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The Stroke Preclinical Assessment Network Multi-Laboratory Model of Thromboembolic Stroke with Thrombolysis: TE-MCAo

Current translational rodent stroke models induce middle cerebral artery occlusion (MCAo) using nylon filaments, injected emboli, intraluminal thrombin, ...

Massachusetts General Hospital

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene () primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation  R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the  therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.

 Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.

Rho-associated protein kinase (ROCK) inhibitors are therapeutic candidates in ischemic stroke and subarachnoid hemorrhage. However, their efficacy in intracerebral hemorrhage (ICH) is unknown. Here, we tested the efficacy of fasudil (10 mg/kg), an isoform-nonselective ROCK inhibitor, and NRL-1049 (10 mg/kg), a novel inhibitor with 43-fold higher selectivity for ROCK2 isoform compared with ROCK1, in a collagenase-induced ICH model in mice. Both short (1-3 days) and prolonged (14 days) therapeutic paradigms were tested using robust sample sizes in both males and females and in active and inactive circadian stages. Outcome readouts included weight loss, mortality, hematoma volume, hemispheric swelling, brain water content, BBB permeability to large molecules, and sensorimotor and cognitive function. We found the treatments safe but not efficacious in improving the hematoma volume, BBB disruption, or neurological deficits in this collagenase-induced ICH model. Intriguingly, however, induction of ICH during the active circadian stage was associated with worse tissue and behavioral outcomes compared with the inactive stage.

Isoform-selective and non-selective rho-kinase inhibitors do not affect collagenase-induced intracerebral hemorrhage outcomes in mice: Influence of sex and circadian cycle.

Ischemic stroke is among the most prevalent diseases, with high death and morbidity. Numerous preclinical studies have reported efficacious interventions in rodent stroke models. However, reperfusion therapies remain the only clinically efficacious intervention to date. Rigor and reproducibility are now recognized as critical to bridge the preclinical-clinical disconnect. Inter-α inhibitor proteins (IαIPs) are a family of structurally related glycoproteins with 2 major forms (inter-α inhibitor and pre-α inhibitor) in blood. Purified human plasma-derived IαIP has beneficial effects in sepsis and hypoxic-ischemic brain injury. More recently, IαIP improved focal ischemic stroke outcomes in mouse models. Here, we tested IαIP efficacy in both transient and permanent stroke mouse models, mimicking previously published study designs and protocols to seek reproducibility.

Examination of Inter-α Inhibitor Proteins in Permanent and Transient Focal Ischemia.

Past failures in translating stroke cerebroprotection provoked calls for a more rigorous methodological approach, leading to the stroke preclinical assessment network SPAN (Stroke Preclinical Assessment Network), where uric acid (UA) treatment exceeded a prespecified efficacy boundary for the primary functional outcome. Still, successful translation to humans requires confirmation of the effect of UA across key biological variables relevant to patients with stroke.

Uric Acid Stroke Cerebroprotection Transcended Sex, Age, and Comorbidities in a Multicenter Preclinical Trial.

The SPAN (Stroke Preclinical Assessment Network) is a confirmatory trial platform to test the efficacy and safety of candidate cerebroprotective interventions in acute stroke. As the largest multicenter preclinical stroke trial to date, the SPAN1 trial (first SPAN) prospectively captured many biological and procedural variables, revealing a high degree of heterogeneity introduced by the multicenter approach that may impact stroke outcomes. Here, we examined the biological and procedural predictors of tissue and neurological outcomes after focal cerebral ischemic stroke in rats.

Determinants of Outcome After Endovascular Middle Cerebral Artery Occlusion in Rats in the SPAN Trial.

The SPAN (Stroke Preclinical Assessment Network) is a confirmatory multicenter trial network to test cerebroprotective interventions in experimental acute stroke. In a first-of-its-kind trial, SPAN tested 6 interventions in a rodent model of transient focal ischemic stroke. Here, we report the efficacy of fasudil, an isoform-nonselective rho-associated kinase inhibitor, on primary and secondary outcomes in the SPAN trial.

Multicenter SPAN Trial of Fasudil in Ischemic Stroke.

Fingolimod is an immunomodulatory drug that has shown promising effects in stroke treatment, including improvements in neurofunctional recovery and a reduction in infarct size. Fingolimod modulates the sphingosine-1-phosphate receptors, which leads to the internalization of sphingosine-1-phosphate receptors on T and B lymphocytes, thereby preventing their egress from secondary lymphoid organs. Here, we report a secondary analysis from the Stroke Preclinical Assessment Network trial. We assessed the effects of fingolimod versus vehicle on stroke outcomes to better evaluate its therapeutic potential.

Fingolimod as a Potential Cerebroprotectant Results From the Stroke Preclinical Assessment Network.

Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.

Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.

Inflammation, particularly mediated through interleukin-6 (IL-6) signaling, plays a critical role in stroke pathophysiology. High levels of IL-6 are associated with poor outcomes in stroke patients. Therapeutic inhibition of IL-6 signaling may offer a novel strategy to mitigate post-stroke damage and improve recovery. This study evaluated the efficacy of tocilizumab (TCZ), a clinically approved monoclonal antibody that blocks IL-6 receptor signaling, using data from the Stroke Preclinical Assessment Network (SPAN), a multi-center, randomized, blinded, placebo-controlled trial in preclinical stroke models.

Article	Year
CADASIL mutations sensitize the brain to ischemia via spreading depolarizations and abnormal extracellular potassium homeostasis. The Journal of clinical investigation\| PubMed ID: 35202003	2022
The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results. Stroke\| PubMed ID: 35354299	2022
Embracing Heterogeneity in The Multicenter Stroke Preclinical Assessment Network (SPAN) Trial. Stroke\| PubMed ID: 36601951	2023
Spreading Depolarizations Suppress Hematoma Growth in Hyperacute Intracerebral Hemorrhage in Mice. Stroke\| PubMed ID: 37610105	2023
A multi-laboratory preclinical trial in rodents to assess treatment candidates for acute ischemic stroke. Science translational medicine\| PubMed ID: 37729432	2023
Biological and Procedural Predictors of Outcome in the Stroke Preclinical Assessment Network (SPAN) Trial. Circulation research\| PubMed ID: 39034919	2024
Hippocampal infarction and generalized seizures predict early mortality after endovascular middle cerebral artery occlusion in mice. Experimental neurology\| PubMed ID: 39079623	2024
Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor. bioRxiv : the preprint server for biology\| PubMed ID: 39605323	2024
Isoform-selective and non-selective rho-kinase inhibitors do not affect collagenase-induced intracerebral hemorrhage outcomes in mice: Influence of sex and circadian cycle. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism\| PubMed ID: 39763388	2025
Examination of Inter-α Inhibitor Proteins in Permanent and Transient Focal Ischemia. Journal of the American Heart Association\| PubMed ID: 39921503	2025
Uric Acid Stroke Cerebroprotection Transcended Sex, Age, and Comorbidities in a Multicenter Preclinical Trial. Stroke\| PubMed ID: 40091742	2025
Determinants of Outcome After Endovascular Middle Cerebral Artery Occlusion in Rats in the SPAN Trial. Stroke\| PubMed ID: 40396268	2025
Multicenter SPAN Trial of Fasudil in Ischemic Stroke. Stroke\| PubMed ID: 40421536	2025
Fingolimod as a Potential Cerebroprotectant Results From the Stroke Preclinical Assessment Network. Stroke\| PubMed ID: 40899256	2025
Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice. Nature biomedical engineering\| PubMed ID: 40935887	2025
Evaluating tocilizumab in ischemic stroke: Findings from the SPAN multicenter trial. Neuropharmacology\| PubMed ID: 41354124	2025

Takahiko Imai

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