Early Brain Injury, an Evolving Frontier in Subarachnoid Hemorrhage Research

Abstracts of Literature

Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage: Mechanism and Therapies

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating neurological disease that has a mortality rate as high as 67% in some series. Traditional research and treatment has focused on addressing the delayed events of cerebral vasospasm following SAH. However, the physiological and cellular events of early brain injury (EBI) make significant contributions to patient outcomes and may even be a more significant factor than delayed cerebral vasospasm. EBI is the result of physiological derangements such as increased intracranial pressure (ICP), decreased cerebral blood flow (CBF), and global cerebral ischemia, which results in blood brain barrier dysfunction, inflammation, and oxidative cascades that lead to neuronal cell death. The consequence of these events to the patient is often death or significant neurological disability. The link between EBI and outcome has come under intense focus with recent studies failing to show improved outcomes following significant inhibition of cerebral vasospasm, and research into the inhibition of EBI cascades is being perused as an effective means of treating SAH patients.

The major causes of death and disability in subarachnoid hemorrhage (SAH) may be early brain injury (EBI) and cerebral vasospasm. Although cerebral vasospasm has been studied and treated by a lot of drugs, the outcome is not improved even if vasospasm is reversed. Based on these data, EBI is considered a primary target for future research, and apoptosis may be involved in EBI after experimental SAH. We reviewed the published literature about the relationship between SAH induced EBI and apoptosis in PubMed. Most available information can be obtained from the endovascular filament perforation animal model. After onset of SAH, intracranial pressure is increased and then cerebral blood flow is reduced. Many factors are involved in the mechanism of apoptotic cell death in EBI after SAH. In the neuronal cells, both intrinsic and extrinsic pathways of apoptosis can occur. Some antiapoptotic drugs were studied and demonstrated a protective effect against EBI after SAH. However, apoptosis in EBI after SAH has been little studied and further studies will provide us more beneficial findings. The study of apoptosis in EBI after experimental SAH may give us new therapies for SAH.

Aneurysmal subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. Devastating post-SAH complications, such as cerebral vasospasm (CVS), delayed cerebral ischemia or seizures to mention a few, are mainly responsible for the poor clinical outcome. Inflammation plays an indispensable role during early brain injury (EBI) and delayed brain injury (DBI) phases over which these complications arise. T helper cells are the major cytokine secreting cells of adaptive immunity that can polarize to multiple functionally unique sub-populations. Here, we investigate different CD4+ T cell subsets during EBI and DBI phases after SAH, and their dynamics during post-SAH complications. Peripheral venous blood from 15 SAH patients during EBI and DBI phases, was analyzed by multicolour flowcytometry. Different subsets of CD3+ CD4+ T cells were characterized by differential cell surface expression of CXCR3 and CCR6 into Th1, Th2, Th17, whereas Tregs were defined by CD25hiCD127lo. The analysis of activation states was done by the expression of stable activation markers CD38 and HLA-DR. Interestingly, compared to healthy controls, Tregs were significantly increased during both EBI and DBI phases. Different activation states of Tregs showed differential significant increase during EBI and DBI phases compared to controls. HLA-DR− CD38+ Tregs were significantly increased during DBI phase compared to EBI phase in SAH patients developing CVS, seizures and infections. However, HLA-DR− CD38− Tregs were significantly reduced during EBI phase in patients with cerebral ischemia (CI) compared to those without CI. HLA-DR− CD38− Th2 cells were significantly increased during EBI phase compared to controls. A significant reduction in Th17/Tregs and HLA-DR− CD38+ Th17/Tregs ratios was observed during both EBI and DBI phases compared to controls. While HLA-DR− CD38− Th17/Tregs and HLA-DR− CD38− Th1/Th2 ratios were impaired only during EBI phase compared to controls. In conclusion, CD4+ T cell subsets display dynamic and unique activation patterns after SAH and during the course of the manifestation of post-SAH complications, which may be helpful for the development of precision neurovascular care. However, to claim this, confirmatory studies with larger patient cohorts, ideally from different ethnic backgrounds, are required. Moreover, our descriptive study may be the grounds for subsequent lab endeavors to explore the underlying mechanisms of our observations.

Aneurysmal subarachnoid hemorrhage (SAH) accounts for about 5 % of all strokes and continues to be a major cause of morbidity and mortality, especially in younger patients (mean age of 52). The global incidence of SAH is 9 per 100, 000 per year with some regional variations [1]. Data from population-based studies suggests a distinct decrease in case fatality (0.9 % per year) over the past four decades to about 30 %, which is likely originated in earlier and more advanced aneurysm repair as well as improved neurocritical care of SAH patients following aneurysm repair [2]. Nevertheless, the incidence of poor functional outcome in SAH patients remains high (about one third of patients). The main determinants for poor outcome after SAH are the degree of early brain injury (EBI) and the incidence of delayed cerebral ischemia (DCI) (see below) [3, 4]. The clinical course of SAH is complex and can be divided into an acute stage (the initial 72 h after SAH ictus) and a subacute stage, which lasts up until 20 days after SAH ictus. The potential main complications in the acute phase are EBI, rebleeding from the ruptured aneurysms, with a frequency of up to 23 % within the first 72 h, and acute hydrocephalus, occurring in about 20 % of the patients, whereas the main complications occurring in the subacute phase are DCI (formerly referred to as Bvasospasm,^ see below), cardiopulmonary or other medical complications, as well as permanent hydrocephalus [5–7]. The term EBI refers not only to direct mechanical damage to the brain tissue due to the hemorrhage but also to a cascade of different pathomechanisms, incl. a temporary increase of intracranial pressure, consecutive reduction of cerebral blood flow and/or microcirculatory spasm, transient global ischemia, neuronal injury or apoptosis, and edema formation [8]. The degree of EBI is a strong and independent predictor for clinical outcome after SAH [9, 3, 4]. The term DCI describes a complex phenomenon as a consequence of several pathomechanisms, incl. (1) macrovascular or angiographic vasospasm, i.e., narrowing of proximal cerebral arteries, (2) microvascular spasm, (3) microthromboembolism, (4) cortical spreading depolarization/ischemia, (5) autoregulatory dysfunction, and (6) inflammation [10–13, 7, 14–17]. The radiological term vasospasm has been widely replaced with the clinical term DCI, which describes a secondary neurological deterioration in the course SAH, excluding other causes of neurological deterioration; an explicit definition for DCI has been proposed by a multidisciplinary research group [16]. This Bparadigm shift^ came along with the insight that angiographic vasospasm occurs in up to 70 % of SAH patients, whereas only 30 % of SAH patients develop clinical features of DCI and, moreover, because pharmaceutical treatments exclusively targeting of angiographic vasospasm failed to improve neurological outcome after SAH [18–20]. However, in those clinical studies where cerebral infarction due to DCI was effectively reduced by experimental pharmaceutical treatments, a significant improvement of clinical outcome was also seen. These data further underlined the concept that DCI comprises several, pro-ischemic pathomechanisms, which ultimately result in cerebral infarction, but the individual importance of each of these pro-ischemic pathomechanisms remains to elucidated [21, 22]. The general shift in scientific focus, i.e., away from ‘vasospasm’ towards DCI and EBI, can also be * Nima Etminan etminan@uni-duesseldorf.de

Intro: Early Brain Injury (EBI) after non-traumatic subarachnoid hemorrhage (SAH) is a significant mediator of neurological injury, but the injury pathways and markers are not well understood. We hypothesized that the inflammatory mediator soluble ST2 (sST2) is associated with markers of EBI. Methods: We studied two independent cohorts, including 190 SAH patients in a discovery cohort and 50 patients in a replication cohort. Clinical markers of EBI included early loss of consciousness, Glasgow Coma Scale (GCS) score at 24 hours, and neurological deterioration within the first 72 hours (defined as a decrease in GCS ≥ 2 points). Radiographic markers included bicaudate index, clinically significant hydrocephalus requiring external ventricular drainage (EVD), and global cerebral edema. Serial plasma sST2 level was measured at early (3.5 ± 1.2 days post-hemorrhage), intermediate (7.8 ± 1.3 days post-hemorrhage), and late (13 ± 2.3 days post-hemorrhage) time points in the discovery cohort and daily during post-hemorrhage days 1-9 in the replication cohort. In the discovery cohort, the relationship between EBI markers and poor functional outcome (90-day modified Rankin Scale; mRS ≥ 3) was assessed using multivariable logistic regression. The association between sST2 level and EBI markers was evaluated using repeated measures analysis. In the replication cohort, associations were examined using an analysis of response profiles. Results: Clinical and radiographic markers of EBI, except for global edema, were each associated with functional outcome in univariate analysis. Of these EBI markers, independent predictors of poor outcome included 24-hour GCS (OR=5.76, 95% CI 2.31-14.4, p<0.001) and clinically significant hydrocephalus (OR=8.74, 95% CI 2.44-31.3, p=0.001). Elevated sST2 was associated with all markers of EBI except for global edema, and independently predicted poor outcome in a multivariate model that included EBI markers. These findings were further replicated in an independent cohort. Conc: Of the potential clinical and radiographic contributors to EBI, poor 24-hour GCS and clinically significant hydrocephalus remain independent predictors of poor outcome. Furthermore, soluble sST2 level is associated with EBI and poor outcome.

Early brain injury (EBI) during the first 72 hours after subarachnoid hemorrhage (SAH) is a key determinant of clinical outcome. A hallmark of EBI, global cerebral ischemia, occurs within seconds of SAH and is thought to be related to increased intracranial pressure (ICP). We tested the hypothesis that ICP elevation and cortical hypoperfusion after SAH are the result of physical blockade of cerebrospinal fluid (CSF) flow pathways by cisternal microthrombi. In mice subjected to SAH, we measured cortical blood volume (CBV) using optical imaging, ICP using pressure transducers and patency of CSF flow pathways using intracisternally injected tracer dye. We then assessed the effects of intracisternal recombinant tissue plasminogen activator (tPA). ICP rose immediately after SAH and remained elevated for 24 hours. This was accompanied by decreased CBV and impaired dye movement . Intracisternal administration of tPA immediately after SAH lowered ICP (from 34.8 ± 3.7 (n=5) to 17.5 ± 3.1 mmHg, (n=6)), increased CBV (from 51.5 ± 8.9 (n=5) to 80 ± 4.7 % of baseline, (n = 6)) and partially restored CSF flow at 24 hours after SAH. Lowering ICP without tPA, by draining CSF, improved CBV at 1, but not 24 hours. These findings suggest that blockade of CSF flow by microthrombi contributes to the early decline in cortical perfusion in an ICP-dependent and independent manner, and that intracisternal tPA may reduce EBI and improve outcome after SAH. Figure 1. tPA reduces ICP and improves perfusion after SAH . A) Representative perfusion images of the cortex at baseline, 1h post SAH/sham (pre-treatment), and 24h post-SAH/Sham (post-treatment). B) ICP at 1h pre-treatment and 24h post-treatment. C) Quantification of perfused CBV 1h pre-treatment and 24h post-treatment. Sham (n =3), SAH vehicle (n =5), SAH tPA (n = 6) * = P <0.05 Scale bar = 500 um

Background There is growing evidence supporting the role of inflammation in early brain injury (EBI) and cerebral vasospasm following subarachnoid hemorrhage (SAH). Matrix Metalloproteinases (MMP) are released by inflammatory cells and can mediate EBI via disruption of the extracellular matrix. MMPs also cleave endothelin-1 (ET-1) into strongly vasoactive fragments and may thereby mediate vasospasm, which can further worsen SAH outcome. We hypothesize that elevated MMP-9 in human cerebrospinal fluid (CSF) is associated with vasospasm and SAH outcome, and therefore a potential clinical biomarker. Methods We prospectively enrolled consecutive SAH subjects, banked serial blood and CSF samples, and evaluated their 3- and 6-month modified Rankins scores (mRS) via telephone follow-up. Angiographic vasospasm was defined as >50% reduction in vessel caliber on angiography on post-SAH day 6-8. Poor outcome was defined as mRS>2. We compared blood and CSF MMP-9 by ELISA on post SAH days 0-1, 2-3, 4-5, 6-8, and 10-14 in a cohort of SAH subjects (N=35) with respect to vasospasm and to 3-month outcome. Continuous variables were compared using student t-test or Wilcoxan rank sum test depending on data normality. Repeated measurements were analyzed using longitudinal regression. Results The study population had a mean age of 53 years and has 54% female. Sixty percent of subjects presented with Hunt and Hess grade of 3 and above. Fifty-four percent developed vasospasm and 32% achieved poor 3-month outcome. Elevation of CSF MMP-9 throughout post SAH days 0-14 was associated with poor 3-month outcome (p=0.008). Specifically, elevated CSF MMP-9 on post SAH day 2-3 (p=0.05) and blood MMP-9 on post SAH day 4-5 (p=0.045) were associated with poor 3-month outcome ( Figure 1 ). Blood MMP-9 correlated strongly with blood leukocyte count (r=0.56, p=0.007). Neither CSF nor blood MMP-9 correlated with vasospasm. Conclusion Early elevation of CSF and blood MMP-9 are associated with poor 3-month outcome but not with vasospasm in SAH. Leukocytes are likely a significant source of blood but not CSF MMP-9. Blood and CSF MMP-9 may mediate neuronal death in SAH via mechanisms independent of angiographic vasospasm. Further studies are necessary to determine the source of CSF MMP-9 and the mechanism by which it mediates poor outcome in SAH. Larger prospective studies are necessary to validate CSF MMP-9 as a predictive biomarker for SAH outcome.

Subarachnoid hemorrhage (SAH), accounting for approximately 5% of all stroke cases, triggers hemorrhagic brain injury where early brain injury (EBI) emerges as a pivotal prognostic determinant. EBI is characterized by multiple pathophysiological processes including oxidative stress (OS), neuroinflammation, neuronal apoptosis, and blood-brain barrier (BBB) disruption following SAH. This study investigated the neuroprotective effects of 3,4-dihydropyrimidin-2(1H)-one derivatives (DHPMs) against neural dysfunction and EBI in a murine SAH model, compared with edaravone (EDA). We hypothesized that DHPMs alleviate SAH-induced neurological damage, dysfunction, and apoptosis through scavenging excess reactive oxygen species (ROS) generated post-SAH, a mechanistic premise systematically validated through experimental analyses. We hypothesized that male C57BL/6 mice weighing between 22 g and 30 g were arbitrarily classified into one of seven cohorts: sham, SAH, SAH + vehicle, SAH + DHPMs 50 mg/kg, SAH + DHPMs 300 mg/kg, SAH + DHPMs 500 mg/kg, and SAH + EDA 10 mg/kg. The intravascular threading method was utilized to induce SAH in the mice. DHPMs were intraperitoneally administered at 50 mg/kg, 300 mg/kg, and 500 mg/kg 15 min after SAH, while EDA was intraperitoneally provided at 10 mg/kg. The modified Garcia score (MGS) and balance beam test (BBT) were employed for behavioral activity assessment at 24 h and 72 h post-SAH, with neurological scores recorded for mice. Subsequently, mice were euthanized to evaluate SAH grade and severity, followed by determination of the optimal drug dosage according to the group exhibiting the most significant behavioral improvement. We computed the brain water content using the dry and wet protocol 24 h post-SAH, while Evans blue was employed to assess BBB leakage. Using Western blot, we also detected pathway-associated proteins, inflammation-related proteins, apoptosis-related proteins, and tight junction (TJ) proteins at the protein level in experimental SAH in mice. Frozen brain tissue sections of mice were prepared for immunofluorescence (IF) staining to observe the changes of microglia, astrocytes, and neurons before and after SAH administration in mice. Our experiment found that the intervention of 300 mg/kg DHPMs after SAH could improve the neurological score of mice, similar to the effects observed with EDA 10 mg/kg intervention. Additionally, DHPMs intervention was found to reduce brain edema and BBB damage 24 h post-SAH, surpassing the efficacy of EDA. Furthermore, the administration of 300 mg/kg DHPMs was linked to augmented Nrf2, apoptosis proteins Bax, Caspase-3, CHOP-1, inflammation proteins interleukin (IL)-1β, IL-18, TJ proteins Occludin, claudin-5, and zonulaoccludens-1 (ZO-1) protein expressions, and diminished heme oxygenase 1 (HO-1) and apoptosis protein Bcl-2 protein expressions. We demonstrated that DHPMs have the potential to safeguard neurological function, mitigate brain edema and BBB disruption, and alleviate OS, inflammation, and apoptosis following SAH.

Subarachnoid hemorrhage (SAH) results from the rupture of an intracranial aneurysm, and the first consequent events are increased intracranial pressure (ICP), reduced cerebral perfusion pressure (CPP), and decreased cerebral blood flow (CBF). The resultant hypoxic state alters autoregulation, ionic homeostasis, and excitotoxicity as well as initiates secondary injuries such as cytotoxic edema, blood-brain barrier (BBB) disruption, inflammation, and apoptotic cell death. Inflammation persists through hemorrhage degradation in the subarachnoid space. Several different aspects of the inflammatory response have been demonstrated in stroke pathogenesis, including cellular response (e.g., leukocyte adherence and microglia activation), expression of adhesion molecules (e.g., selectins, integrins, and immunoglobulin superfamily), production of inflammatory mediators (e.g., cytokines, nitric oxide/nitric oxide synthase (NO/NOS), and free radicals), and accumulation of platelet aggregates. Since all of these inflammatory aspects lead to brain edema and cell death, inflammation could be a particularly important target for designing therapeutic strategies against secondary injuries after SAH. Given these inflammatory contributions could be seen in large vessels, a plethora of research has been intended to reduce cerebral vasospasm (CVS) after SAH. The main research field, however, is moving toward studying early brain injury (EBI) because some human research demonstrated the morphological alleviation of CVS alone might not improve the functional recovery in patients after SAH. This chapter provides the current knowledge of the inflammatory response, translational research, and human clinical trials in SAH as well as discusses emerging opportunities for novel therapeutic strategies for clinical management of SAH.

Blood-brain barrier (BBB) disruption and neural apoptosis are thought to promote early brain injury (EBI) after subarachnoid hemorrhage (SAH). Previous studies have demonstrated that valproic acid (VPA) decreased brain injury in a prechiasmatic injection model of SAH in mice. It should be noted that the beneficial effects of VPA and the underlying mechanisms have not been fully elucidated. To characterize the effects of VPA on BBB disruption and neural apoptosis and to determine mechanisms involved in EBI after SAH. An endovascular perforation model was used to induce SAH in rats. VPA (300 mg/kg) was promptly administered after SAH induction, and the same dose was given 12 hours later. Quercetin (100 mg/kg), an inhibitor of heat shock protein 70 (HSP70), was injected into the peritoneum 2 hours before SAH induction. Mortality, SAH grades, neurological function, Evans Blue extravasation, brain edema, transmission electron microscopy, Western blot, double fluorescence labeling, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining also were used. VPA treatment decreased BBB disruption and brain edema, attenuated neural apoptosis, and improved neurobehavioral functions in EBI after SAH. Double fluorescence labeling indicated that matrix metallopeptidase 9 (MMP-9) was located predominately in neurons and endothelial cells. VPA upregulated the expression of HSP70, effectively decreased the expression and activity of MMP-9, and reduced claudin-5 and occludin degradation. Meanwhile, VPA also upregulated the expression of phosphorylated Akt and bcl-2. Both the anti-BBB disruption and antiapoptotic effects of VPA were abolished by quercetin. VPA prevented BBB disruption and alleviated neural apoptosis after SAH. The action of VPA appeared to be mediated though the HSP70/MMPs and HSP70/Akt pathways. BBB, blood-brain barrierEBI, early brain injuryHSP, heat shock proteinMMP, matrix metalloproteinasePBS, phosphate-buffered salineSAH, subarachnoid hemorrhageTUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelingVPA, valproic acid.

OBJECT.: Aneurysmal subarachnoid hemorrhage (SAH) causes devastating rates of mortality and morbidity. Accumulating studies indicate that early brain injury (EBI) greatly contributes to poor outcomes after SAH and that oxidative stress plays an important role in the development of EBI following SAH. Astaxanthin (ATX), one of the most common carotenoids, has a powerful antioxidative property. However, the potential role of ATX in protecting against EBI after SAH remains obscure. The goal of this study was to assess whether ATX can attenuate SAH-induced brain edema, blood-brain barrier permeability, neural cell death, and neurological deficits, and to elucidate whether the mechanisms of ATX against EBI are related to its powerful antioxidant property. Two experimental SAH models were established, including a prechiasmatic cistern SAH model in rats and a one-hemorrhage SAH model in rabbits. Both intracerebroventricular injection and oral administration of ATX were evaluated in this experiment. Posttreatment assessments included neurological scores, body weight loss, brain edema, Evans blue extravasation, Western blot analysis, histopathological study, and biochemical estimation. It was observed that an ATX intracerebroventricular injection 30 minutes post-SAH could significantly attenuate EBI (including brain edema, blood-brain barrier disruption, neural cell apoptosis, and neurological dysfunction) after SAH in rats. Meanwhile, delayed treatment with ATX 3 hours post-SAH by oral administration was also neuroprotective in both rats and rabbits. In addition, the authors found that ATX treatment could prevent oxidative damage and upregulate the endogenous antioxidant levels in the rat cerebral cortex following SAH. These results suggest that ATX administration could alleviate EBI after SAH, potentially through its powerful antioxidant property. The authors conclude that ATX might be a promising therapeutic agent for EBI following SAH.

Early brain injury (EBI) during the first 72 h after subarachnoid hemorrhage (SAH) is an important determinant of clinical outcome. A hallmark of EBI, global cerebral ischemia, occurs within seconds of SAH and is thought to be related to increased intracranial pressure (ICP). We tested the hypothesis that ICP elevation and cortical hypoperfusion are the result of physical blockade of cerebrospinal fluid (CSF) flow pathways by cisternal microthrombi. In mice subjected to SAH, we measured cortical blood volume (CBV) using optical imaging, ICP using pressure transducers, and patency of CSF flow pathways using intracisternally injected tracer dye. We then assessed the effects of intracisternal injection of recombinant tissue plasminogen activator (tPA). ICP rose immediately after SAH and remained elevated for 24 h. This was accompanied by a decrease in CBV and impaired dye movement. Intracisternal administration of tPA immediately after SAH lowered ICP, increased CBV, and partially restored CSF flow at 24 h after SAH. Lowering ICP without tPA, by draining CSF, improved CBV at 1 h, but not 24 h after SAH. These findings suggest that blockade of CSF flow by microthrombi contributes to the early decline in cortical perfusion in an ICP-dependent and ICP-independent manner and that intracisternal tPA may reduce EBI and improve outcome after SAH.

Oxidative stress was shown to play a crucial role in the diverse pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Microcirculatory dysfunction is thought to be an important and fundamental pathological change in EBI. However, other than blood-brain barrier (BBB) disruption, the influence of oxidative stress on microvessels remains to be elucidated. The aim of this study was to investigate the role of oxidative stress on microcirculatory integrity in EBI. SAH was induced in male Sprague-Dawley rats using an endovascular perforation technique. A free radical scavenger, edaravone, was administered prophylactically by intraperitoneal injection. SAH grade, neurological score, brain water content, and BBB permeability were measured at 24h after SAH induction. In addition, cortical samples taken at 24h after SAH were analyzed to explore oxidative stress, microvascular mural cell apoptosis, microspasm, and microthrombosis. Edaravone treatment significantly ameliorated neurological deficits, brain edema, and BBB disruption. In addition, oxidative stress-induced modifications and subsequent apoptosis of microvascular endothelial cells and pericytes increased after SAH induction, while the administration of edaravone suppressed this. Consistent with apoptotic cell inhibition, microthromboses were also inhibited by edaravone administration. Oxidative stress plays a pivotal role in the induction of multiple pathological changes in microvessels in EBI. Antioxidants are potential candidates for the treatment of microvascular disturbances after SAH.