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.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes.

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.