Results of the present study demonstrate that heat shock preconditioning elevates Hsp27 and Hsp70 expression in zebrafish XAV939 embryos and reduces both embryo mortality and apoptotic cell death in brain and eye of zebrafish embryos after HR. In addition, our results demonstrate that HSF1 is required for preconditioning dependent and independent resistance to HR. LDN-193189 However, although heat shock protein expression was induced by HR alone as well as by heat shock preconditioning, HSF1 knockdown resulted in only a moderate decrease in inducible Hsp70 expression and no detectable reduction in Hsp27 expression in embryos subject to HR without heat shock preconditioning. These results establish the zebrafish embryo as a model for the study of ischemic injury in brain and eye tissues and suggest an unconventional role for HSF1 in cellular resistance to ischemia/reperfusion injury. Previous studies have characterized the survival of zebrafish embryos subjected to low oxygen conditions up to 48 hours post fertilization . As significant development occurs after that time, we examined the response of older embryos to HR. 48, 60, and 72 hpf embryos were incubated in fish water containing low oxygen and survival, defined as the absence of opaque tissues and the presence of a beating heart, was assessed 24 hours post treatment. Resulting survival curves are shown in Figure 1. LD50s are approximately 145 min for 48 hpf embryos, 110 min for 60 hpf embryos, and 80 min for 72 hpf embryos, indicating an increasing susceptibility to HR with increasing age. Heat shock preconditioning is considered to be a hallmark of HSF1-dependent cytoprotection . However, the ability of heat shock preconditioning to protect zebrafish embryos against HR injury has not been previously established. To address this, 34 hpf embryos were preconditioned at 37uC for 90 min and allowed to recover at 28uC for 14 hours. Embryos were then subjected to 160 min of hypoxia at 28.5uC and returned to normoxic conditions for another 24 hours. Over five independent experiments, average survival of non-preconditioned embryos was 8.3%, whereas average survival of preconditioned embryos was 61.0% . Therefore, heat shock preconditioning produced a dramatic and statistically significant increase in survival of embryos subjected to subsequent HR.