Reduced Infarct Volume Research Articles

Green synthesis, chemical characterization, and protective potentials of silver nanoparticles on the development of cerebrovascular diseases in rat cerebral ischemia reperfusion injury

Thrombolysis or mechanical recanalization are two methods that can be used to restore blood flow after an ischemic stroke. However, it is important to note that in certain situations, reperfusion can actually exacerbate the initial damage caused by ischemia, leading to a condition known as “cerebral reperfusion injury.” This injury involves several pathological processes, including the infiltration of leukocytes, activation of platelets and complement, postischemic hyperperfusion, and disruption of the blood–brain barrier. The main objective of research conducted globally is to create a novel therapeutic medication for stroke. In light of the nano revolution, it is imperative to merge nanoscience and medicine. Knowledge acquired from nature has paved the path for the development of biogenic technologies, facilitating the production of cutting-edge nanomaterials. We have recently conducted a study to evaluate the neuroprotective properties of a blend of Mellisa officinalis leaf extract and green-mediated silver nanoparticles on the development of cerebrovascular diseases in rat cerebral ischemia reperfusion injury. The silver nanoparticles were thoroughly examined using UV–Vis, FE-SEM, and EDX analyses to ensure comprehensive characterization. The Ag NPs were observed to have various polymorphic shapes and were found to be aggregated. Also, the particles average size was measured by ImageJ software and it is about 29.15 nm. The UV–Vis measurements showed that the spectra displayed the highest absorption at around 432 nm. The stroke occurred as a result of the occlusion of the middle cerebral artery for 1.5 h, followed by reperfusion for 2 days. Before the ischemia reperfusion, the rats received an intravenous injection of AgNPs at doses of 40, 80 and 160 µg/kg once daily for a consecutive period of 3 days. The evaluation of the cell damage index involved assessing various factors such as immunoglobulin G (IgG) extravasation into the cerebral parenchyma, lactate dehydrogenase efficacy, serum MDA level, infarct volume, and learning and memory function. The results of the study showed that pretreatment with AgNPs had a protective effect on stroke in animals. Moreover, AgNPs effectively alleviated the memory and learning impairments caused by stroke. Additionally, AgNPs significantly reduced infarct volume, IgG extravasation, serum MDA level, and lactate dehydrogenase efficacy.

Electroacupuncture regulates histone acetylation of Bcl-2 and Caspase-3 genes to improve ischemic stroke injury

BackgroundImbalances between Bcl-2 and caspase-3 are significant evidence of apoptosis, which is considered an influential factor in rapidly occurring neuronal cell death and the decline of neurological function after stroke. Studies have shown that acupuncture can reduce poststroke brain cell damage via either an increase in Bcl-2 or a reduction in caspase-3 exposure. The current study aimed to investigate whether acupuncture could modulate Bcl-2 and caspase-3 expression through histone acetylation modifications, which could potentially serve as a neuroprotective mechanism. MethodsThis study used TTC staining, Nissl staining, Clark neurological system score, and Evans Blue (EB) extravasation to evaluate neurological damage following stroke. The expression of Bcl-2/caspase-3 mRNA was detected by real-time fluorescence quantification of PCR (real-time PCR), whereas the protein expression levels of Bcl-2, Bax, caspase-3, and cleaved caspase-3 were assessed using western blotting. TUNEL staining of the ischemic cortical neurons determined apoptosis in the ischemic cortex. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, along with the protein performance of AceH3, H3K9ace, and H3K27ace, were detected to evaluate the degree of histone acetylation. The acetylation enrichment levels of H3K9 and K3K27 in the Bcl-2/caspase-3 gene were assessed using Chromatin Immunoprecipitation (ChIP) assay. ResultsOur data demonstrated that electroacupuncture (EA) exerts a significant neuroprotective effect in middle cerebral artery occlusion (MCAO) rats, as evidenced by a reduction in infarct volume, neuronal damage, Blood-Brain Barrier (BBB) disruption, and decreased apoptosis of ischemic cortical neurons. EA treatment can promote the mRNA and protein expression of the Bcl-2 gene in the ischemic brain while reducing the mRNA and protein expression levels of caspase-3 and effectively decreasing the protein expression levels of Bax and cleaved caspase-3. More importantly, EA treatment enhanced the level of histone acetylation, including Ace-H3, H3K9ace, and H3K27ace, significantly enhanced the occupancy of H3K9ace/H3K27ace at the Bcl-2 promoter, and reduced the enrichment of H3K9ace and H3K27ace at the caspase-3 promoter. However, the Histone Acetyltransferase inhibitor (HATi) treatment reversed these effects. ConclusionsOur data demonstrated that EA mediated the expression levels of Bcl-2 and caspase-3 in MCAO rats by regulating the occupancy of acetylated H3K9/H3K27 at the promoters of these two genes, thus exerting a cerebral protective effect in ischemic reperfusion (I/R) injury.

Potensi Citicoline sebagai Terapi Neuroproteksi dan Neuroregenerasi pada Stroke Iskemik Akut

Ischemic stroke, caused by a blockage in the brain’s blood vessels, is a critical emergency that requires immediate treatment. This study examined the efficacy of citicoline as an adjunct therapy to improve outcomes in acute ischemic stroke. A comprehensive search of four databases (Cochrane Library, ScienceDirect, PubMed, and Google Scholar) was conducted, identifying a total of 5265 articles. Three clinical trials were selected for critical appraisal. Agarwal demonstrated a larger reduction in stroke infarct volume in the citicoline group (4.22 cm3) compared to the placebo group (2.63 cm3) (p=0.483). On day 90, the citicoline group exhibited superior results in mRS scores of 0-2 (p=0.732), NIHSS scores of 0-2 (p=0.934), Barthel index ≥95 (p=0.564), and patient mortality (p=0.468). Seifaddini’s study revealed improved peak systolic velocity and mean arterial flow velocity in the cerebrovascular system. Enrico’s study showed improved mean short afferent latency inhibition in the cholinergic circuits of the citicoline group (p=0.004). Citicoline proves effective as an adjuvant neuroprotective and neuroregenerative therapy for acute ischemic stroke.

Inhibition of leukocyte migration after ischemic stroke by VE-cadherin mutation in a mouse model leads to reduced infarct volumes and improved motor skills.

To distinguish between the genuine cellular impact of the ischemic cascade by leukocytes and unspecific effects of edema and humoral components, two knock-in mouse lines were utilized. Mouse lines Y731F and Y685F possess point mutations in VE-cadherin, which lead to a selective inhibition of transendothelial leukocyte migration or impaired vascular permeability. Ischemic stroke was induced by a model of middle cerebral artery occlusion. Analysis contained structural outcomes (infarct volume and extent of brain edema), functional outcomes (survival analysis, rotarod test, and neuroscore), and the extent and spatial distribution of leukocyte migration (heatmaps and fluorescence-activated cell sorting (FACS) analysis). Inhibition of transendothelial leukocyte migration as in Y731F mice leads to smaller infarct volumes (52.33±4719 vs. 70.43±6483mm3 , p=.0252) and improved motor skills (rotarod test: 85.52±13.24s vs. 43.06±15.32s, p=.0285). An impaired vascular permeability as in Y685F mice showed no effect on structural or functional outcomes. Both VE-cadherin mutations did not influence the total immune cell count or spatial distribution in ischemic brain parenchyma. Selective inhibition of transendothelial leukocyte migration by VE-cadherin mutation after ischemic stroke in a mouse model leads to smaller infarct volumes and improved motor skills.

FDA compound library screening Baicalin upregulates TREM2 for the treatment of cerebral ischemia-reperfusion injury

Acute ischemic stroke (AIS) is a leading cause of global incidence and mortality rates. Oxidative stress and inflammation are key factors in the pathogenesis of AIS neuroinjury. Therefore, it is necessary to develop drugs that target neuroinflammation and oxidative stress in AIS. The Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), primarily expressed on microglial cell membranes, plays a critical role in reducing inflammation and oxidative stress in AIS. In this study, we employed a high-throughput screening (HTS) strategy to evaluate 2625 compounds from the (Food and Drug Administration) FDA library in vitro to identify compounds that upregulate the TREM2 receptor on microglia. Through this screening, we identified Baicalin as a potential drug for AIS treatment. Baicalin, a flavonoid compound extracted and isolated from the root of Scutellaria baicalensis, demonstrated promising results. Next, we established an in vivo mouse model of cerebral ischemia-reperfusion injury (MCAO/R) and an in vitro microglia cell of oxygen-glucose deprivation reperfusion (OGD/R) to investigate the role of Baicalin in inflammation injury, oxidative stress, and neuronal apoptosis. Our results showed that baicalin effectively inhibited microglia activation, reactive oxygen species (ROS) production, and inflammatory responses in vitro. Additionally, baicalin suppressed neuronal cell apoptosis. In the in vivo experiments, baicalin not only improved neurological functional deficits and reduced infarct volume but also inhibited microglia activation and inflammatory responses. Overall, our findings demonstrate the efficacy of Baicalin in treating MCAO/R by upregulating TREM2 to reduce inflammatory responses and inhibit neuronal apoptosis.

Blood-Brain Barrier-Penetrating and Lesion-Targeting Nanoplatforms Inspired by the Pathophysiological Features for Synergistic Ischemic Stroke Therapy.

Ischemic stroke is a dreadful vascular disorder that poses enormous threats to the public health. Due to its complicated pathophysiological features, current treatment options after ischemic stroke attack remains unsatisfactory. Insufficient drug delivery to ischemic lesions impeded by the blood-brain barrier (BBB) largely limits the therapeutic efficacy of most anti-stroke agents. Herein, inspired by the rapid BBB penetrability of 4T1 tumor cells upon their brain metastasis and natural roles of platelet in targeting injured vasculatures, a bio-derived nanojacket is developed by fusing 4T1 tumor cell membrane with platelet membrane, which further clothes on the surface of paeonol and polymetformin-loaded liposome to obtain biomimetic nanoplatforms (PP@PCL) for ischemic stroke treatment. The designed PP@PCL could remarkably alleviate ischemia-reperfusion injury by efficiently targeting ischemic lesion, preventing neuroinflammation, scavenging excess reactive oxygen species (ROS), reprogramming microglia phenotypes, and promoting angiogenesis due to the synergistic therapeutic mechanisms that anchor the pathophysiological characteristics of ischemic stroke. As a result, PP@PCL exerts desirable therapeutic efficacy in injured PC12 neuronal cells and rat model of ischemic stroke, which significantly attenuates neuronal apoptosis, reduces infarct volume, and recovers neurological functions, bringing new insights into exploiting promising treatment strategies for cerebral ischemic stroke management.

Dl-3-n-butylphthalide attenuates cerebral ischemia/reperfusion injury in mice through AMPK-mediated mitochondrial fusion.

Introduction: NBP is a compound isolated from celery seeds, which was approved by the National Medical Products Administration in 2002 for clinical treatment of ischemic stroke. However, in brain ischemia/reperfusion (I/R) injury, the related research on mitochondrial dynamics and its mechanism of action of NBP still need to be further studied. The aim of this study was to assess NBP on cerebral pathology in ischemic stroke in vivo, with a specific focus on the molecular mechanisms of how NBP promotes mitochondrial fusion. Methods: Male C57BL/6 mice were utilized in this study and were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Pre-ischemia, NBP was administered through intraperitoneal (i.p.) injection for 7days. Results: Our findings demonstrated that NBP effectively reduced infarct volume, improved neurological dysfunction, enhanced cerebral blood flow, and promoted mitochondrial fusion in mice subjected to MCAO/R. More importantly, the pro-fusion effects of NBP were found to be linked to the activation of AMPK/Mfn1 pathway, and with the activation of neurological function, which was partially eliminated by inhibitors of AMPK. Discussion: Our results revealed that NBP is a novel mitochondrial fusion promoter in protecting against ischemic stroke through the AMPK-mediated Mfn1. These findings contribute to the understanding of novel mechanisms involved in the protection of neurological function following NBP treatment for ischemic stroke.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid during reperfusion promotes multifaceted brain and vascular protection in spontaneously hypertensive rats with transient ischaemic stroke

Hypertension is the most prevalent modifiable risk factor for stroke and is associated with worse functional outcomes. Pharmacological inhibition of histone deacetylases by suberoylanilide hydroxamic acid (SAHA) modulates gene expression and has emerged as a promising therapeutic approach to reduce ischaemic brain injury. Here, we have tested the therapeutic potential of SAHA administered during reperfusion in adult male spontaneously hypertensive (SHR) rats subjected to transient middle cerebral artery occlusion (tMCAO; 90 min occlusion/24 h reperfusion). Animals received a single dose of SAHA (50 mg/kg) or vehicle i.p. at 1, 4, or 6 h after reperfusion onset. The time-course of brain histone H3 acetylation was studied. After tMCAO, drug brain penetrance and beneficial effects on behavioural outcomes, infarct volume, oedema, angiogenesis, blood-brain barrier integrity, cerebral artery oxidative stress and remodelling, and brain and vascular inflammation were evaluated. SAHA increased brain histone H3 acetylation from 1 to 6 h after injection, reaching the ischaemic brain administered during reperfusion. Treatment given at 4 h after reperfusion onset improved neurological score, reduced infarct volume and oedema, attenuated microglial activation, prevented exacerbated MCA angiogenic sprouting and blood-brain barrier breakdown, normalised MCA oxidative stress and remodelling, and modulated brain and cerebrovascular cytokine expression. Overall, we demonstrate that SAHA administered during early reperfusion exerts robust brain and vascular protection after tMCAO in hypertensive rats. These findings are aligned with previous research in ischaemic normotensive mice and help pave the way to optimise the design of clinical trials assessing the effectiveness and safety of SAHA in ischaemic stroke.

Neuroprotective effects of galectin‑1 on cerebral ischemia/reperfusion injury by regulating oxidative stress.

Oxidative stress contributes to the pathology of cerebral ischemia/reperfusion (I/R) injury. Galectin-1 has shown an anti-oxidative stress effect. The present study investigated whether this anti-oxidative stress effect can account for the neuroprotective actions of galectin-1 induced by cerebral I/R injury. A cerebral I/R injury model was created in C57Bl/6 mice by transient occlusion of the middle cerebral artery, after which the mice were treated with galectin-1 for 3 days. Infarct volumes were measured. A rotarod test and neurological deficit score assessment was performed to evaluate the neurological deficits. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and lipid peroxidation malondialdehyde (MDA), while the anti-oxidative stress status was assessed by measuring molecules such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidation enzyme (GSH-Px) in the ischemic cerebral hemisphere of mice. The inflammatory cytokines, including Interleukin 1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α) were measured, and the expression of microglia was evaluated by immunohistochemistry in the ischemic cerebral hemisphere of mice. Galectin-1 treatment ameliorated neurological deficits and reduced infarct volumes in the mice model with cerebral I/R injury. Moreover, it was demonstrated that galectin-1 can significantly alleviate cerebral I/R injury in the ischemic cerebral hemisphere by decreasing the production of ROS and MDA, but increasing the production of CAT, SOD and GSH-Px. Galectin-1 treatment decreased microglia expression, and IL-1, IL-6 and TNF-α levels in the ischemic cerebral hemisphere of mice. Galectin-1 could improve the outcome of cerebral I/R injury by alleviating oxidative stress. Moreover, the neuroprotective effect of galectin-1 in cerebral ischemia could be related to its anti-oxidative stress effect.

Systematic review of melatonin in cerebral ischemia-reperfusion injury: critical role and therapeutic opportunities.

Cerebral ischemia-reperfusion (I/R) injury is the predominant causes for the poor prognosis of ischemic stroke patients after reperfusion therapy. Currently, potent therapeutic interventions for cerebral I/R injury are still very limited. Melatonin, an endogenous hormone, was found to be valid in preventing I/R injury in a variety of organs. However, a systematic review covering all neuroprotective effects of melatonin in cerebral I/R injury has not been reported yet. Thus, we perform a comprehensive overview of the influence of melatonin on cerebral I/R injury by collecting all available literature exploring the latent effect of melatonin on cerebral I/R injury as well as ischemic stroke. In this systematic review, we outline the extensive scientific studies and summarize the beneficial functions of melatonin, including reducing infarct volume, decreasing brain edema, improving neurological functions and attenuating blood-brain barrier breakdown, as well as its key protective mechanisms on almost every aspect of cerebral I/R injury, including inhibiting oxidative stress, neuroinflammation, apoptosis, excessive autophagy, glutamate excitotoxicity and mitochondrial dysfunction. Subsequently, we also review the predictive and therapeutic implications of melatonin on ischemic stroke reported in clinical studies. We hope that our systematic review can provide the most comprehensive introduction of current advancements on melatonin in cerebral I/R injury and new insights into personalized diagnosis and treatment of ischemic stroke.

Upregulation of TRPC1 in microglia promotes neutrophil infiltration after ischemic stroke

Neutrophil infiltration has been linked to worse clinical outcomes after ischemic stroke. Microglia, a key type of immune-competent cell, engage in cross-talk with the infiltrating immune cells in the inflamed brain area, yet the molecular mechanisms involved remain largely unexplored. In this study, we investigated the mechanisms of how canonical transient receptor potential 1 (TRPC1) modulated neutrophil infiltration in male mouse cerebral ischemia and reperfusion injury (CIRI) models. Our findings revealed a notable upregulation of TRPC1 in microglia within both middle cerebral artery occlusion reperfusion (MCAO/R) and in vitro oxygen-glucose deprivation/regeneration (OGD/R) model. Conditional Trpc1 knockdown in microglia markedly reduced infarct volumes and alleviated neurological deficits. Microglia conditional Trpc1 knockdown mice displayed less neutrophil infiltration in peri-infarct area. Trpc1 knockdown microglia exhibited a reduced primed proinflammatory phenotype with less secretion of CC-Chemokines ligand (CCL) 5 and CCL2 after MCAO/R. Blocking CCL5/2 significantly mitigated neutrophil infiltration in microglia/neutrophil transwell co-culture system upon OGD/R condition. Trpc1 knockdown markedly reduced store-operated calcium entry and nuclear factor of activated T-cells c1 (NFATc1) level in OGD/R treated microglia. Overexpression of Nfatc1 reversed the CCL5/2 reducing effect of Trpc1 knockdown, which is mediated by small interfering RNA in BV2 cells upon OGD/R. Our data indicate that upregulation of TRPC1 in microglia stimulates the production of CCL5/2 through the Ca2+/NFATc1 pathway. Upregulated CCL5/2 leads to an increase in neutrophil infiltration into the brain, thereby aggravating reperfusion injury. Our results demonstrate the importance of TRPC1 in microglia-mediated neuroinflammation and suggest a potential means for reducing CIRI induced neurological injury.

GPR55 Inactivation Diminishes Splenic Responses and Improves Neurological Outcomes in the Mouse Ischemia/Reperfusion Stroke Model.

Although strokes are frequent and severe, treatment options are scarce. Plasminogen activators, the only FDA-approved agents for clot treatment (tissue plasminogen activators (tPAs)), are used in a limited patient group. Moreover, there are few approaches for handling the brain's inflammatory reactions to a stroke. The orphan G protein-coupled receptor 55 (GPR55)'s connection to inflammatory processes has been recently reported; however, its role in stroke remains to be discovered. Post-stroke neuroinflammation involves the central nervous system (CNS)'s resident microglia activation and the infiltration of leukocytes from circulation into the brain. Additionally, splenic responses have been shown to be detrimental to stroke recovery. While lymphocytes enter the brain in small numbers, they regularly emerge as a very influential leukocyte subset that causes secondary inflammatory cerebral damage. However, an understanding of how this limited lymphocyte presence profoundly impacts stroke outcomes remains largely unclear. In this study, a mouse model for transient middle cerebral artery occlusion (tMCAO) was used to mimic ischemia followed by a reperfusion (IS/R) stroke. GPR55 inactivation, with a potent GPR55-specific antagonist, ML-193, starting 6 h after tMCAO or the absence of the GPR55 in mice (GPR55 knock out (GPR55ko)) resulted in a reduced infarction volume, improved neurological outcomes, and decreased splenic responses. The inhibition of GPR55 with ML-193 diminished CD4+T-cell spleen egress and attenuated CD4+T-cell brain infiltration. Additionally, ML-193 treatment resulted in an augmented number of regulatory T cells (Tregs) in the brain post-tMCAO. Our report offers documentation and the functional evaluation of GPR55 in the brain-spleen axis and lays the foundation for refining therapeutics for patients after ischemic attacks.

In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel

Background Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. Methods We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. Results In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. Conclusion M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Abstract TP199: Prominent Laterality of the Posterior Cerebral Artery on MR Angiography Before Mechanical Thrombectomy in Patients With the Middle Cerebral Artery Occlusion Predicts Long-Term Functional Outcome

Background and Purpose: Well-developed collateral flow is reported to be associated with a reduction of infarct volume, successful reperfusion, and better functional outcome in acute ischemic stroke (AIS). Relatively increased visualization of posterior cerebral artery (PCA) ipsilateral to the ischemic lesion on MR angiography (MRA), a sign of prominent PCA laterality (PCAL), represents the development of collateral flow from PCA to middle cerebral artery (MCA) territory. However, the prognostic value of PCAL on functional outcome in AIS patients having MCA occlusion and undergoing mechanical thrombectomy, is not well known. Methods: We retrospectively reviewed initial MRA scans of pre-morbidly independent patients treated with mechanical thrombectomy for M1-segment occlusion from September 2017 to May 2022. PCAL was defined as better visualization of ipsilateral PCA, compared to contralateral PCA, by one or more segments on MRA. We divided patients into two groups, those presenting PCAL and not, and compared the background characteristics, in-hospital management, and functional outcome between the two groups. Factors associated with favorable outcome (mRS score 0-2) at 3 months after onset were assessed using multivariate logistic regression analysis. Results: In total, 172 patients (76 female [44%], median age 77 [IQR 74.5-78.0] years, median NIHSS score 16 [15-18]) were included. Of these, 86 patients (50%) presented PCAL on initial MRA. There is no significant difference between the two groups in age, sex, major vascular risk factors, NIHSS on admission, DWI-ASPECTS, onset-to-recanalization time (OTR), and preclinical modified Rankin scale. Multivariate logistic regression analysis showed an adjusted odds ratio of 2.11 for a favorable outcome in patients with PCAL (95% CI: 0.986 to 4.53, P=0.054). When focused on the cases with OTR >6 hours (n=53), PCAL was independently associated with favorable outcome (OR 5.77, 95% CI: 1.28 to 26.0, P=0.022). Conclusion: PCAL on initial MRA in patients with M1-segment occlusion predicts favorable long-term functional outcome after mechanical thrombectomy, especially in the case taking longer time from onset to recanalization.

Abstract TP301: Tmem30a - Mediated Mitigation of Phosphatidylserine Exposure May Be a Novel Therapeutic Target for Brain Protection in Ischemic Stroke

Cellular pathways of self-preservation are highly conserved and may represent powerful ways to protect tissue against injury and disease. Here, we use a combination of cell, mouse, rat, nonhuman primate, and human models to demonstrate the feasibility of evoking endogenous self-preservation pathways to protect brain tissue in stroke. The transmembrane protein 30A ( Tmem30a ), the β-subunit of P4-ATPases, helps maintain the asymmetric distribution of phospholipids in plasma membrane, especially phosphatidylserine (PS) in the inner leaflet. Tmem30a transcripts were increased in the penumbra in two types of animal models of stroke. The increased Tmem30a transcripts were associated with less PS exposure and neuron death in vitro and improved neurological outcomes and extended therapeutic window in vivo. Mechanistically, Tmem30a upregulation led to decreased levels of apoptosis-related markers. Moreover, we found that recombinant Annexin V protein can decrease PS exposure, reduce infarct volumes, and improve neurological impairments in wild mice after ischemia/reperfusion, but not in Tmem30a knockout mice and those receiving siRNA- Tmem30a . Lastly, we also observed that high Annexin V levels were associated with better outcomes in stroke patients with salvageable penumbra, but not in those without penumbral tissues. Overall, our findings reveal a previously unappreciated role of maintaining asymmetric distribution of phospholipids in protecting the penumbra in the context of clinically relevant reperfusion. Tmem30a is essential for PS-blocking dependent neuroprotection and might represent a potential new strategy for evoking endogenous self-preservation pathways to protect brain tissue after stroke.

Abstract WMP119: CD13 Facilitates Immune Cell Migration and Aggravates Acute Injury but Promotes Chronic Post-Stroke Recovery

Introduction: Acute stroke leads to the activation of myeloid cells. Activated myeloid cells express CD13, which facilitates their migration into the site of injury. Chronically after stroke, repair processes, including angiogenesis, are activated and enhance post-stroke recovery. However, angiogenic blood vessels which play a role in recovery also express CD13. Overall, the specific contribution of CD13 to acute and chronic stroke outcomes is unknown. Methods: CD13 expression was estimated in mice and humans after the ischemic stroke. Young (8-12 weeks) male wild-type (WT) and global CD13 knockout (KO) mice were used for this study. Mice underwent 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. For acute studies, the mice were euthanized at either 24- or 72 hours post-stroke. For chronic studies, the Y maze, Barnes maze, and the open field were performed on day 7 and day 28 post-stroke. Mice were euthanized at day 30 post-stroke and the brains were collected for assessment of inflammation, white matter injury, tissue loss, and angiogenesis. Flow cytometry was performed on days 3 and 7 post-stroke to quantify infiltrated monocytes and neutrophils and CXCL12/CXCR4 signaling. Results: Brain CD13 expression and infiltrated CD13 + monocytes and neutrophils increased acutely after the stroke. The brain CD13 + lectin + blood vessels increased on day 15 after the stroke. Similarly, an increase in the percentage area CD13 was observed in human stroke patients at the subacute time after stroke. Deletion of CD13 resulted in reduced infarct volume and improved neurological recovery after acute stroke. However, CD13KO mice had significantly worse memory deficits, amplified gliosis, and white matter damage compared to WT animals at chronic time points. CD13KO mice had an increased percentage of CXCL12 + cells but a reduced percentage of CXCR4 + cells and decreased angiogenesis at day 30 post-stroke. Conclusions: CD13 is involved in the transmigration of monocytes and neutrophils after stroke, and acutely, led to decreased infarct size and improved behavioral outcomes. However, loss of CD13 led to reductions in post-stroke angiogenesis by reducing CXCL12/CXCR4 signaling.

Abstract WMP111: Intra-Arterial Administration of Cerebral Endothelial Cell Derived Small Extracellular Vesicles Reduces Neurovascular Damage and Improves Neurological Outcome After Acute Ischemia in the Aged Rat

Stroke is a leading cause of death and disability worldwide, mainly affecting the elderly. Endovascular thrombectomy (EVT) is a standard of care therapy for stroke patients with large-vessel occlusion, however, the majority of these patients do not achieve full functional improvement. The present study tested the hypothesis that intra-arterial (IA) administration of cerebral endothelial cell derived small extracellular vesicles (CEC-sEVs) following transient middle cerebral artery occlusion (tMCAO) improves stroke outcome in the aged rat. CEC-sEVs (1x10 11 particles/injection) isolated from healthy adult rats were employed. Male aged rats (18 months, n=10/group) subjected to 3h tMCAO were randomly divided into the experimental groups. To examine whether IA injection of CEC-sEVs improves stroke outcome, CEC-sEVs were administered via the internal carotid artery (ICA) immediately following reperfusion by withdrawal of the occlusion filament. To examine whether intravenous administration (IV) of CEC-sEVs during tMCAO and then followed by IA CEC-sEVs upon reperfusion further enhances stroke outcome, CEC-sEVs were given at 30 min of tMCAO via a tail vein and upon reperfusion via the ICA. The ischemic rats treated with IA saline were used as a control. Neurological outcome and infarct volume were assessed in a blinded manner. IA CEC-sEVs upon reperfusion significantly reduced infarct volume (24±5% vs 31±6% in saline, p<0.05) which was associated with robust improvement of neurological function from weeks 2 to 4 after tMCAO compared to saline treated rats. Moreover, rats treated with CEC-sEVs IV at 30min followed by IA treatment upon reperfusion further reduced infarct volume (16±6%) compared to rats treated with IA saline or CEC-sEVs. These rats also exhibited robust and early improvement of neurological function starting at 1d after tMCAO. In conclusion, IA administration of CEC-sEVs upon reperfusion significantly reduces ischemic damage, while an additional early (30min post stroke) dose of IV CEC-sEVs further enhances and accelerates the therapeutic effect of IA CEC-sEVs in the aged rats after tMCAO. Our data suggest that CEC-sEVs could potentially may be a therapy to amplify the efficacy of EVT even for elderly patients with acute ischemic stroke.

Abstract WMP89: Reperfusion Pattern as a Predictor of Final Infarct Growth in Patients With Anterior Large Vessel Occlusion Ischemic Stroke

Aim: The first pass effect (FPE) and complete sudden recanalization (cSR) have been identified as an indicator of favorable clinical outcome. We aimed to compare final infarct volume (FIV) according to different reperfusion pattern. Methods: We included consecutive patients admitted with an anterior LVO stroke (intracranial internal carotid artery, MCA-M1) who underwent endovascular treatment between January 2018 and April 2022. Patients with partial or no-reperfusion (eTICI 0-2b) were excluded. Final complete reperfusion (TICI 2c-3) was categorized as achieved at first pass (FPE) or in subsequent passes as sudden (cSR: from TICI 0-1 to TICI 2c-3 in a single pass) or progressive (PR: interim partial recanalization). FIV was quantified using an automatic segmentation tool (Methinks AI) on 24-hour NCCT. Aggregated infarct distribution heatmaps were generated to compare FIV distribution. Results: Of 316 consecutive patients, complete reperfusion was achieved in 202 patients (63.9%). Reperfusion patterns were as follows: FPE (109/202, 54.0%), cSR (28/202, 13.9%) and PR (65/202, 32.1%). The mean FIV was 28.1mL (95%CI 19.1-37.1), 49.5mL (95%CI 18.8-80.2) and 58.4mL (95% CI 37.6-79.2) in FPE patients, cSR and PR patients, respectively (p=0.014). After adjusting for confounders, FPE and cSR patterns were independently associated with a reduced FIV (FPE: -61.7%, 95%CI -77.2% to -35.8%, p<0.001; cSR: -39.7%, 95%CI -63.8% to 0.6%, p=0.05). The rate of excellent clinical outcome (mRS 90d 0-1) was higher in FPE (36.1%) and cSR (35.7%) groups compared to the PR group (20.3%, p=0.081). Conclusion: Improved clinical outcomes in patients with FPE and cSR patterns seem to be influenced by reduced infarct volume in a stepwise manner (FIV FPE<cSR<PR). PR may reflect clot fragmentation and embolization, contributing to infarct growth and worse outcomes. Reperfusion patterns should be explored for selecting target populations for adjunctive neuroprotective therapies during EVT.

Abstract TMP109: Elovanoid Precursors Are Neuroprotective in Experimental Ischemic Stroke

Objective: Ischemic stroke triggers oxidative and biochemical responses that disrupt cell homeostasis. We previously demonstrated that Elovanoids (ELVs), a novel class of homeostatic lipid mediators, reduced infarct volumes and improved neurologic function when administered IV following middle cerebral artery occlusion (MCAo). The present study defines neuroprotection by very long-chain polyunsaturated fatty acid (VLC-PUFA) ELV precursors (C-32:6 and C-34:6) delivered intranasally following experimental ischemic stroke. Methods: Male SD rats underwent 2 hours of MCAo. Treatments with C-32:6, C-34:6 (1μg/μl, 20 μg per rat), or vehicle were administered at 1, 24, and 48h after onset of MCAo. Behavior testing was performed on days 1, 2, 3, and 7. Ischemic core and penumbra were computed from T2WI on day 7 followed by immunohistochemistry. RT-qPCR gene expression was investigated in a separate cohort of rats treated as above at 72 h. Results: C-32:6 and C-34:6 improved behavioral function on days three by (41% and 20%) and seven by (45% and 24%); T2WI total lesion volumes were reduced by (87% and 61%), respectively, compared to the vehicle. In the ischemic penumbra, C-32:6 and C-34:6 reduced TMEM119 + microglia by (38% and 29%), increased NeuN + neurons by (87% and 110%), reduced IgG staining by (54% and 25%), and increased the number of SMI-71 + vessels by (50% and 26%). In the core, C-32:6 and C-34:6 reduced TMEM119 + microglia by (12% and 15%), increased NeuN + neurons by (251% and 89%) and increased the number of SMI-71 + vessels by (31% and 70%). Anti-inflammatory marker genes for protective microglia and astrocyte phenotypes Tm4sf1, Thbs1, Ptx3, Gpc4, Fizz1, Cd14, Msr1, Manf , and Osmr were upregulated with C-32:6 and genes Fcrls, S1pr3, and Vim were downregulated with C-34:6. Conclusion: These data suggest that IN administered C-32:6 C-34:6 improved behavior, protected the ischemic penumbra, increased neurogenesis and angiogenesis, and reduced the number of microglia cells. The overall gene expression changes observed from free fatty acid C-32:6 indicate that its modulatory effects promote the biological activity of astrocytes, microglia, and cerebrovascular cells involved in plasticity.

Abstract 27: A Novel Adjuvant Strategy After Endovascular Therapy: Arterial Glyceryl Trinitrate in Acute Ischemic Stroke for Neuroprotection (AGAIN)

Introduction: Although mechanical thrombectomy (MT) demonstrates robust clinical efficacy in acute ischemic stroke (AIS), not all stroke patients benefit from successful reperfusion. The protective effect of NO donors has been shown to prevent ischemia-reperfusion injury through reduction of reactive oxygen species formation in cardiovascular studies. Glyceryl Trinitrate (GTN), a Food and Drug Administration-approved vasodilator, is one of the widely used exogenous NO donors used in the clinic and has been trialed for neuroprotection in AIS without successful clinical translation. The recent advances in MT for AIS have paved the way for promising new opportunities to supplementary neuroprotective therapies to patients after reperfusion. This study evaluated the safety, feasibility, and preliminary efficacy of intra-arterial administration of GTN after MT for neuroprotection. Methods: This is a prospective randomized controlled study (ID: ChiCTR2100045254). Eligible patients were randomized to receive 800μg GTN or same volume of normal saline through the catheter after recanalization. The primary outcome was symptomatic intracranial hemorrhage (sICH), while secondary outcomes included mortality, functional outcome, infarction volume, complications, and blood nitrate index (NOx). Results: A total of 40 patients were enrolled and randomized with no participants being lost to follow-up. There was no significant difference in the proportion of sICH between GTN and control groups. No significant difference was observed in mortality or rates of neurological deterioration and other complications. Favorable trends for both functional independence (mRS 0-2, 75.0% vs. 65.0%) at 90 days and reduction in final infarct volume (33.2 vs. 38.9 ml) have been observed in GTN group, although they did not reach the significant levels. Moreover, the concentration of blood NO X was increased (p<0.05) at 2 hours after GTN administration (26.2 vs 18.0 μmol/l). Conclusion: The AGAIN study suggests intra-arterial administration of GTN post MT is safe and feasible in AIS patient. The discernible positive trends may be due to the raised NO X levels, which potentially highlight the mechanistic reinforcement for these observed benefits.