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.

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.

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.

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.

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.

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.

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.

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.

Rap1 signal modulators control the maintenance of hematopoietic progenitors in bone marrow and adult long-term hematopoiesis.

Adult long-term hematopoiesis depends on sustaining hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) niches, where their balance of quiescence, self-renewal, and hematopoietic differentiation is tightly regulated. Although various BM stroma cells that produce niche factors have been identified, regulation of the intrinsic responsiveness of HSPC to the niche factors remains elusive. We previously reported that mice deficient for Sipa1, a Rap1 GTPase-activating protein, develop diverse hematopoietic disorders of late onset. Here we showed that transplantation of BM cells expressing membrane-targeted C3G (C3G-F), a Rap1 GTP/GDP exchanger, resulted in the progressive decline of the numbers of HSPC repopulated in BM with time and impaired long-term hematopoiesis of all cell lineages. C3G-F/HSPC were sustained for months in spleen retaining hematopoietic potential, but these cells inefficiently contributed to overall hematopoietic reconstitution. C3G-F/HSPC showed enhanced proliferation and differentiation with accelerated progenitor cell exhaustion in response to stem cell factor (SCF). Using a Ba/F3 cell line, we confirmed that the increased basal Rap1GTP levels with C3G-F expression caused a markedly prolonged activation of c-Kit receptor and downstream signaling through SCF ligation. A minor population of C3G-F/HSPC also showed enhanced proliferation in the presence of thrombopoietin (TPO) compared to Vect/HSPC. Current results suggest an important role of basal Rap1 activation status of HSPC in their maintenance in BM for sustaining long-term adult hematopoiesis.

A novel free radical scavenger, NSP-116, ameliorated the brain injury in both ischemic and hemorrhagic stroke models.

Reperfusion injury is a serious problem in ischemic stroke therapy, which leads to neuronal damage and intracranial hemorrhage (ICH). A novel free radical scavenger, NSP-116, has anti-oxidative effect and may ameliorate reperfusion injury. The purpose of this study was to investigate the effects of NSP-116 on both ischemic and hemorrhagic stroke models. First, we assessed whether NSP-116 has protective effects in vitro. Pre-treatment of NSP-116 decreased neuronal cell damage induced by H2O2 or LPS. Moreover, NSP-116 also suppressed mitochondria damage and apoptosis in H2O2-induced neuronal injury model. Based on these results, we used a middle cerebral artery occlusion (MCAO)-induced ischemic stroke model or a collagenase-induced ICH model. Using the MCAO model, we evaluated the cerebral blood flow (CBF), neurological deficit, and infarct volume. Hematoma volume was assessed at 3 days after ICH. In the MCAO model, oral administration of NSP-116 at 30 mg/kg attenuated the reduction of CBF, neurological deficits, and infarct formation. Interestingly, NSP-116 also ameliorated hematoma expansion and neurological deficits in the ICH model. Additionally, pre-treatment of NSP-116 suppressed the brain microvascular endothelial cell death induced by collagenase treatment. Collectively, our findings indicated that oral administration of NSP-116 attenuates both ischemic and hemorrhagic brain injuries after stroke.

Nrf2 activator ameliorates hemorrhagic transformation in focal cerebral ischemia under warfarin anticoagulation.

BACKGROUND AND PURPOSE: Oxidative stress has been reported to be a main cause of neuronal cell death in ischemia reperfusion injury (IRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important factor involved in anti-oxidative responses. We previously reported that bardoxolone methyl (BARD), an Nrf2 activator, prevented damage induced by IRI. In this study, we investigated the effect of BARD on hemorrhagic transformation in the context of blood brain barrier (BBB) protection. METHODS: Mice received pre-treatment with warfarin (4.0 mg/kg, p.o.). IRI was subsequently induced 18 h after the warfarin administration by transient middle cerebral artery occlusion (MCAO) for 6 h. BARD (0.06, 0.2, 0.6 or 2.0 mg/kg) or saline was injected intravenously immediately after reperfusion. The infarct volume, neurological score, intracranial hemorrhage volume, and BBB permeability were evaluated 24 h after MCAO. The survival rate and behavioral functional recovery were evaluated for 7 days following IRI. Furthermore, the effects of BARD on BBB components were investigated by western blotting and immunostaining analysis. RESULTS: BARD suppressed warfarin-mediated increases in the intracranial hemorrhage volume without affecting the infarct volume. BBB permeability was also suppressed by administration of BARD. Western blotting showed that BARD increased expression of BBB components such as endothelial cells, pericytes, and tight junction proteins. Furthermore, immunostaining showed that BARD induced localization of Nrf2 to endothelial cells and pericytes. CONCLUSIONS: BARD suppressed the exacerbation hemorrhage caused by warfarin pretreatment and ameliorated BBB disruption by protecting endothelial cells, pericytes, and tight junction protein expressions. These results indicate that Nrf2 activators may be an effective therapy against hemorrhagic transformation caused by anticoagulant drugs.

Non-muscle myosin IIA is a functional entry receptor for herpes simplex virus-1.

Herpes simplex virus-1 (HSV-1), the prototype of the α-herpesvirus family, causes life-long infections in humans. Although generally associated with various mucocutaneous diseases, HSV-1 is also involved in lethal encephalitis. HSV-1 entry into host cells requires cellular receptors for both envelope glycoproteins B (gB) and D (gD). However, the gB receptors responsible for its broad host range in vitro and infection of critical targets in vivo remain unknown. Here we show that non-muscle myosin heavy chain IIA (NMHC-IIA), a subunit of non-muscle myosin IIA (NM-IIA), functions as an HSV-1 entry receptor by interacting with gB. A cell line that is relatively resistant to HSV-1 infection became highly susceptible to infection by this virus when NMHC-IIA was overexpressed. Antibody to NMHC-IIA blocked HSV-1 infection in naturally permissive target cells. Furthermore, knockdown of NMHC-IIA in the permissive cells inhibited HSV-1 infection as well as cell-cell fusion when gB, gD, gH and gL were coexpressed. Cell-surface expression of NMHC-IIA was markedly and rapidly induced during the initiation of HSV-1 entry. A specific inhibitor of myosin light chain kinase, which regulates NM-IIA by phosphorylation, reduced the redistribution of NMHC-IIA as well as HSV-1 infection in cell culture and in a murine model for herpes stromal keratitis. NMHC-IIA is ubiquitously expressed in various human tissues and cell types and, therefore, is implicated as a functional gB receptor that mediates broad HSV-1 infectivity both in vitro and in vivo. The identification of NMHC-IIA as an HSV-1 entry receptor and the involvement of NM-IIA regulation in HSV-1 infection provide an insight into HSV-1 entry and identify new targets for antiviral drug development.

Vaginal memory T cells induced by intranasal vaccination are critical for protective T cell recruitment and prevention of genital HSV-2 disease.

UNLABELLED: Protective immunity against genital pathogens causing chronic infections, such as herpes simplex virus 2 (HSV-2) or human immunodeficiency virus, requires the induction of cell-mediated immune responses locally in the genital tract. Intranasal immunization with a thymidine kinase-deficient (TK(-)) mutant of HSV-2 effectively induces HSV-2-specific gamma interferon (IFN-γ)-secreting memory T cell production and protective immunity against intravaginal challenge with wild-type HSV-2. However, the precise mechanism by which intranasal immunization induces protective immunity in the distant genital mucosa more effectively than does systemic immunization is unknown. Here, we showed that intranasal immunization with live HSV-2 TK(-) induced the production of effector T cells and their migration to, and retention in, the vaginal mucosa, whereas systemic vaccination barely established a local effector T cell pool, even when it induced the production of circulating memory T cells in the systemic compartment. The long-lasting HSV-2-specific local effector T cells induced by intranasal vaccination provided superior protection against intravaginal wild-type HSV-2 challenge by starting viral clearance at the entry site earlier than with intraperitoneal immunization. Intranasal immunization is an effective strategy for eliciting high levels of cell-mediated protection of the genital tract by providing long-lasting antigen (Ag)-specific local effector T cells without introducing topical infection or inflammation. IMPORTANCE: Intranasal (i.n.) vaccines against sexually transmitted diseases that are caused by viruses such as herpes simplex virus 2 (HSV-2) have long been in development, but no vaccine candidate is currently available. Understanding the cellular mechanisms of immune responses in a distant vaginal mucosa induced by i.n. immunization with HSV-2 will contribute to designing such a vaccine. Our study demonstrated that i.n. immunization with an attenuated strain of HSV-2 generated long-lasting IFN-γ-secreting T cells in vaginal mucosa more effectively than systemic immunization. We found that these vaginal effector memory T cells are critical for the early stage of viral clearance at natural infection sites and prevent severe vaginal inflammation and herpes encephalitis.

Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness.

Us3 is a serine-threonine protein kinase that is encoded by herpes simplex virus 1 (HSV-1). In experimental animal models of HSV infection, peripheral and intracranial inoculations can be used to study viral pathogenicity in peripheral sites (e.g., eyes and vagina) and central nervous systems (CNSs), respectively. In addition, peripheral inoculation can be used to investigate this virus' ability to invade the CNS (neuroinvasiveness) from peripheral sites. HSV-1 Us3 has previously been shown to be critical for viral pathogenicity in both peripheral sites and CNSs of mice. However, the role of HSV-1 Us3 in viral neuroinvasiveness has not yet been elucidated. In the present study, the yields of a Us3 null mutant virus and its repaired virus in the eyes, trigeminal ganglia, and brains of mice following ocular inoculation were examined. It was found that, although the repaired virus appeared in the brains of mice 3 days after infection, peak replication occurring 7 days after infection, no viral replication of the Us3 null mutant virus was detectable. These findings indicate that HSV-1 Us3 plays a crucial role in the ability of the virus to invade the brain from the eyes. Thus, HSV-1 Us3 is a significant neuroinvasiveness factor in vivo.

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.

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.

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.