transient focal cerebral ischemia in mice

The phosphodiesterase (PDE) superfamily comprises enzymes responsible for the cAMP and cGMP degradation to AMP and GMP. PDEs are abundant in the brain, where they are involved in several neuronal functions. High PDE10A abundance was previously observed in the striatum; however its consequences for stroke recovery were unknown. Herein, we evaluated the effects of PDE10A deactivation by TAK-063 (0.3 or 3 mg/kg, initiated 72 h post-stroke) in mice exposed to intraluminal middle cerebral artery occlusion. We found that PDE10A deactivation over up to eight weeks dose-dependently increased long-term neuronal survival, angiogenesis, and neurogenesis in the peri-infarct striatum, which represents the core of the middle cerebral artery territory, and reduced astroglial scar formation, whole brain atrophy and, more specifically, striatal atrophy. Functional motor-coordination recovery and the long-distance plasticity of pyramidal tract axons, which originate from the contralesional motor cortex and descend through the contralesional striatum to innervate the ipsilesional facial nucleus, were enhanced by PDE10A deactivation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a set of dopamine receptor-related and neuronal plasticity-related PDE10A targets, which were elevated (e.g., protein phosphatase-1 regulatory subunit 1B) or reduced (e.g., serine/threonine protein phosphatase 1α, β-synuclein, proteasome subunit α2) by PDE10A deactivation. Our results identify PDE10A as a therapeutic target that critically controls post-ischemic brain tissue remodeling and plasticity.

N-Methyl-D-Aspartate Receptors Antagonist Prevents Secondary Ischemic Brain Injury Associated With Lipopolysaccharide-Induced Sepsis-Like State Presumably via Immunomodulatory Actions.

Infection is a major reason for poor stroke outcomes, and sepsis is a major cause of stroke-elated deaths. We herein examined whether NMDA receptor blockade, which was reported to exert anti-inflammatory actions, protects against the deleterious consequences of lipopolysaccharide (LPS)-induced sepsis-like state in adult male NMRI mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO). At 24 h post-ischemia, vehicle or Escherichia coli LPS (2 or 4 mg/kg) was intraperitoneally administered, whereas 30 min later vehicle or ketamine (10 mg/kg), which is a non-competitive NMDA receptor antagonist, was intraperitoneally applied. Delivery of LPS at a dosage of 4 mg/kg induced a sepsis-like state characterized by a rectal temperature reduction by ∼4.0°C, increased neurological deficits in Clark score, cylinder and open-field tests, increased brain infarct volume and reduced neuronal survival in the previously ischemic tissue. Notably, additional treatment with ketamine (10 mg/kg) significantly attenuated the sepsis-associated rectal temperature reduction by ∼1.5°C, reduced neurological deficits, reduced infarct volume, and promoted neuronal survival. Ketamine alone did not influence infarct volume or neurological deficits. Real-time PCR data analysis showed that GFAP, CD86, CD206, IL-1β, and IL-10 mRNA levels were significantly increased in ischemic brains of LPS-treated compared with vehicle-treated mice. Additional treatment with ketamine significantly decreased IL-1β and IL-10, but not GFAP, CD86, and CD206 mRNA levels. Our data show that ketamine at a dose that on its own does not confer neuroprotection reverses the adverse effects of LPS-induced sepsis-like state post-ischemia, presumably via immunomodulatory actions.

RXRγ attenuates cerebral ischemia-reperfusion induced ferroptosis in neurons in mice through transcriptionally promoting the expression of GPX4.

Cerebral ischemia is a common cerebrovascular disease with high mortality and disability rate. Exploring its mechanism is essential for developing effective treatment for cerebral ischemia. Therefore, this study aims to explore the regulatory effect and mechanism of retinoid X receptor γ (RXRγ) on cerebral ischemia-reperfusion (I/R) injury. A mouse intraluminal middle cerebral artery occlusion model was established, and PC12 cells were exposed to anaerobic/reoxygenation (A/R) as an in vitro model in this study. Cerebral I/R surgery or A/R treatment induced ferroptosis, downregulated RXRγ and GPX4 (glutathione peroxidase 4) levels, upregulated cyclooxygenase-2 (COX-2) level and increased ROS (reactive oxygen species) level in A/R induced cells or I/R brain tissues in vivo or PC12 cells in vitro. Knockdown of RXRγ downregulated GPX4 and increased COX-2 and ROS levels in A/R induced cells. RXRγ overexpression has the opposite effect. GPX4 knockdown reversed the improvement of RXRγ overexpression on COX-2 downregulation, GPX4 upregulation and ferroptosis in PC12 cells. Furthermore, chromatin immunoprecipitation (ChIP) and luciferase reporter gene assays revealed that RXRγ bound to GPX4 promoter region and activated its transcription. Overexpression of RXRγ or GPX4 alleviated brain damage and inhibited ferroptosis in I/R mice. In conclusion, RXRγ-mediated transcriptional activation of GPX4 might inhibit ferroptosis during I/R-induced brain injury.

Cerebroprotein hydrolysate injection is involved in promoting long-term angiogenesis, vessel diameter and density after cerebral ischemia in mice

AimsIn this study, we aimed investigate the impacts of CH-I on angiogenesis, effects for vascular structure changes and long-term neurological recovery after ischemic stroke as well as the potential mechanisms. Main methodsYoung male mice subjected to intraluminal middle cerebral artery occlusion were administrated with CH-I once daily from day 1 to day 14 after stroke. The infarct volume was evaluated by TTC staining at day 7 after stroke. Neurological deficits were measured 1 to 28 days after stroke. Microvascular density, astrocyte coverage, and angiogenesis were assessed by IF, qRT-PCR, and WB at regular intervals after stroke. LSCI and TPMI measured changes in blood flow and vascular density and width from the day after stroke to day 28. Key findingsCompared with the dMCAO group, CH-I treatment significantly improved neurological recovery and reduced the infarct at day 7 after stroke. CH-I treatment increased the expression of the CD31, BrdU+/CD31+ microvessels and GFAP positive vessels in the peri-infarct cortex at day 7 to 28 after stroke. The expression of protein and gene were enhanced in CH-I group. CH-I significantly improved cerebral blood flow at day 7 after stroke. CH-I increased the vascular density and vascular width at day 14 after stroke. SignificanceCH-I has been shown to restore nerve function, reduce the rate of cerebral infarction, increase microvascular density, and promote angiogenesis. CH-I improved cerebral blood flow, protected blood vessels from postoperative stenosis, and improved vascular plasticity.

Tenascin-C restricts reactive astrogliosis in the ischemic brain

Cellular responses in glia play a key role in regulating brain remodeling post-stroke. However, excessive glial reactivity impedes post-ischemic neuroplasticity and hampers neurological recovery. While damage-associated molecular patterns and activated microglia were shown to induce astrogliosis, the molecules that restrain astrogliosis are largely unknown. We explored the role of tenascin-C (TnC), an extracellular matrix component involved in wound healing and remodeling of injured tissues, in mice exposed to ischemic stroke induced by transient intraluminal middle cerebral artery occlusion. In the healthy adult brain, TnC expression is restricted to neurogenic stem cell niches. We previously reported that TnC is upregulated in ischemic brain lesions. We herein show that the de novo expression of TnC post-stroke is closely associated with reactive astrocytes, and that astrocyte reactivity at 14 days post-ischemia is increased in TnC-deficient mice (TnC−/−). By analyzing the three-dimensional morphology of astrocytes in previously ischemic brain tissue, we revealed that TnC−/− reduces astrocytic territorial volume, branching point number, and branch length, which are presumably hallmarks of the homeostatic regulatory astrocyte state, in the post-acute stroke phase after 42 days. Interestingly, TnC−/− moderately increased aggrecan, a neuroplasticity-inhibiting proteoglycan, in the ischemic brain tissue at 42 days post-ischemia. In vitro in astrocyte-microglia cocultures, we showed that TnC−/− reduces the microglial migration speed on astrocytes and elevates intercellular adhesion molecule 1 (ICAM1) expression. Post-stroke, TnC−/− did not alter the ischemic lesion size or neurological recovery, however microglia-associated ICAM1 was upregulated in TnC−/− mice during the first week post stroke. Our data suggest that TnC plays a central role in restraining post-ischemic astrogliosis and regulating astrocyte-microglial interactions.

Repetitive xenon treatment improves post-stroke sensorimotor and neuropsychiatric dysfunction

Stroke is a life-changing event as stroke survivors experience changes in personality, emotions and mood. We investigated the effect of xenon gas encapsulated in liposomes on stroke-generated sensorimotor impairments, and anxiety- and depression-like phenotypes. Ischemic stroke was created by the intraluminal middle cerebral artery occlusion (MCAO) for 6 h followed by reperfusion in rats. Xenon-liposome (6 mg/kg, intravenous) treatment was given multiple times starting at 2 h post-ischemia through 6 h (5X), and once-daily for next 3 days. Rats underwent ischemic injury displayed sensorimotor deficits in the adhesive removal, vibrissae-evoked forelimb placement and rotarod tests. These animals also made lesser entries and spent less time on open arms of the elevated-plus maze and swam more in passive mode in the forced swimming test, indicating anxiety- and depression-like behaviors at 28- and 35-days post-injury, respectively. Repeated intravenous treatment with xenon-liposomes ameliorated these behavioral aberrations (p < 0.05). Gut microbiome analysis (16S ribosomal-RNA gene sequencing) showed a decrease in the Clostridium clusters XI, XIVa, XVIII and Lactobacillus bacterium, and increase of the Prevotella in the xenon-liposome group. No microbiota communities were majorly affected across the treatments. Moreover, xenon treatment group showed augmented plasma levels of IL-6 cytokines (∼5 fold) on day-35 post-ischemia, while no change was noticed in the IL-1β, IL-4, IL-10, IL-13 and MCP-1 levels. Our data highlights the safety, behavioral recovery and reversal of post-stroke brain injury following xenon-liposome treatment in an extended ischemic model. These results show the potential for this treatment strategy to be translated to patients with stroke.

Transient Middle Cerebral Artery Occlusion with an Intraluminal Suture Enables Reproducible Induction of Ischemic Stroke in Mice.

Ischemic stroke is a leading cause of mortality and chronic disability worldwide, underscoring the need for reliable and accurate animal models to study this disease's pathology, molecular mechanisms of injury, and treatment approaches. As most clinical strokes occur in regions supplied by the middle cerebral artery (MCA), several experimental models have been developed to simulate an MCA occlusion (MCAO), including transcranial MCAO, micro- or macro-sphere embolism, thromboembolisation, photothrombosis, Endothelin-1 injection, and - the most common method for ischemic stroke induction in murine models - intraluminal MCAO. In the intraluminal MCAO model, the external carotid artery (ECA) is permanently ligated, after which a partially-coated monofilament is inserted and advanced proximally to the common carotid artery (CCA) bifurcation, before being introduced into the internal carotid artery (ICA). The coated tip of the monofilament is then advanced to the origin of the MCA and secured for the duration of occlusion. With respect to other MCAO models, this model offers enhanced reproducibility regarding infarct volume and cognitive/functional deficits, and does not require a craniotomy. Here, we provide a detailed protocol for the surgical induction of unilateral transient ischemic stroke in mice, using the intraluminal MCAO model. Graphic abstract: Overview of the intraluminal monofilament method for transient middle cerebral artery occlusion (MCAO) in mouse.

Exacerbated VEGF up-regulation accompanies diabetes-aggravated hemorrhage in mice after experimental cerebral ischemia and delayed reperfusion

Reperfusion therapy is the preferred treatment for ischemic stroke, but is hindered by its short treatment window, especially in patients with diabetes whose reperfusion after prolonged ischemia is often accompanied by exacerbated hemorrhage. The mechanisms underlying exacerbated hemorrhage are not fully understood. This study aimed to identify this mechanism by inducing prolonged 2-hour transient intraluminal middle cerebral artery occlusion in diabetic Ins2Akita/+ mice to mimic patients with diabetes undergoing delayed mechanical thrombectomy. The results showed that at as early as 2 hours after reperfusion, Ins2Akita/+ mice exhibited rapid development of neurological deficits, increased infarct and hemorrhagic transformation, together with exacerbated down-regulation of tight-junction protein ZO-1 and up-regulation of blood-brain barrier-disrupting matrix metallopeptidase 2 and matrix metallopeptidase 9 when compared with normoglycemic Ins2+/+ mice. This indicated that diabetes led to the rapid compromise of vessel integrity immediately after reperfusion, and consequently earlier death and further aggravation of hemorrhagic transformation 22 hours after reperfusion. This observation was associated with earlier and stronger up-regulation of pro-angiogenic vascular endothelial growth factor (VEGF) and its downstream phospho-Erk1/2 at 2 hours after reperfusion, which was suggestive of premature angiogenesis induced by early VEGF up-regulation, resulting in rapid vessel disintegration in diabetic stroke. Endoplasmic reticulum stress-related pro-apoptotic C/EBP homologous protein was overexpressed in challenged Ins2Akita/+ mice, which suggests that the exacerbated VEGF up-regulation may be caused by overwhelming endoplasmic reticulum stress under diabetic conditions. In conclusion, the results mimicked complications in patients with diabetes undergoing delayed mechanical thrombectomy, and diabetes-induced accelerated VEGF up-regulation is likely to underlie exacerbated hemorrhagic transformation. Thus, suppression of the VEGF pathway could be a potential approach to allow reperfusion therapy in patients with diabetic stroke beyond the current treatment window. Experiments were approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong [CULATR 3834-15 (approval date January 5, 2016); 3977-16 (approval date April 13, 2016); and 4666-18 (approval date March 29, 2018)].

Postischemic Neuroprotection Associated With Anti-Inflammatory Effects by Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles in Aged Mice.

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce ischemic neuroprotection in mice by modulating the brain infiltration of leukocytes and, specifically polymorphonuclear neutrophils. So far, effects of MSC-sEVs were only studied in young ischemic rodents. We herein examined the effects of MSC-sEVs in aged mice. Male and female C57Bl6/j mice (8-10 weeks or 15-24 months) were exposed to transient intraluminal middle cerebral artery occlusion. Vehicle or sEVs (equivalent of 2×106 MSCs) were intravenously administered. Neurological deficits, ischemic injury, blood-brain barrier integrity, brain leukocyte infiltration, and blood leukocyte responses were evaluated over up to 7 days. MSC-sEV delivery reduced neurological deficits, infarct volume, brain edema, and neuronal injury in young and aged mice of both sexes, when delivered immediately postreperfusion or with 6 hours delay. MSC-sEVs decreased leukocyte and specifically polymorphonuclear neutrophil, monocyte, and macrophage infiltrates in ischemic brains of aged mice. In peripheral blood, the number of monocytes and activated T cells was significantly reduced by MSC-sEVs. MSC-sEVs induce postischemic neuroprotection and anti-inflammation in aged mice.

Post-ischemic protein restriction induces sustained neuroprotection, neurological recovery, brain remodeling, and gut microbiota rebalancing

BackgroundModerate dietary protein restriction confers neuroprotection when applied before ischemic stroke. How a moderately protein-reduced diet influences stroke recovery when administered after stroke, is a clinically relevant question. This question has not yet been investigated. MethodsMale C57BL6/J mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately after the stroke, mice were randomized to two normocaloric diets: a moderately protein-reduced diet containing 8% protein (PRD) or normal diet containing 20% protein (ND). Post-stroke neurological deficits were evaluated by a comprehensive test battery. Antioxidant and neuroinflammatory responses in the brain and liver were evaluated by Western blot and RTqPCR. Stroke-induced brain injury, microvascular integrity, glial responses, and neuroplasticity were assessed by immunohistochemistry. Fecal microbiota analysis was performed using 16S ribosomal RNA amplicon sequencing. ResultsWe show that PRD reduces brain infarct volume after three days and enhances neurological and, specifically, motor-coordination recovery over six weeks in stroke mice. The recovery-promoting effects of PRD were associated with increased antioxidant responses and reduced neuroinflammation. Histochemical studies revealed that PRD increased long-term neuronal survival, increased peri-infarct microvascular density, reduced microglia/macrophage accumulation, increased contralesional pyramidal tract plasticity, and reduced brain atrophy. Fecal microbiota analysis showed reduced bacterial richness and diversity in ischemic mice on ND starting at 7 dpi. PRD restored bacterial richness and diversity at these time points. ConclusionModerate dietary protein restriction initiated post-ischemic stroke induces neurological recovery, brain remodeling, and neuroplasticity in mice by mechanisms involving antiinflammation and, in the post-acute phase, commensal gut microbiota rebalancing.

Senolytic Therapy for Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke is one of the leading causes of death, and even timely treatment can result in severe disabilities. Reperfusion of the ischemic stroke region and restoration of the blood supply often lead to a series of cellular and biochemical consequences, including generation of reactive oxygen species (ROS), expression of inflammatory cytokines, inflammation, and cerebral cell damage, which is collectively called cerebral ischemia-reperfusion (IR) injury. Since ROS and inflammatory cytokines are involved in cerebral IR injury, injury could involve cellular senescence. Thus, we investigated whether senolytic therapy that eliminates senescent cells could be an effective treatment for cerebral IR injury. To determine whether IR induces neural cell senescence in vitro, astrocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). OGD/R induced astrocyte senescence and senescent cells in OGD/R-injured astrocytes were effectively eliminated in vitro by ABT263, a senolytic agent. IR in rats with intraluminal middle cerebral artery occlusion induced cellular senescence in the ischemic region. The senescent cells in IR-injured rats were effectively eliminated by intravenous injections of ABT263. Importantly, ABT263 treatment significantly reduced the infarct volume and improved neurological function in behavioral tests. This study demonstrated, for the first time, that senolytic therapy has therapeutic potential for cerebral IR injury.

Multimodality Imaging of the Brain-Heart-Axis after Ischemic Stroke Reveals Acute and Chronic Cardiac Dysfunction after Cerebral Ischemia

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG; Ev Studienwerk Villigst Introduction Cerebral ischemia remains a major contributor to global morbidity and mortality. Beyond neurological dysfunction, cerebral stroke also increases risk of subsequent cardiac events and development of chronic heart failure. The mechanisms underlying this brain-heart communication are poorly characterized, but systemic inflammation is thought to play a role. Purpose We hypothesized that cerebral ischemic stroke evokes systemic inflammation contributing to acute cardiac stress and persistent contractile dysfunction, which may be monitored by serial molecular imaging by positron emission tomography (PET). Methods C57Bl6 mice underwent transient (30min) intraluminal middle cerebral artery occlusion (MCAo, n = 88) to induce stroke or sham surgery (n = 18). Serial 18F-GE180 PET at 24h, 7d, and 21d after injury identified translocator protein (TSPO) in brain microglia and peripheral macrophages as a marker of inflammation. Magnetic resonance imaging (MRI) determined stroke size and characterized cardiac function at 1 and 3wk after MCAo. Imaging signals were confirmed by autoradiography, immunohistochemistry and fluorescence-activated cell sorting. Results MCAo in mice induced cerebral stroke of variable size (median 44.68mm3, range:5.12-139.60mm3). Localized inflammation in the injured hemisphere was observed by TSPO PET covering territory consistent to the stroke location, with marked increase in the neuroinflammatory signal at 7d relative to the contralateral hemisphere (% injected dose (ID)/g max: 3.82 ± 0.84 vs 2.51 ± 0.32, p &amp;lt; 0.001). TSPO signal was similarly elevated in the stroke region compared to sham animals (%ID/g max: 3.82 ± 0.84 vs 2.59 ± 0.72, p &amp;lt; 0.001). Corresponding with neuroinflammation, a heightened global myocardial TSPO signal was observed 7d after MCAo compared to sham (%ID/g: 7.75 ± 2.79 vs 5.61 ± 1.98, p = 0.041). TSPO PET signal remained elevated 21d after stroke in brain (3.20 ± 1.15 vs 2.46 ± 0.37, p = 0.012) and heart (8.62 ± 2.43 vs 5.85 ± 0.66, p = 0.022). Cerebral stroke was associated with reduced contractile function compared to sham at 1wk (ejection fraction, EF: 49.58 ± 16.90 vs 62.17 ± 5.25%, p = 0.025) and 3wk (54.30 ± 5.70 vs 66.06 ± 3.48%, p = 0.009). Larger stroke size identified early after injury by T2 weighted MRI, reflecting the focal injury and penumbra edema, correlated with worse subsequent cardiac function at 21d (r = 0.745, p = 0.009). Despite increased TSPO signal, flow cytometry showed comparable CD45+ leukocyte content in the left ventricle after MCAo compared to sham at 7d (p = 0.749). Conclusions Ischemic stroke evoked parallel cerebral and cardiac TSPO upregulation and persistent contractile dysfunction, beginning early after injury. The extent and severity of brain injury predicts left ventricle dysfunction, but the role of inflammation requires further investigation. Molecular imaging may identify risk and guide novel therapies to improve cardiac outcome after stroke.