Occlusion Model Research Articles

Noninvasive Detection of Chorioretinal Hypoxia via Poly(lactic‐co‐glycolic acid) Nanoparticles Embedded with Purely Organic Phosphors

Ischemia‐induced hypoxia is a critical complication in retinal diseases, leading to significant vision impairment and blindness due to disrupted blood flow and oxygen delivery. Currently, there is no effective method to assess oxygen levels in extravascular retinal tissue. Traditional hypoxia detection methods, such as oxygen‐sensitive microelectrodes, magnetic resonance imaging, and retinal oximetry, have limitations including invasiveness, low spatial resolution, and lack of real‐time monitoring. Herein, a noninvasive hypoxia detection method is proposed by utilizing lipid‐polymer nanoparticles (NPs) with purely organic room‐temperature phosphorescence materials for real‐time detection with high spatial and temporal resolution. To enhance biocompatibility and efficacy, NPs were fabricated using biodegradable poly(lactic‐co‐glycolic acid) (PLGA) and SeCO as a phosphor. PLGA degrades into nontoxic by‐products, while the excitation wavelength of SeCO at 393 nm minimizes damage from short wavelengths and enhances tissue penetration. Furthermore, the NPs’ size is optimized to improve cellular uptake and reduce bodily accumulation, as smaller NPs are preferred for biocompatibility. Herein, synthesis, characterization, and evaluation of these PLGA‐based phosphorescent NPs in rabbit models of retinal vein occlusion and choroidal vascular occlusion are involved. This approach represents a significant advancement in noninvasive biomedical imaging, improving the diagnosis and management of ischemic retinal diseases.

Thrombolytic efficacy and safety of recombinant scu-PA in a rabbit retinal vein occlusion model.

Retinal vein occlusion (RVO) has become the second most common retinal vascular disease after diabetic retinopathy. Existing therapeutic approaches, including intravitreal injection of antivascular endothelial growth factors (anti-VEGFs) and/or glucocorticoids and laser therapy, primarily address secondary macular edema and neovascularisation. However, these strategies do not address the underlying cause of the disease and may have harmful side effects. There is an urgent need for therapies that have a better prognosis and include the administration of thrombolytics at an early stage. Therefore, in the present study, we investigated the thrombolytic effect of treatment with recombinant human Single-chain urokinase-type plasminogen activators (scu-PA)and the differences in its efficacy at different doses in a rabbit RVO model. In addition, through a series of ophthalmological examinations, such as optical coherence tomography (OCT) and electrophysiology, conducted to ascertain the effects of treatment with scu-PA on the ocular fibrinolytic system, we noted a definitive safety window for the vitreous administration of scu-PA. Therefore, this study is the first to confirm that an intravenous or vitreous cavity injection of scu-PA has definitive potential for treating RVO; however, additional clinical studies are needed for further validation.

A novel cerebral ischemia-reperfusion model using intraluminal filament in rabbit

Abstract Introduction Stroke models are based on permanent or transient occlusion of cerebral arteries. Despite transient occlusion model tends to produce more variable results, it can investigate reperfusion. Although ischemia-reperfusion model is well established in rat using a silicone-filament by exposition of carotid artery, the endovascular approach in large animal is still a challenge. The purpose was to develop a novel cerebral ischemia-reperfusion model in rabbit using an intraluminal wire by means of an endovascular approach and subsequent assessed by MRI. Methods Four New Zealand rabbits (4-5 kg) underwent cerebral ischaemia-reperfusion. A 4Fr introducer sheath was inserted into the femoral artery. Under fluoroscopy, a 4Fr Cobra catheter was placed into the common carotid artery. A 1.9Fr microcatheter was inserted coaxially into the internal carotid artery, and a 0.014” microwire was advanced into the middle cerebral artery (MCA). Alternatively, the posterior cerebral artery (PCA) was occluded through the vertebral artery when the above approach failed. After 1 hour, reperfusion was performed by withdrawing the microwire. MRI examinations were conducted from 1 to 3 hours later. Results Technical success was achieved in three out of four animals, with the MCA reached in two rabbits and the PCA in one. In the rabbit with technical failure, it was impossible to advance the microwire into either the MCA or PCA due to the tortuosity of the vessels and vasospasm. Conclusion Ischemia-reperfusion model in rabbit though an intraluminal wire may provide a new model using an endovascular approach for the earlier research of transient stroke.

Optimized PLGA encapsulated SA-2 nanosuspension exhibits sustained intraocular pressure reduction in the mouse microbead occlusion model of ocular hypertension.

Elevated intraocular pressure (IOP) is implicated in the structural and functional damage to the retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). Topical IOP lowering agents provide short-term relief, necessitating frequent dosing. Moreover, non-adherence to frequent eyedrops administration contributes significantly to visual field loss and worsens the disease outcome. We optimized the poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulation of hybrid antioxidant-nitric oxide donor SA-2 (SA-2NP), investigated its bioavailability, duration of IOP lowering efficacy, and effects on retinal function in the microbead model of ocular hypertension (OHT). SA-2 was bioavailable in the anterior and posterior segments after 1, 8, and 24 h post-single topical eyedrop administration. SA-2NP significantly lowered IOP (∼25-34%) and preserved the RGC function after weekly eyedrop administration for 3 weeks in C57BL/6J mice. In conclusion, the optimized SA-2NP formulation demonstrated optimal bioavailability, ocular safety, and prolonged IOP-lowering efficacy in the mouse microbead occlusion model of OHT.

A Method for Imaging the Ischemic Penumbra with MRI using IVIM.

In acute ischemic stroke, the amount of "local" CBF distal to the occlusion, i.e. all blood flow within a region whether supplied antegrade or delayed and dispersed through the collateral network, may contain valuable information regarding infarct growth rate and treatment response. DSC CBF using a local arterial input function (AIF) is one method of quantifying local CBF (local-qCBF) and correlates with collaterals. Similarly, intravoxel incoherent motion MRI (IVIM) is "local", with excitation and readout in the same plane, and a potential alternative way to measure local-qCBF. The purpose of this work was to compare IVIM local-qCBF against DSC local-qCBF in the ischemic penumbra, compare measurement of perfusion-diffusion mismatch (PWI/DWI), and examine if local-qCBF may improve prediction of final infarct. Eight experiments in a pre-clinical canine model of middle cerebral artery occlusion were performed; native collateral circulation was quantified via x-ray DSA 30 minutes post-occlusion, and collateralization was subsequently enhanced in a subset of experiments with simultaneous pressor and vasodilator. IVIM and DSC MRI were acquired 2.5hr post-occlusion. IVIM was post-processed to return local-qCBF from fD*, water transport time (WTT) from D*, diffusion from D, and the PWI/DWI mismatch. These were compared with DSC parameters processed first with a standard global-AIF and then with a local-AIF. These DSC parameters included time-to-maximum, local MTT, standard-qCBF, local-qCBF and PWI/DWI mismatch. Infarct volume was measured with DWI at 2.5hrs and 4hrs post-occlusion. 2.5hr post-occlusion, IVIM local-qCBF in the non-infarcted ipsilateral territory strongly correlated with DSC local-qCBF (slope=1.00, R2=0.69, Lin's CCC=0.71). Correlation was weaker between IVIM local-qCBF and DSC standard-qCBF (R2=0.13). DSC localqCBF and IVIM local-qCBF in the non-infarcted ipsilateral territory both returned strong prediction of final infarct volume (R2=0.78, R2=0.61 respectively). DSC standard-qCBF was a weaker predictor (R2=0.12). The hypoperfused lesion from DSC local-qCBF and from IVIM local-qCBF both predicted final infarct volume with good sensitivity and correlation (slope=2.08, R2=0.67, slope=2.50, R2=0.68 respectively). The IVIM PWI/DWI ratio was correlated with infarct growth (R2=0.70) and WTT correlated with DSC MTT (R2=0.60). Non-contrast IVIM measurement of local-qCBF and PWI/DWI mismatch may include collateral circulation and improve prediction of infarct growth. AIF: arterial input function, IVIM: intravoxel incoherent motion, qCBF: quantitative cerebral blood flow, WTT: water transport time, MCAO: middle cerebral artery occlusion, MD: mean diffusivity.

Exosomes derived from highly scalable and regenerative human progenitor cells promote functional improvement in a rat model of ischemic stroke.

Globally, there are 15 million stroke patients each year who have significant neurological deficits. Today, there are no treatments that directly address these deficits. With demographics shifting to an older population, the problem is worsening. Therefore, it is crucial to develop feasible therapeutic treatments for stroke. In this study, we tested exosomes derived from embryonic endothelial progenitor cells (eEPC) to assess their therapeutic efficacy in a rat model of ischemic stroke. Importantly, we have developed purification methods aimed at producing robust and scalable exosomes suitable for manufacturing clinical grade therapeutic exosomes. We characterized exosome cargos including RNA-seq, miRNAs targets, and proteomic mass spectrometry analysis, and we found that eEPC-exosomes were enhanced with angiogenic miRNAs (i.e., miR-126), anti-inflammatory miRNA (i.e., miR-146), and anti-apoptotic miRNAs (i.e., miR-21). The angiogenic activity of diverse eEPC-exosomes sourced from a panel of eEPC production lines was assessed in vitro by live-cell vascular tube formation and scratch wound assays, showing that several eEPC-exosomes promoted the proliferation, tube formation, and migration in endothelial cells. We further applied the exosomes systemically in a rat middle cerebral artery occlusion (MCAO) model of stroke and tested for neurological recovery (mNSS) after injury in ischemic animals. The mNSS scores revealed that recovery of sensorimotor functioning in ischemic MCAO rats increased significantly after intravenous administration of eEPC-exosomes and outpaced recovery obtained through treatment with umbilical cord stem cells. Finally, we investigated the potential mechanism of eEPC-exosomes in mitigating ischemic stroke injury and inflammation by the expression of neuronal, endothelial, and inflammatory markers. Taken together, these data support the finding that eEPCs provide a valuable source of exosomes for developing scalable therapeutic products and therapies for stroke and other ischemic diseases.

Astragalus mongholicus and Scutellaria baicalensis Extracts Mixture Target Pyroptosis in Ischemic Stroke via the NLRP3 Pathway.

Ischemic stroke, caused by blocked cerebral blood flow, requires prompt intervention to prevent severe motor and cognitive impairments. Despite extensive drug development efforts, the failure rate of clinical trials remains high, highlighting the need for novel therapeutic approaches. This study investigated the therapeutic potential of a natural herbal extract mixture of Astragalus mongholicus Bunge (AM) and Scutellaria baicalensis Georgi (SB), traditionally used in Eastern Asian herbal medicine (EAHM) for ischemic stroke treatment. Using transient middle cerebral artery occlusion (tMCAO) and photothrombotic (PTB) mouse models, oral administration of the AM-SB mixture was evaluated during both acute and chronic phases. Results showed that AM-SB significantly reduced infarction volume, inflammation (IL-1β, TNF-α), and pyroptosis-related markers (NLRP3, GSDMD, ASC, Caspase-1), while decreasing gliosis and improving cerebral metabolites. Behavioral assessments revealed that early and sustained AM-SB intervention enhanced motor and cognitive functions, as measured by mNSS, Rotarod, Novel Object Recognition, and Passive Avoidance tests. These findings suggest that AM-SB extract is a promising alternative therapy for ischemic stroke management.

Comprehensive multi-omics, behavioral and morphological analysis of the hazards of nano-plastics in mice with internal carotid artery occlusion.

Accumulation of nanoplastics (NPs) poses a severe threat to the homeostasis of the internal environment in patients with chronic diseases. The effects of NP contamination on health in chronically ill populations must urgently be elucidated. In this study, NPs injected via the tail vein were distributed in the brain and internal organs in a mouse model of chronic internal carotid occlusion. Mice with chronic internal carotid artery occlusion exposed to NPs showed behavioral abnormalities, such as depression and anxiety, thus indicating detrimental effects of NPs on the brain. Subsequently, we used proteomics and metabolomics to analyze the specific mechanisms underlying the damaging effects of NP deposition in the brain. The findings helped explain the differences in the underlying biochemical responses at the microscopic level in mice after NP exposure. The NPs not only accumulated in the brain and caused pathologic damage, but also contributed to accelerating atherosclerosis in the mouse model of internal carotid artery occlusion. This work confirms the risk of NPs in a model of internal carotid artery occlusion and elucidates the mechanism underlying this harm; moreover, it provides theoretical support for developing strategies to decrease microplastic intake in patients with internal carotid artery occlusion.

Electroacupuncture inhibits neuronal pyroptosis in ischemic brain injury through modulating SIRT5-mediated NEK7 succinylation.

Ischemic stroke is a leading cause of global death. The treatment of this disease can inevitably result in reperfusion, thereby triggering cerebral ischemia-reperfusion injury (IRI) and neuronal pyroptosis. Electroacupuncture derived from traditional acupuncture has been proven to have favorable effects on ameliorating brain IRI and pyroptosis. Hence, the goal of the current research was to elucidate the mechanism governing electroacupuncture in cerebral IRI. We employed middle cerebral artery occlusion (MCAO) model to induce brain IRI. Our results revealed that electroacupuncture attenuated IRI in MCAO mice by minishing brain damage and hindering neuronal pyroptosis. Strikingly, it was discovered that electroacupuncture provoked the decrease of succinylation level and enhanced expression of SIRT5. Then, we demonstrated that knockdown of SIRT5 reversed the role of electroacupuncture in cerebral infarct injury and pyroptosis. In terms of mechanism, SIRT5 impeded the succinylation modification of NEK7 at K81 site to downregulate its expression level. Eventually, overexpression of NEK7 abrogated the impacts of electroacupuncture on MCAO mice. In conclusion, this study provided the compelling evidence that electroacupuncture restrained neuronal pyroptosis to mitigate ischemic brain damage via desuccinylating NEK7 in a SIRT5-dependent manner.

Danhong injection modulates microglial polarization and neuroinflammation via the JUNB/NF-κB pathway in ischemic stroke.

Ischemic stroke (IS) is a leading cause of death and disability in China. Danhong Injection (DHI) is a traditional Chinese medicine preparation made from Salvia miltiorrhiza var. miltiorrhiza and Carthamus tinctoriusL. It is used for treating stroke in China with proven safety and efficacy. Microglia M1/M2 polarization is a key factor in IS inflammatory response. However, the key transcription factors that regulate microglia polarization are unknown. It is also not clear how DHI exerts its mechanism in the treatment of IS. This research aimed to investigate the effect of DHI on microglial polarization and neuroinflammation associated with IS and to elucidate the underlying mechanisms, with an emphasis on the JUNB/NF-κB signaling pathway. An oxygen-glucose deprivation (OGD) damage cell model and a permanent middle cerebral artery occlusion (pMCAO) model in C57BL/6 mice were employed. Neurological deficits, cerebral infarct volume, and microglial activation were assessed. Non-targeted metabolomics analysis with UHPLC-QE-MS and molecular biology methods, including RT-qPCR and Western blot, were applied to investigate the mechanisms. In vivo, DHI decreased inflammation, reduced brain damage, and enhanced neurological function. DHI also ameliorated microglial activation and OGD-induced apoptosis in vitro. Metabolomics analysis identified significant metabolic changes, particularly in amino acid metabolism. Additionally, DHI treatment decreased the upregulated mRNA levels of ASS1 and ASL after OGD, indicating an influence on the arginine biosynthesis pathway, which is crucial for microglial function. DHI modulated the M1 to M2 phenotypes of microglial polarization and regulated microglial polarization through the JUNB/NF-κB signaling pathway. This was confirmed by JUNB silencing experiments. DHI exhibits neuroprotective effects via suppressing ASS1 through the JUNB/NF-κB pathway, promoting the M2 state of microglia, and lowering the expression of inflammatory cytokines. This research unveils the potential therapeutic target of JUNB for IS treatment and sheds light on the novel intervention mechanism of DHI in microglial cells.

Single-Cell Insights Into Cellular Response in Abdominal Aortic Occlusion-Induced Hippocampal Injury.

Ischemia-reperfusion of the abdominal aorta often results in damage to distant organs, such as the heart and brain. This cellular heterogeneity within affected tissues complicates the roles of specific cell subsets in abdominal aorta occlusion model (AAO) injury. However, cell type-specific molecular pathology in the hippocampus after ischemia is poorly understood. In this study, we adopted a mouse AAO to investigate the single-cell transcriptome in the hippocampi in AAO mice. Male C57BL/6 mice (8 weeks old) were used to create an AAO model, with animals divided into Sham and I/R groups. The I/R group was subjected to 2 h of ischemia followed by 24 h of reperfusion, after which hippocampal tissues were collected for single-cell RNA sequencing and histological analysis. Behavioral tests, including the Rotarod, Y-maze, and new object recognition tests, were performed daily for 28 days post-surgery to evaluate neurological function. A total of 62,624 cells were corresponding 7 cell types with neuronal, glial, and vascular lineages. We next analyzed cell-specific gene alterations in AAO mice and the function of these cell-specific Genes. AAO injury upregulated astrocyte and oligodendrocyte precursor cell (OPC) proportions (p-value < 0.05). Astrocytes showed unique gene expression related to neurogenesis and mRNA processing. Five distinct astrocyte subtypes emerged post-injury. OPCs exhibited enhanced synapse organization. Microglia activation and the elevated expression level of the epithelial cell oxidative phosphorylation protein-protein interaction (PPI) module indicate an inflammatory response and metabolic changes in response to AAO injury. Our scRNA-seq analysis provides insights into transcriptional changes at the single-cell level in response to AAO-induced hippocampal injury. This study illustrates how the hippocampal region responds to such injury and identifies potential therapeutic targets for intervention, thereby paving the way for future research and treatment strategies.

Zinc pretreatment for protection against intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (I/R) injury (II/RI) is a critical condition that results in oxidative stress, inflammation, and damage to multiple organs. Zinc, an essential trace element, offers protective benefits in several tissues during I/R injury, but its effects on intestinal II/RI remain unclear. To investigate the effects of zinc pretreatment on II/RI and associated multiorgan damage. C57BL/6 mice were pretreated with zinc sulfate (ZnSO4, 10 mg/kg) daily for three days before I/R injury was induced via superior mesenteric artery occlusion (SMAO) and abdominal aortic occlusion (AAO) models. Tissue and serum samples were collected to evaluate intestinal, liver, and kidney damage using Chiu's score, Suzuki score, and histopathological analysis. Caco-2 cells and intestinal organoids were used for in vitro hypoxia-reoxygenation injury models to measure reactive oxygen species (ROS) and superoxide dismutase (SOD) levels. Zinc pretreatment significantly reduced intestinal damage in the SMAO and AAO models (P < 0.001). The serum levels of liver enzymes (alanine aminotransferase, aspartate aminotransferase) and kidney markers (creatinine and urea) were lower in the zinc-treated mice than in the control mice, indicating reduced hepatic and renal injury. In vitro, zinc decreased ROS levels and increased SOD activity in Caco-2 cells subject to hypoxia-reoxygenation injury. Intestinal organoids pretreated with zinc exhibited enhanced resilience to hypoxic injury compared to controls. Zinc pretreatment mitigates II/RI and reduces associated multiorgan damage. These findings suggest that zinc has potential clinical applications in protecting against I/R injuries.

Inhibition of Circ0001679 Alleviates Ischemia/Reperfusion-induced Brain Injury via miR-216/TLR4 Regulatory Axis

Background: Stroke, primarily known as ischemic stroke, is a leading cause of mortality and disability worldwide. Reperfusion after the ischemia stroke resolves is necessary for maintaining the health of brain tissues; however, it also induces inflammation and oxidative stress, resulting in brain injury. This study aimed to investigate the role of circ0001679 in the pathology of I/R (Ischemia/Reperfusion)-induced brain injury and explore its therapeutic potential for I/R injury. Methods: The Oxygen-Glucose Deprivation/Re-oxygenation (OGD/R) model was employed in primary mouse astrocytes, and the Middle Cerebral Artery Occlusion (MCAO) model was established in mice to mimic ischemia-reperfusion-induced injury. Si-circ0001679, anti-miR- 216, and TLR4 ORF-clone were transfected either in cells or mice to study the molecular mechanisms during I/R-induced injury. Inflammation and oxidative stress were monitored after treatment. Results: Upregulated gene expression of circ0001679 was noticed in both OGD/R-treated primary mouse astrocytes and MCAO-induced mouse brain tissue. Silencing circ0001679 reduced cellular damage, inflammation, and oxidative stress induced by OGD/R treatment. Knocking down of circ0001679 alone with either miR-216 inhibition or TLR4 overexpression increased the inflammation response and oxidative stress compared to circ0001679 silencing only. Moreover, inhibition of circ0001679 attenuated brain injury in MCAO-treated mice via reduced infarction, neuronal damage, apoptosis, inflammation, and oxidative stress. Conclusion: This study unveiled a novel regulatory axis of circ0001679-miR-216-TLR4 in I/Rinduced brain injury. Targeting circ0001679 may represent a promising therapeutic strategy for I/R-induced brain injury. result: Upregulated gene expression of circ0001679 was noticed in both OGD/R-treated primary mouse astrocytes and MCAO-induced mouse brain tissue. Silencing circ0001679 reduced cellular damage, inflammation, and oxidative stress induced by OGD/R treatment. Knocking down of circ0001679 alone with either miR-216 inhibition or TLR4 overexpression increased the inflammation response and oxidative stress compared to circ0001679 silencing only. Moreover, inhibition of circ0001679 attenuated brain injury in MCAO-treated mice via reduced infarction, neuronal damage, apoptosis, inflammation, and oxidative stress.

Oxygen-glucose-deprived peripheral blood mononuclear cells act on hypoxic lesions after ischemia-reperfusion injury.

Despite advances in reperfusion therapies, ischemic stroke remains a major cause of long-term disability due to residual hypoxic lesions persisting after macrovascular reperfusion. These residual hypoxic lesions, caused by microvascular dysfunction, represent an important therapeutic target. We previously demonstrated that oxygen-glucose-deprived peripheral blood mononuclear cells (OGD-PBMCs) migrate to ischemic brain regions and promote functional recovery after stroke. This recovery occurs through mechanisms involving hypoxia-inducible factor-1α, exosomal miR-155-5p, and vascular endothelial growth factor (VEGF). However, it remains unclear whether OGD-PBMCs target hypoxic regions. We evaluated cerebral blood flow using a laser speckle flow imaging system. Next, we utilized pimonidazole to investigate the presence of hypoxic lesions after ischemia-reperfusion injury in a rat suture occlusion model in immunohistochemical analyses. We also compared levels of a cell surface receptor in human PBMCs by flow cytometric analysis under normoxic and OGD conditions. We found persistent pimonidazole-positive hypoxic lesions at 10- and 28-days post-reperfusion despite restored gross cerebral perfusion. Treatment with the C-X-C motif chemokine receptor 4 (CXCR4) inhibitor AMD3100 before and after OGD-PBMCs administration reduced the number of OGD-PBMCs in the brain parenchyma compared to the control group (P = 0.018). Administered OGD-PBMCs localized within these hypoxic regions via the stromal cell-derived factor-1/CXCR4 chemotactic axis. OGD-PBMCs enhanced VEGF expression, specifically within hypoxic lesions, compared to the phosphate-buffered saline group (P < 0.01). Furthermore, OGD-PBMCs reduced the number of pimonidazole-positive hypoxic cells in the ischemic core on 28 days. These findings demonstrate that OGD-PBMCs selectively migrate to and modulate the microenvironment of hypoxic lesions following cerebral ischemia-reperfusion injury. Targeting these residual hypoxic regions may underline the therapeutic effects of OGD-PBMC treatment and represent a promising strategy for improving stroke recovery despite successful recanalization.

Connectome-based prediction of functional impairment in experimental stroke models.

Experimental rat models of stroke and hemorrhage are important tools to investigate cerebrovascular disease pathophysiology mechanisms, yet how significant patterns of functional impairment induced in various models of stroke are related to changes in connectivity at the level of neuronal populations and mesoscopic parcellations of rat brains remain unresolved. To address this gap in knowledge, we employed two middle cerebral artery occlusion models and one intracerebral hemorrhage model with variant extent and location of neuronal dysfunction. Motor and spatial memory function was assessed and the level of hippocampal activation via Fos immunohistochemistry. Contribution of connectivity change to functional impairment was analyzed for connection similarities, graph distances and spatial distances as well as the importance of regions in terms of network architecture based on the neuroVIISAS rat connectome. We found that functional impairment correlated with not only the extent but also the locations of the injury among the models. In addition, via coactivation analysis in dynamic rat brain models, we found that lesioned regions led to stronger coactivations with motor function and spatial learning regions than with other unaffected regions of the connectome. Dynamic modeling with the weighted bilateral connectome detected changes in signal propagation in the remote hippocampus in all 3 stroke types, predicting the extent of hippocampal hypoactivation and impairment in spatial learning and memory function. Our study provides a comprehensive analytical framework in predictive identification of remote regions not directly altered by stroke events and their functional implication.

Wilms' tumor 1-associated protein aggravates ischemic stroke by promoting M1 polarization of microglia by enhancing PTGS2 mRNA stability in an m6A-dependent manner.

Mounting evidence indicates the involvement of N6-methyladenosine (m6A) alterations in diverse neurological disorders and the activation of microglia. However, the role of m6A methyltransferase Wilms' tumor 1-associated protein (WTAP) in regulating microglial polarization during ischemic stroke (IS) remains unknown. We performed bioinformatics analysis to identify m6A-related differentially expressed genes in IS and validated these genes in a mouse middle cerebral artery occlusion model and a BV2 cell oxygen-glucose deprivation/reperfusion model. We found that microglial m6A modification was increased, and that WTAP was the most significantly differentially expressed m6A regulator during IS. High expression of WTAP is closely correlated with microglia-mediated neuroinflammation in IS. Mechanistically, WTAP promoted m6A modification, which promoted prostaglandin endoperoxide synthase-2 (PTGS2) by enhancing its mRNA stability. WTAP promoted M1 microglial polarization by elevating PTGS2 expression via m6A modification of PTGS2 mRNA in the oxygen-glucose deprivation/reperfusion model. In conclusion, WTAP is a crucial posttranscriptional regulator that contributes to post-IS neuroinflammation. WTAP knockdown confers cerebral protection by shifting the microglial phenotype from M1 to M2, primarily by reducing PTGS2 mRNA stability in an m6A-dependent manner.

The Phenotype Changes of Astrocyte During Different Ischemia Conditions.

Dementia is becoming a major health problem in the world, and chronic brain ischemia is an established important risk factor in predisposing this disease. Astrocytes, as one major part of the blood-brain barrier (BBB), are activated during chronic cerebral blood flow hypoperfusion. Reactive astrocytes have been classified into phenotype pro-inflammatory type A1 or neuroprotective type A2. However, the specific subtype change of astrocyte and the mechanisms of chronic brain ischemia are still unknown. In order to depict the phenotype changes and their possible roles during this process, a rat bilateral common carotid artery occlusion model (BCAO) was employed in the present study. Meanwhile, the signaling pathways that possibly regulate these changes were investigated as well. After four-week occlusion, astrocytes in the cortex of BCAO rats were shown to be the A2 phenotype, identified by the significant up-regulation of S100a10 accompanied by the down-regulation of Connexin 43 (CX43) protein. Next, we established in vitro hypoxia models, which were set up by stimulating primary astrocyte cultures from rat cortex with cobalt chloride, low glucose, or/and fibrinogen. Consistent with in vivo data, the cultured astrocytes also transformed into the A2 phenotype with the up-regulation of S100a10 and the down-regulation of CX43. In order to explore the mechanism of CX43 protein changes, C6 astrocyte cells were handled in both hypoxia and low-glucose stimulus, in which decreased pERK and pJNK expression were found. In conclusion, our data suggest that in chronic cerebral ischemia conditions, the gradual ischemic insults could promote the transformation of astrocytes into A2 type instead of A1 type, and the phosphorylation of CX43 was negatively regulated by the phosphorylation of ERK and JNK. Also, our data could provide some new evidence of how to leverage the endogenous astrocytes phenotype changes during CNS injury by promoting them to be "protector" and not "culprit".

ACTH-like Peptides Compensate Rat Brain Gene Expression Profile Disrupted by Ischemia a Day After Experimental Stroke.

Background: Ischemic stroke results from a disruption of cerebral blood flow. Adrenocorticotropic hormone (ACTH) serves as the basis for the creation of synthetic peptides as neuroprotective agents for stroke therapy. Previously, using RNA-Seq we first revealed differential expressed genes (DEGs) associated with ACTH(4-7)PGP (Semax) and ACTH(6-9)PGP peptides under cerebral ischemia conditions. Analysis was carried out at 4.5 h after transient middle cerebral artery occlusion (tMCAO) model in the ipsilateral frontal cortex of a rat brain. Methods: Here, we analyzed the penumbra-associated frontal cortex of rats and actions under the same peptides at 24 h after tMCAO using RNA-Seq. Results: 3774 DEGs (fold change > 1.5 and Padj < 0.05) were identified under ischemia conditions, whereas 1539 and 2066 DEGs were revealed under Semax and ACTH(6-9)PGP peptides at 24 h after tMCAO. Furthermore, both peptides significantly reduced expression distortions caused by ischemia for 1171 genes associated with immune and neurosignaling pathways. Concomitantly, there were 32 DEGs under ACTH(6-9)PGP versus Semax administration at 24 h after tMCAO. Besides, neurogenesis-, angiogenesis-, protein kinase- and growth factor-related DEGs were revealed under peptides action. Previously, we observed the neuroprotective effect of peptides at the histological level in rat brains at 24 h after tMCAO. Thus, here we demonstrate the transcriptome manifestation of this histological effect. Furthermore, comparison with previous data at the 4.5 h post-tMCAO time point showed that the pattern of peptide action on the transcriptome depends on the time elapsed after tMCAO. Conclusions: We revealed that the effect of ACTH(6-9)PGP was more similar to Semax than different from it a day after tMCAO. At this time point, ACTH-like peptides compensated rat brain gene expression profiles disrupted by ischemia. Thus, our results may be useful for selecting more effective structures for future anti-stroke drugs and appropriate post-stroke time points for their testing.

Exosomes derived from umbilical cord mesenchymal stem cells ameliorate ischemic brain injury in mice by regulating AAK1 via miR-664a-5p

Objective To identify the molecular targets of MSC-derived exosomes in treating cerebral ischemia and elucidate their therapeutic mechanisms. Methods We utilized a mouse model of middle cerebral artery occlusion (MCAO) and treated mice with umbilical cord mesenchymal stem cells (uc-MSCs) derived exosomes. Proteomic analysis identified AAK1(AP2 associated kinase 1) as a key target protein. Functional studies confirmed that AAK1 modulates the NF-κB signaling pathway in ischemic stroke. MicroRNA profiling, bioinformatic prediction, and cell experiments identified miR-664a-5p as the specific microRNA regulating AAK1 expression. Finally, we validated the therapeutic effects of uc-MSCs-derived exosomes using engineered miR-664a-5p-deficient exosomes. Results Our findings demonstrate that uc-MSCs-derived exosomes exert neuroprotective effects in ischemic stroke by modulating the AAK1/NF-κB axis via miR-664a-5p. Conclusion This study provides novel insights into the therapeutic mechanism of MSC-derived exosomes in ischemic stroke, highlighting their potential for developing exosome-based therapies.

Early and concomitant administration of norepinephrine and ilomedin improves microcirculatory perfusion without impairing macrocirculation in an intestinal ischemia-reperfusion injury swine model : a randomized experimental trial.

Intestinal ischemia-reperfusion injury is associated with both macrocirculatory and microcirculatory failure. Association of a vasoconstrictor in combination with a vasodilator such as ilomedin may improve macrocirculation parameters, microcirculation perfusion and reduce endothelial dysfunction. The primary objective was to demonstrate a difference in mean arterial pressure (MAP) after intestinal reperfusion with the concomitant administration of norepinephrine and ilomedin during ischemia compared with traditional hemodynamic treatment strategies (fluid resuscitation and vasopressors only). Secondary objectives were to demonstrate an improvement in peripheral and intestinal microcirculatory perfusion and endothelial dysfunction after intestinal reperfusion using this association. We conducted a randomized preclinical trial in twenty-one large white pigs, in which a 2-hour small bowel ischemia was performed using a segmental mesenteric occlusion model, followed by a 2-hour reperfusion. Pigs were randomized into 3 groups: goal directed fluid therapy, early administration of norepinephrine before reperfusion and early administration of ilomedin and norepinephrine before reperfusion. Macrocirculatory (MAP and Cardiac Index (CI)), microcirculatory (Sublingual with SideStream Dark Field system and intestinal hemoglobin oxygen saturation with hyperspectral imaging (HSI)) measurements and biological analysis (biomarkers of endothelial dysfunction) were performed. There were no significant differences in the MAP (p = 0.499) and the CI (p = 0.659) between the 3 groups. Perfused Vessel Density (PVD) in sublingual microcirculation was significantly higher immediately after reperfusion and 2 hours after reperfusion in the early administration of ilomedin and norepinephrine group compared with the other 2 groups (p < 0.05). Hemoglobin oxygen saturation measured at the intestinal level was significantly higher immediately after reperfusion in the early administration of ilomedin and norepinephrine group compared with the other 2 groups (p < 0.01). There were no significant differences in biomarkers of endothelial dysfunction between the 3 groups. Creatinine, AST and alkaline phosphatases increased significantly 2 hours after reperfusion in the early administration of ilomedin and norepinephrine group compared with baseline (p < 0.05). Early administration of norepinephrine and ilomedin during ischemia improved short-term post-reperfusion sublingual and intestinal microcirculation without worsening macrocirculatory parameters in an intestinal ischemia-reperfusion injury model. However, use of this strategy seemed to worsen both liver and kidney function.