Despite increasing knowledge over the last several decades of the factors contributing to delayed cerebral ischemia (DCI), it remains a significant cause of morbidity after aneurysmal subarachnoid hemorrhage (aSAH). The hypercoagulable state induced by aneurysm rupture is critical to most proposed mechanisms of DCI either through its direct contribution to microthrombi formation, or through the indirect effects of the products of coagulation on vasospasm, and inflammation. In this review we summarize clinical evidence of hypercoagulability after aSAH as well as both the clinical and pre-clinical evidence of its role in DCI, highlighting the role that microthrombi and NETosis are thought to play.

Approximately 30% of aneurysmal subarachnoid hemorrhage (aSAH) patients who survive the rupture develop delayed cerebral ischemia (DCI) 4 to 10 days following aSAH. Multiple clinical studies indicate the cause of DCI is multifactorial, but identifying which patients are at-risk for developing DCI is lacking. Previous studies have identified numerous biomarkers, such as coagulation and platelet factors, which may predict patients at-risk for DCI, but to-date, no markers are widely utilized in the clinical setting. Thus, we sought to investigate various biomarkers of platelets to identify which factors are predictive of patients at-risk for DCI. aSAH patients were prospectively enrolled and had blood collected 1, 2, 4, 7, and 10 days post-aSAH. DCI was adjudicated by three independent clinicals. Blood was used from 148 aSAH patients (n=101 no DCI, N=47 DCI) for analysis of TEG, platelet receptors for activation (CD62p) and aggregation (PAC-1) via flow cytometry, platelet aggregation via Chrono-log, and 22 aSAH (n=17 no DCI, n=5 DCI) patients were used to analyze the platelet activation status (i.e. morphology, see Figures) via microscopy. Six healthy patients were used as controls. aSAH caused hypercoagulability in all aSAH patients. However, neither TEG nor Chrono-log parameters were reliable in predicting which aSAH patients would develop DCI. Similarly, the markers for platelet activation (CD62p) and aggregation (PAC-1) were not able to distinguish which aSAH patients would get DCI. Interestingly, morphological analysis of platelets revealed significantly higher platelet activation two days after aSAH in the aSAH patients who later developed DCI (see Figure). Although this preliminary study involves a small sample size of 22 patients, the results suggest that platelet morphology rather than other platelet tests may identify individuals at-risk for DCI following aSAH. However, further research with a larger patient cohort is necessary to validate these findings.

Neutrophils are reported to be critical mediators of to poor outcome after subarachnoid hemorrhage (SAH). Following SAH, neutrophils cause vascular occlusion via neutrophil extracellular traps (NETs) and NETs have been identified as a therapeutic target to prevent delayed cerebral ischemia in mice (DCI) with SAH. In this study, our hypothesis was that markers of NETs are higher in aneurysmal SAH patients developing DCI compared to SAH patients not developing DCI. Moreover, we hypothesized NETs occlude blood vessels in the brain of SAH patients developing DCI. Using aneurysmal SAH patient blood collected at 1, 2, 4, 7, and 10 days post-SAH, we used ELISA kits to measure three markers of NETs: neutrophil elastase, citrullinated histone H3, and H3.1 nucleosome. We collected autopsy samples to examine for the presence of NETs in the brain vessels. Finally, as neutrophil counts (and the ratio of neutrophils to lymphocytes) are reported to be a biomarker for DCI, we assessed these measures too. Two hundred and sixty-nine aneurysmal SAH patients were included in our analyses: 193 patients did not develop DCI and 76 patients developed DCI. In our study, neutrophil elastase and citrullinated histone H3 were NETs markers that were able to distinguish between SAH patients who developed DCI and those who did not develop DCI. Specifically, neutrophil elastase was elevated in SAH patients developing DCI (compared to that of patients not developing DCI) and neutrophil elastase levels could predict which SAH patients were at high-risk for developing DCI as early as day 2 post-SAH. Citrullinated histone H3 displayed an elevated level in DCI patients vs no DCI patients on day 10. Moreover, NETs were observed occluding the brain blood vessels in SAH patients. None of the other measures had any predictive value for identifying which SAH patients were likely to develop DCI: neutrophil count, neutrophil to lymphocyte ratio, H3.1 nucleosome. The findings of this study suggest NETs factors may be useful biomarkers to predict which aneurysmal SAH patients are at-risk for DCI. As NETs were observed occluding brain vessels, NETs may also be a therapeutic target.

During cortical development, newly born neurons migrate radially or tangentially from their origin to expand the cortex. Simultaneously, neuron-derived factors support angiogenesis, and an elaborate network of blood cerebral vessels develops in the cortex. Traditionally, blood cerebral vessels were considered to support the growing cortex or migrating neurons by providing nutrients and oxygen. However, recent studies have shed light on Endothelial cells’ influence on cortical development; they guide neuronal migration by providing molecular cues and structural support. Here, we review the current understanding of how CNS cerebral vessels support neurogenesis, neuronal migration, and the formation of the six-layer cortical structure during development. Additionally, we explore current knowledge regarding the vascular role in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder, and schizophrenia.

Therapeutic angiogenesis is essential for regenerating brain tissue damaged by stroke, yet it remains an unmet clinical challenge. During brain development, pro-angiogenic genes drive the formation of vascular networks, with their expression tightly regulated in later stages. We found that in adult CNS endothelial cells (ECs), angiogenesis-related genes are epigenetically silenced through histone deacetylase 2 (HDAC2) and the polycomb repressive complex 2 (PRC2). Conditional deletion of Hdac2 in ECs reactivated pro-angiogenic signaling, including Wnt/β-catenin target genes, leading to functional neovascularization with preserved blood-brain barrier (BBB) integrity in the adult brain. In contrast, Ezh2 (PRC2 subunit) deletion reduced vessel density and compromised BBB function. Deletion of Hdac2 and Ezh2 immediately after transient ischemic stroke conferred vascular protection by modulating stroke-induced transcriptional programs in CNS ECs. In contrast, delayed deletion, initiated seven days post-stroke, after significant neuronal loss in the infarct region, induced robust revascularization and promoted post-stroke neurogenesis, with differentiation into both excitatory and inhibitory neurons. These findings highlight CNS EC HDAC2 as a promising therapeutic target for inducing adult brain angiogenesis, facilitating revascularization, and supporting neuronal regeneration following stroke.

Subarachnoid hemorrhage (SAH), characterized by the presence of hemoglobin (Hb) in the subarachnoid space, significantly impacts cerebral vessels, leading to various pathological outcomes. The toxicity of cell-free Hb released from erythrocytes and its metabolites after SAH causes vasoconstriction and neuronal damage, and correlates with delayed ischemic neurological deficits (DIND). While animal models have provided substantial and invaluable data in the research of aneurysmal SAH, the specific effects of subarachnoid blood on cerebral arteries remain greatly understudied. Here, we describe the changes in the genetic profile of human cerebral arteries exposed to free Hb for 48 h. We performed an ex vivo exposure, followed by mRNA sequencing of the vessels. Compared to controls 54 genes were downregulated, and 53 genes were upregulated in human cerebral arteries after Hb exposure. Enrichment analysis identified the ferroptosis pathway as the most significantly affected. Further lipid peroxidation (LPO) assays and elevated ACSL4 gene expression support a ferroptosis pathway. Additionally, Hb exposure altered key signaling pathways essential for vascular stability (PI3K-Akt, MAPK), modified G-protein signaling mediated by RGS1/2, and suppressed key transcription factors such as KLF5, NR4A1, and FOS. Our results underscore the critical role of Hb in driving pathological responses in brain vessels. Furthermore, our dataset could be valuable for developing interventions after SAH and may help identify the underlying causes of vascular injury.

INTRODUCTION: Neutrophil extracellular traps (NETs) are a hallmark of the immune system, activating during times of cellular stress and neovascular events. Within SAH pathophysiology, NETs have the ability to impede healing and obstruct vasculature by inducing inflammation and releasing factors that cause vasoconstriction. METHODS: SAH was induced within C57 male mice via endovascular perforation. Female mice were injected with blood. Mice were then administered either GSK106 (vehicle) or GSK484 (treatment) Mice were assessed behaviorally daily through composite neurobehavior exams. Microthrombi count was assessed at -2 from Bregma in the whole slice. In–vivo MRI was used to evaluate cerebral blood flow and infarct volume 1 day and 5 days post SAH. Behavior was analyzed using a parametric two-way ANOVA test and MRI data were analyzed using a two-way ANOVA with post hoc testing. NET formation was counted post-SAH in both male and female mice. RESULTS: In both males and females, SAH groups exhibited an increase in neuroscore that was near that of the sham group, showing recovery of negative outcomes following SAH. Similarly, the mean infarct volume for male and female mice exhibited a decrease when treated with the PAD4 inhibitor. Overall, the NET count displayed a large increase for the vehicle group and showed a level similar to the sham group when looking at mice treated with the inhibitor. CONCLUSIONS: The PAD4 inhibitor, GSK484, has been shown to improve recovery in SAH-aged mice and may be a viable therapy for positive outcomes in SAH animals.

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is notoriously known for its high mortality and morbidity. Approximately one-third of the patients who survive aneurysm rupture are reported to develop delayed cerebral ischemia (DCI), which contributes to a poor clinical outcome. Currently, there are no biomarkers for identifying which aSAH patients are at risk of developing DCI. We aimed to determine the feasibility of cerebrospinal fluid (CSF) exosomal microRNAs (miRNAs) for predicting DCI post-aSAH. Methods: aSAH patients were prospectively enrolled, and CSF samples were collected at two time points (<24 h and 72 h post-aSAH) from individuals undergoing external ventricular drainage. Exosomal miRNAs were isolated from the CSF for analysis. In the initial group of patients (discovery cohort), an exploratory analysis was conducted using a CSF panel containing 84 miRNAs, assessed by quantitative real-time PCR (RT-qPCR). Based on this analysis, 27 miRNAs were selected for further evaluation in a second group of patients (validation cohort). Among these, 10 miRNAs had previously been reported in SAH-related CSF studies, supporting their relevance for continued investigation. Results: In this study, RT-qPCR analysis of 84 miRNAs in CSF samples from aSAH patients (n = 10 DCI, n = 16 no DCI) and non-aSAH controls (n = 5) identified 9 upregulated and 13 downregulated miRNAs in the DCI group, and 7 upregulated and 18 downregulated miRNAs in the no-DCI group, compared to the controls. When comparing DCI to no-DCI patients, 13 miRNAs were found to be upregulated in the DCI group. Additionally, seven miRNAs showed temporal upregulation in DCI patients between early (<24 h/T1) and later (72 h/T3) time points across both discovery and validation cohorts. However, no miRNAs were uniquely expressed in either DCI or no-DCI groups, limiting their potential as specific biomarkers for DCI. Conclusions: Despite analyses in both the discovery and validation phases, no miRNAs emerged as consistent and reliable biomarkers for distinguishing DCI from no-DCI patients. However, the identified miRNAs are involved in the key KEGG pathways that regulate vascular integrity, neuronal survival, and inflammatory processes central to DCI pathophysiology. These findings highlight the complexity of miRNA regulation following aSAH, as reflected by the variability in differentially expressed miRNAs between cohorts. This variability may be influenced by factors such as limited sample size, patient heterogeneity, individual biological differences, and experimental variability. Comprehensive profiling using larger, well-characterized cohorts, along with rigorous validation, is essential to determine the predictive value and mechanistic significance of candidate miRNAs in DCI.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating neurological disease, and one of the primary drivers of morbidity after aneurysm rupture is the phenomenon of delayed cerebral ischemia (DCI). Significant knowledge has been gained over the past two decades of the impact of neuroinflammation in DCI; and neutrophils are now believed to play a major role. There is significant human subject data showing the rise of neutrophil related inflammatory markers and neutrophil’s association with poor outcome after aSAH, but as of yet no trials involving human subjects have been done specifically targeting neutrophils. There is however a growing body of evidence in animals models that targeting neutrophils, or their byproducts such as neutrophil extracellular traps improves outcomes. This review summarizes the available evidence of neutrophil’s impact in both human subjects and animal models of aSAH and should serve as an impetuous to explore clinical trials in human subjects.

Free hemoglobin’s release into CSF from blood breakdown is a primary instigator of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH). Early clearance of subarachnoid blood with intrathecal (IT) fibrinolytics has shown potential to decrease incidence of DCI. However, the dosage of fibrinolytic needed is not known. We investigated the ability of low dose tissue plasminogen activator (tPA) to rapidly remove subarachnoid clot. We performed a single center retrospective review of aSAH patients who received IT tPA. Dosing consistent with the CLEAR III trial for IVH was utilized—tPA (1 mg) administered every 8 h via an external ventricular drain (EVD) for up to three doses. CT imaging was obtained before initiation and after the final dose. Subarachnoid clot was quantified using the Hijdra score on initial and follow-up scans. The IT tPA group was compared to a large retrospective cohort without IT tPA. Eight aSAH patients received IT tPA treatment for the purpose of increasing blood removal after aSAH. CT imaging indicated that the Hijdra Score had a 70–100

INTRODUCTION: Microthrombosis (platelet aggregation) has been suggested as a major factor contributing to delayed neurological deterioration in patients after subarachnoid hemorrhage (SAH). However, experimental studies on the role of microthrombi in delayed deficits after SAH have not been investigated in depth. Similarly, the only clinically existing antiplatelet therapy is nimodipine, with other therapies being invasive. METHODS: SAH was induced in adult male and female C57BL/6 mice via endovascular perforation. Mice were randomly assigned into sham (n=6/sex) or SAH groups (n=22-24/sex). A neurobehavior exam was performed on days 1-3, 5, and 7 post-SAH using a composite neuroscore. Animals were sacrificed on the day of delayed deficits or 7 days post-SAH. Microthrombi count and vessel diameters (for vasospasm) were measured using H&E stained brain slices. All outcomes were performed and all data were analyzed by a blinded investigator. RESULTS: Seventeen percent (4/24) of male mice and thirty-six percent (8/22) of female mice developed delayed deficits on days 3-5 post-SAH. Those mice which developed delayed deficits had significantly more microthrombi in their brains than mice which did not develop delayed deficits; vasospasm did not correlate with delayed deficits. Additionally, female SAH mice develop delayed deficits at a higher frequency than males. CONCLUSIONS: This work found that microthrombi correlated with delayed deficits. The data within this study suggests that preventing platelets and microthrombi may improve functional recovery and reduce the risk of delayed neurological deficits. Future directions include testing therapeutics such as tirofiban and other platelet antagonists.

Impaired cerebral circulation, induced by blood vessel constrictions and microthrombi, leads to delayed cerebral ischemia after subarachnoid hemorrhage (SAH). 12/15-Lipooxygenase (12/15-LOX) overexpression has been implicated in worsening early brain injury outcomes following SAH. However, it is unknown if 12/15-LOX is important in delayed pathophysiological events after SAH. Since 12/15-LOX produces metabolites that induce inflammation and vasoconstriction, we hypothesized that 12/15-LOX leads to microvessel constriction and microthrombi formation after SAH, and thus, 12/15-LOX is an important target to prevent delayed cerebral ischemia. SAH was induced in C57BL/6 and 12/15-LOX−/− mice of both sexes by endovascular perforation. Expression of 12/15-LOX was assessed in brain tissue slices and in vitro. C57BL/6 mice were administered either ML351 (12/15-LOX inhibitor) or vehicle. Mice were evaluated for daily neuroscore and euthanized on day 5 to assess cerebral 12/15-LOX expression, vessel constrictions, platelet activation, microthrombi, neurodegeneration, infarction, cortical perfusion, and development of delayed deficits. Finally, the effect of 12/15-LOX inhibition on platelet activation was assessed in SAH patient samples using a platelet spreading assay. In SAH mice, 12/15-LOX was upregulated in brain vascular cells, and there was an increase in 12-S-HETE. Inhibition of 12/15-LOX improved brain perfusion on days 4–5 and attenuated delayed pathophysiological events, including microvessel constrictions, microthrombi, neuronal degeneration, and infarction. Additionally, 12/15-LOX inhibition reduced platelet activation in human and mouse blood samples. Cerebrovascular 12/15-LOX overexpression plays a major role in brain dysfunction after SAH by triggering microvessel constrictions and microthrombi formation, which reduces brain perfusion. Inhibiting 12/15-LOX may be a therapeutic target to improve outcomes after SAH.

The blood-brain barrier (BBB) controls the movement of molecules into and out of the central nervous system (CNS). Since a functional BBB forms by mouse embryonic day E15.5, we reasoned that gene cohorts expressed in CNS endothelial cells (EC) at E13.5 contribute to BBB formation. In contrast, adult gene signatures reflect BBB maintenance mechanisms. Supporting this hypothesis, transcriptomic analysis revealed distinct cohorts of EC genes involved in BBB formation and maintenance. Here, we demonstrate that epigenetic regulator's histone deacetylase 2 (HDAC2) and polycomb repressive complex 2 (PRC2) control EC gene expression for BBB development and prevent Wnt/β-catenin (Wnt) target genes from being expressed in adult CNS ECs. Low Wnt activity during development modifies BBB genes epigenetically for the formation of functional BBB. As a Class-I HDAC inhibitor induces adult CNS ECs to regain Wnt activity and BBB genetic signatures that support BBB formation, our results inform strategies to promote BBB repair.

BACKGROUND:After subarachnoid hemorrhage (SAH), neutrophils are deleterious and contribute to poor outcomes. Neutrophils can produce neutrophil extracellular traps (NETs) after ischemic stroke. Our hypothesis was that, after SAH, neutrophils contribute to delayed cerebral ischemia (DCI) and worse outcomes via cerebrovascular occlusion by NETs.METHODS:SAH was induced via endovascular perforation, and SAH mice were given either a neutrophil-depleting antibody, a PAD4 (peptidylarginine deiminase 4) inhibitor (to prevent NETosis), DNAse-I (to degrade NETs), or a vehicle control. Mice underwent daily neurological assessment until day 7 and then euthanized for quantification of intravascular brain NETs (iNETs). Subsets of mice were used to quantify neutrophil infiltration, NETosis potential, iNETs, cerebral perfusion, and infarction. In addition, NET markers were assessed in the blood of aneurysmal SAH patients.RESULTS:In mice, SAH led to brain neutrophil infiltration within 24 hours, induced a pro-NETosis phenotype selectively in skull neutrophils, and caused a significant increase in iNETs by day 1, which persisted until at least day 7. Neutrophil depletion significantly reduced iNETs, improving cerebral perfusion, leading to less neurological deficits and less incidence of DCI (16% versus 51.9%). Similarly, PAD4 inhibition reduced iNETs, improved neurological outcome, and reduced incidence of DCI (5% versus 30%), whereas degrading NETs marginally improved outcomes. Patients with aneurysmal SAH who developed DCI had elevated markers of NETs compared with non-DCI patients.CONCLUSIONS:After SAH, skull-derived neutrophils are primed for NETosis, and there are persistent brain iNETs, which correlated with delayed deficits. The findings from this study suggest that, after SAH, neutrophils and NETosis are therapeutic targets, which can prevent vascular occlusion by NETs in the brain, thereby lessening the risk of DCI. Finally, NET markers may be biomarkers, which can predict which patients with aneurysmal SAH are at risk for developing DCI.

Endovascular thrombectomy (EVT) is one of the most effective therapies for acute ischemic stroke attributable to large‐vessel occlusion but, despite successful treatment, there remains a significant number of patients with disability. The phenomenon of incomplete microcirculatory reperfusion or no reflow is thought to underlie a substantial proportion of cases with unfavorable outcome. This phenomenon likely arises from platelet aggregation and endothelial edema impacting the cerebral microvasculature, vessels that are below the resolution of digital subtraction angiography. Although incomplete microcirculatory reperfusion prevents tissue recovery and poses a significant clinical challenge, there are multiple therapeutic options administered early after recanalization that have been shown to be promising. In this review, we discuss incomplete microcirculatory reperfusion after EVT and highlight various treatment approaches with a particular focus on antiplatelet therapy, including inhibition of the glycoprotein IIb/IIIa receptor pathway. We also review the rigor of previous studies exploring the use of intravenous and intraarterial administration of tirofiban in neurologic disease before EVT, during EVT, after EVT, or as rescue therapy to determine its effect on clinical outcomes.