Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial.

BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.

Hippocampal infarction and generalized seizures predict early mortality after endovascular middle cerebral artery occlusion in mice.

Endovascular middle cerebral artery occlusion (MCAO) is a widely used experimental ischemic stroke model. However, the model carries high early mortality. Our aim was to investigate the factors that influence early mortality within 48 h of reperfusion after transient MCAO. Using C57BL/6 mice, we induced 1-hour endovascular filament MCAO. To introduce heterogeneity of infarct volumes, a subset of animals had additional tandem common carotid artery occlusion (MCAO+CCAO). Continuous video monitoring was used to gain insight into the cause of death. Mortality within 48 h was 25% in the pooled cohort. All animals with early mortality suffered from infarcts in the hippocampus, sometimes accompanied by infarcts in the thalamus and midbrain, which occurred exclusively in the MCAO+CCAO group. All animals with early mortality developed convulsive seizures captured on video monitoring. None of the animals that did not develop convulsive seizures died. Among the three regions, hippocampal infarction appeared necessary for convulsive seizures and early mortality. Our data highlight seizures as the primary cause of mortality within the first 48 h after endovascular filament MCAO, linked to hippocampal infarction. Since hippocampal blood supply is mainly from the posterior cerebral artery (PCA), avoiding concurrent PCA ischemia can decrease mortality in proximal MCAO models.

A multi-laboratory preclinical trial in rodents to assess treatment candidates for acute ischemic stroke.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Spreading Depolarizations Suppress Hematoma Growth in Hyperacute Intracerebral Hemorrhage in Mice.

BACKGROUND: Spreading depolarizations (SDs) occur in all types of brain injury and may be associated with detrimental effects in ischemic stroke and subarachnoid hemorrhage. While rapid hematoma growth during intracerebral hemorrhage triggers SDs, their role in intracerebral hemorrhage is unknown. METHODS: We used intrinsic optical signal and laser speckle imaging, combined with electrocorticography, to investigate the effects of SD on hematoma growth during the hyperacute phase (0-4 hours) after intracortical collagenase injection in mice. Hematoma expansion, SDs, and cerebral blood flow were simultaneously monitored under normotensive and hypertensive conditions. RESULTS: Spontaneous SDs erupted from the vicinity of the hematoma during rapid hematoma growth. We found that hematoma growth slowed down by >60% immediately after an SD. This effect was even stronger in hypertensive animals with faster hematoma growth. To establish causation, we exogenously induced SDs (every 30 minutes) at a remote site by topical potassium chloride application and found reduced hematoma growth rate and final hemorrhage volume (18.2±5.8 versus 10.7±4.1 mm3). Analysis of cerebral blood flow using laser speckle flowmetry revealed that suppression of hematoma growth by spontaneous or induced SDs coincided and correlated with the characteristic oligemia in the wake of SD, implicating the vasoconstrictive effect of SD as one potential mechanism of action. CONCLUSIONS: Our findings reveal that SDs limit hematoma growth during the early hours of intracerebral hemorrhage and decrease final hematoma volume.

Embracing Heterogeneity in The Multicenter Stroke Preclinical Assessment Network (SPAN) Trial.

The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.

Protective effects of alpha-mangostin encapsulated in cyclodextrin-nanoparticle on cerebral ischemia.

The production of reactive oxygen species (ROS) during and after the onset of an ischemic stroke induces neuronal cell death and severely damages brain function. Therefore, reducing ROS by administrating antioxidant compounds is a promising approach to improving ischemic symptoms. Alpha-mangostin (α-M) is an antioxidant compound extracted from the pericarp of the mangosteen fruit. Reportedly, α-M decreases neuronal toxicity in primary rat cerebral cortical neurons. In this study, we investigated the neuroprotective activity of α-M in both in vitro and in vivo assays. Pretreatment with α-M inhibited excessive cellular ROS production after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro using an SH-SY5Y (human neuroblastoma) cell line. In addition, α-M maintained mitochondrial membrane potential and suppressed mitochondrial-specific ROS production induced by OGD/R. Meanwhile, the low bioavailability of α-M due to its poor water solubility has been an insuperable obstruction impeding extensive investigations of the biological functions of α-M and its medical applications. To overcome this problem, we synthesized a cyclodextrin-based nanoparticle (CDNP) that is known to increase the loading efficiency and binding constant of α-M, compared with cyclodextrins themselves. This nano-formulated α-M (α-M/CDNP) was optimized for an in vivo ischemic stroke model. Our results indicated that α-M/CDNP (25 mg/kg/injection) reduced infarct volume and improved neurological behavior (p = 0.036 and p = 0.046, respectively). These in vivo results suggest that α-M appears to cross the blood-brain barrier (BBB) with the help of a nano-formulation with CDNP. Combining an in vitro BBB model and a physicochemical binding assay between α-M and albumin, it is speculated that α-M released from CDNP would interact with albumin during its prolonged circulation in the blood, and the resultant α-M/albumin complex may cross the BBB through the absorptive-mediated transcytosis pathway. These findings suggest the potential clinical application of α-M in ischemic stroke treatment.

The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

CADASIL mutations sensitize the brain to ischemia via spreading depolarizations and abnormal extracellular potassium homeostasis.

Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL) is the most common monogenic form of small vessel disease characterized by migraine with aura, leukoaraiosis, strokes, and dementia. CADASIL mutations cause cerebrovascular dysfunction in both animal models and humans. Here, we showed that 2 different human CADASIL mutations (Notch3 R90C or R169C) worsen ischemic stroke outcomes in transgenic mice; this was explained by the higher blood flow threshold to maintain tissue viability compared with that in wild type (WT) mice. Both mutants developed larger infarcts and worse neurological deficits compared with WT mice, regardless of age or sex after filament middle cerebral artery occlusion. However, full-field laser speckle flowmetry during distal middle cerebral artery occlusion showed comparable perfusion deficits in mutants and their respective WT controls. Circle of Willis anatomy and pial collateralization also did not differ among the genotypes. In contrast, mutants had a higher cerebral blood flow threshold, below which infarction ensued, suggesting increased sensitivity of brain tissue to ischemia. Electrophysiological recordings revealed a 1.5- to 2-fold higher frequency of peri-infarct spreading depolarizations in CADASIL mutants. Higher extracellular K+ elevations during spreading depolarizations in the mutants implicated a defect in extracellular K+ clearance. Altogether, these data reveal a mechanism of enhanced vulnerability to ischemic injury linked to abnormal extracellular ion homeostasis and susceptibility to ischemic depolarizations in CADASIL.

Nafamostat protects against early brain injury after subarachnoid hemorrhage in mice.

This study aimed to evaluate the effects of nafamostat, a serin protease inhibitor, in the management of subarachnoid hemorrhage (SAH). SAH was induced by endovascular perforation in male mice. Nafamostat was administered intraperitoneally four times immediately after SAH induction. Cerebral blood flow, neurological behavior tests, SAH grade and protein expression were evaluated at 24 h after SAH induction. In the in vitro model, human brain microvascular endothelial cells (HBMVECs), HBVECs were exposed to thrombin and hypoxia for 24 h; nafamostat was administered and the protein expression was evaluated. Eighty-eight mice were included in the in vivo study. Fifteen mice (17%) were excluded because of death or procedure failure. Nafamostat exerted no significant effect on the SAH grade or cerebral blood flow; however, it improved the neurological behavior and suppressed the thrombin and MMP-9 expression. In addition, nafamostat suppressed the ICAM-1 expression and p38 phosphorylation in the in vitro study. Nafamostat has a protective effect against HBMVEC after exposure to thrombin and hypoxia, suggesting its role in improving the neurological outcomes after SAH. These findings indicate that nafamostat has the potential to be a novel therapeutic drug in the management of SAH.

MYCL promotes iPSC-like colony formation via MYC Box 0 and 2 domains.

Human induced pluripotent stem cells (hiPSCs) can differentiate into cells of the three germ layers and are promising cell sources for regenerative medicine therapies. However, current protocols generate hiPSCs with low efficiency, and the generated iPSCs have variable differentiation capacity among different clones. Our previous study reported that MYC proteins (c-MYC and MYCL) are essential for reprogramming and germline transmission but that MYCL can generate hiPSC colonies more efficiently than c-MYC. The molecular underpinnings for the different reprogramming efficiencies between c-MYC and MYCL, however, are unknown. In this study, we found that MYC Box 0 (MB0) and MB2, two functional domains conserved in the MYC protein family, contribute to the phenotypic differences and promote hiPSC generation in MYCL-induced reprogramming. Proteome analyses suggested that in MYCL-induced reprogramming, cell adhesion-related cytoskeletal proteins are regulated by the MB0 domain, while the MB2 domain regulates RNA processes. These findings provide a molecular explanation for why MYCL has higher reprogramming efficiency than c-MYC.

Involvement of Cerebral Blood Flow on Neurological and Behavioral Functions after Subarachnoid Hemorrhage in Mice.

OBJECTIVE: Cerebral Blood Flow (CBF) change after Subarachnoid Hemorrhage (SAH) is strongly associated with brain injuries such as early brain injury and delayed cerebral ischemia. We evaluated the correlation between CBF using Laser Speckle Flow Imaging (LSFI) after SAH and neurological findings in the sub-acute phase. METHOD: An SAH was induced by endovascular perforation in male mice. CBF was quantitatively measured by using LSFI at six time points, immediately to 14 days after SAH induction. Behavior tests and survival rate were evaluated. The mice were divided into recovery and hypo-perfusion groups according to their CBF at 1 day after the procedure. RESULT: Forty mice were included in this study. Five mice (20%) were included in the hypo-perfusion group, and the remaining 20 (80%) mice were classified as the recovery group. The decrease of CBF in the recovery group was observed until 1 day after the procedure. However, the decrease of CBF in the hypo-perfusion group was prolonged until 7 days after the procedure. Neurological findings and survival rates in the hypo-perfusion group were significantly worse than those in the recovery group. The low alternation cases (≤ 50%) in the Y-maze test in the recovery group (n = 5) had significantly lower CBF at 1 day after the procedure. CONCLUSION: Low blood flow at 1 day after SAH was associated with worse survival rate, neurological findings, and memory disturbance. Early improvement in CBF may be associated with an improved prognosis after SAH.

Guanabenz and Clonidine, α2-Adrenergic Receptor Agonists, Inhibit Choroidal Neovascularization.

BACKGROUND: Neovascular age-related macular degeneration (AMD) with choroidal neovascularization (CNV) is a leading cause of blindness in elderly people. Anti-vascular endothelial growth factor (anti-VEGF)-drugs are used to treat AMD patients; however, some patients are resistant to these therapies. OBJECTIVE: The purpose of this study was to investigate the anti-angiogenic effects of α2-adrenergic agonists, including guanabenz and clonidine. METHODS: We evaluated the anti-angiogenic effects of α2-adrenergic agonists in human retinal microvascular endothelial cells (HRMECs). A proliferation assay was conducted, and the migration ratio was evaluated. In a laser-induced CNV model, guanabenz and clonidine were delivered via intraperitoneal injection or implantation of an osmotic pump device. Fourteen days following CNV induction, CNV lesion size and fundus fluorescein angiography (FFA) were evaluated. RESULTS: Guanabenz and clonidine inhibited VEGF-induced retinal endothelial cell growth and migration. In the CNV model mice, CNV lesion sizes were reduced by intraperitoneal administration of guanabenz or clonidine. Data, including body weight, systolic blood pressure, and heart rate showed that guanabenz (0.5 and 2.0 mg/kg/day) had little effect on these parameters; conversely, a high dose of clonidine (1.0 mg/kg/day) did affect these parameters. Additionally, clonidine did not affect CNV size, but continuous administration of guanabenz attenuated both CNV size and leakage from neovessels. CONCLUSION: Our study suggests a key role for α2-adrenergic receptors during CNV formation. Therefore, we suggest that α2-adrenergic receptor agonists may represent novel therapeutic drugs for patients with neovascular AMD.

Potential therapeutic effects of Nrf2 activators on intracranial hemorrhage.

Intracranial hemorrhage (ICH) is a devastating disease which induces high mortality and poor outcomes including severe neurological dysfunctions. ICH pathology is divided into two types: primary brain injury (PBI) and secondary brain injury (SBI). Although there are numerous preclinical studies documenting neuroprotective agents in experimental ICH models, no effective drugs have been developed for clinical use due to complicated ICH pathology. Oxidative and inflammatory stresses play central roles in the onset and progression of brain injury after ICH, especially SBI. Nrf2 is a crucial transcription factor in the anti-oxidative stress defense system. Under normal conditions, Nrf2 is tightly regulated by the Keap1. Under ICH pathological conditions, such as overproduction of reactive oxygen species (ROS), Nrf2 is translocated into the nucleus where it up-regulates the expression of several anti-oxidative phase II enzymes such as heme oxygenase-1 (HO-1). Recently, many reports have suggested the therapeutic potential of Nrf2 activators (including natural or synthesized compounds) for treating neurodegenerative diseases. Moreover, several Nrf2 activators attenuate ischemic stroke-induced brain injury in several animal models. This review summarizes the efficacy of several Nrf2 activators in ICH animal models. In the future, Nrf2 activators might be approved for the treatment of ICH patients.

Excess adiponectin in eyes with progressive ocular vascular diseases.

Anti-vascular endothelial growth factor (VEGF) therapies are now the first-line treatment for many ocular diseases, but some patients are non-responders to these therapies. The purpose of this study was to determine whether the level of adiponectin increased the pathogenesis of retinal edema and neovascularization in the retina of progressive ocular vascular diseases. We examined the role played by adiponectin in two types of cells and animal models which are retinal vein occlusion (RVO) and oxygen-induced retinopathy (OIR) mice. Our results showed that an injection of anti-adiponectin antibody ameliorated the retinal edema and ischemia through the depression of the expression level of VEGF-related factors and tight junction-related proteins in the retina of RVO mice. The intravitreal injection of anti-adiponectin antibody also decreased the degree of retinal neovascularization in an OIR mice. In addition, exposure of human retinal microvascular endothelial cells and human brain microvascular pericytes in culture to adiponectin increased both the vascular permeability and neovascularization through the increase of inflammatory factor and the dropout of the pericytes. These findings indicate that adiponectin plays a critical role in retinal edema and neovascularization, and adiponectin is a potential therapeutic target for the treatment of diabetic macular edema, proliferative diabetic retinopathy, and RVO.

Deferasirox, a trivalent iron chelator, ameliorates neuronal damage in hemorrhagic stroke models.

PURPOSE: Intracranial hemorrhage (ICH) is a devastating disease with high mortality and morbidity. After ICH, iron released from the hematoma plays a crucial role in secondary brain injury. Deferasirox (DFR) is a trivalent iron chelator, which was approved to treat iron overload syndrome after transfusion. The aim of the present study was to investigate the protective effects of DFR in both in vitro and in vivo ICH models. METHODS: Using a hemin-induced SH-SY5Y cell damage model, we performed an intracellular bivalent iron (Fe2+) accumulation assay, cell death assay, oxidative stress assessments, and Western blotting analysis. Moreover, the effects of DFR intraventricular administration on hematoma, neurological deficits, and histological alteration were evaluated in an in vivo ICH mouse model by collagenase. RESULTS: DFR significantly suppressed the intracellular Fe2+ accumulation and cell death caused by hemin exposure. These effects were related to the suppression of both reactive oxygen species and lipid peroxidation over-production. In Western blotting analysis, hemin increased the expression of ferritin (an iron storage protein), LC3 and p62 (autophagy-related markers), phosphorylated p38 (a stress response protein), and cleaved-caspase3 and cleaved-poly (adenosine diphosphate ribose) polymerase (PARP) (apoptosis-related makers). However, DFR suppressed the increase of these proteins. In addition, DFR attenuated the neurological deficits until 7 days after ICH without affecting hematoma and injury area. Furthermore, DFR also suppressed microglia/macrophage activation in peri-hematoma area at 3 days after ICH. CONCLUSION: These findings indicate that DFR might be a useful therapeutic agent for the therapy of ICH.

Therapeutic Effects of Iron Chelation in Atorvastatin-Induced Intracranial Hemorrhage of Zebrafish Larvae.

OBJECTIVE: Intracranial hemorrhage (ICH) catastrophically damages the cerebral vasculature, and severely compromises blood-brain barrier (BBB) function. The prognosis of ICH is poor due to the drastic and rapid progression of its pathology, and the lack of effective treatments presents a significant unmet clinical need. The present paper provides several evidences about the relationship between ICH bleeding status and mortality and the potential therapeutic effects of an iron chelator for ICH. METHODS: Zebrafish are a highly transparent animal model, allowing live imaging of the complex cerebral vasculature. Thus, to further elucidate ATV-induced ICH, we investigated the concentration- and time-dependent phenotypes of ATV-induced ICH with zebrafish larvae. RESULTS: The effects of ATV on mortality and ICH incidence in zebrafish larvae were concentration-dependent. Further, ATV treatment decreased vascular density of the hindbrain in a concentration-dependent manner, and hematoma volume was inversely correlated with ATV concentration. The number of cranial TUNEL-positive apoptotic cells was markedly increased 3 days post-fertilization. Importantly, the iron chelator deferasirox (DFR) decreased the incidence of ATV-induced ICH in zebrafish larvae. CONCLUSION: These findings provided insight into the pathology and regulatory mechanism of ATV-induced ICH, and demonstrated the therapeutic effects of iron chelators.

Importance of CBF measurement to exclude concomitant cerebral infarction in the murine endovascular perforation SAH model.

OBJECTIVE: Concomitant cerebral infarction (CI) is could be a potential concern in experimental subarachnoid hemorrhage (SAH) induced by endovascular perforation. We propose a noninvasive method for excluding CI in a murine SAH model by using Laser speckle flow imaging (LSFI). METHODS: An SAH was induced with endovascular perforation (EVP) in male ddY mice. The cerebral blood flow (CBF) was quantitatively measured in the bilateral cerebral cortex was performed by using LSFI at five timepoints (preprocedure, immediately after, and 3 hours, 6 hours, and 24 hours after the procedure). The mice were then euthanized, and the SAH grade and volume of the CI were evaluated. The mice were divided into the SAH group and the SAH + CI group. Differences between the groups were assessed. RESULTS: Forty-eight mice were used in this study. Six were the sham control group. Five SAH mice died within 24 hours after the procedure. A large CI on the ipsilateral side occurred in 15 (40.5%) mice (i.e., SAH + CI group). The remaining 22 (59.5%) mice were classified as the SAH group. The SAH grading score was not significantly different between the groups. The neurological score and CBF of the ipsilateral hemisphere were significantly higher in the SAH group than in the SAH + CI group (neurological score: 12.3 vs. 8, p < 0.01; CBF: 343.1 vs. 205.5; p < 0.01). The cut-off modified neurological score for excluding CI was 8 (area under the curve [AUC]: 0.77) and CBF at 24 hours after the procedure was 279.2 (AUC:0.856). CONCLUSIONS: Using LSFI is less invasive and effectively excludes concomitant CI in experimental SAH. This methodological protocol may ad in improving the quality of the EVP-SAH model.

Antitumour Effects of Astaxanthin and Adonixanthin on Glioblastoma.

Several antitumour drugs have been isolated from natural products and many clinical trials are underway to evaluate their potential. There have been numerous reports about the antitumour effects of astaxanthin against several tumours but no studies into its effects against glioblastoma. Astaxanthin is a red pigment found in crustaceans and fish and is also synthesized in Haematococcus pluvialis; adonixanthin is an intermediate product of astaxanthin. It is known that both astaxanthin and adonixanthin possess radical scavenging activity and can confer a protective effect on several damages. In this study, we clarified the antitumour effects of astaxanthin and adonixanthin using glioblastoma models. Specifically, astaxanthin and adonixanthin showed an ability to suppress cell proliferation and migration in three types of glioblastoma cells. Furthermore, these compounds were confirmed to transfer to the brain in a murine model. In the murine orthotopic glioblastoma model, glioblastoma progression was suppressed by the oral administration of astaxanthin and adonixanthin at 10 and 30 mg/kg, respectively, for 10 days. These results suggest that both astaxanthin and adonixanthin have potential as treatments for glioblastoma.

The Mitochondria-targeted Peptide, Bendavia, Attenuated Ischemia/Reperfusion-induced Stroke Damage.

After ischemic stroke, oxygen and nutrition depletion induce mitochondrial dysfunction, which aggravates brain injury. Bendavia, a mitochondria-targeted tetra-peptide, has anti-oxidative and anti-inflammatory activities. We previously reported that bendavia protected human brain microvascular endothelial cells against oxygen/glucose deprivation (OGD)-induced damage via preserving mitochondrial function. The effects of bendavia on mitochondrial function include the inhibition of reactive oxygen species (ROS) production, inhibition of apoptosis, and restoration of adenosine tri-phosphate synthesis. However, the influence of bendavia on the blood-brain barrier (BBB) and neurons after brain ischemia/reperfusion damage is unclear. The aim of this study was to investigate whether bendavia has protective effects against ischemia/reperfusion damage using both in vivo and in vitro models. The in vivo experiments were conducted in mice, which were subjected to transient middle cerebral occlusion (t-MCAO) to induce brain ischemia/reperfusion damage. After t-MCAO, the cerebral blood flow (CBF), neurological deficits, infarct volume, BBB permeability, and microglia/macrophage activation were assessed. Compared to the vehicle group, bendavia administration (administered twice; immediately after reperfusion and 4 h later) attenuated the sensori-motor dysfunction and infarct formation independent of CBF variation. In addition, bendavia decreased BBB hyper-permeability and microglia/macrophage activation. The in vitro experiments were conducted utilizing two models: (1) OGD/re-oxygenation (OGD/R) or (2) hydrogen peroxide (H2O2)-induced neuron damage. In both models, bendavia inhibited neuronal cell death induced by OGD/R or H2O2. These findings indicated that bendavia attenuated brain ischemia/reperfusion damage and has direct neuroprotective effects against cell injury. Therefore, bendavia may be a novel therapeutic agent to improve ischemic stroke patient outcome.