Therapeutic hypothermia (TH) benefits survivors of cardiac arrest and neonatal hypoxic–ischemic injury and may benefit stroke patients. Large TH clinical trials, however, have shown mixed results. Given the substantial pre-clinical literature supporting TH, we explored possible mechanisms for clinical trial variability. Using a standard rodent stroke model ( n = 20 per group), we found smaller infarctions after 2 h pre- or post-reperfusion TH compared to 4 h. To explore the mechanism of this discrepancy, we used primary cell cultures of rodent neurons, astrocytes, or endothelial cells subjected to oxygen–glucose deprivation (OGD). Then, cells were randomly assigned to 33℃, 35℃ or 37℃ for varying durations after varying delay times. Both 33 and 35℃ TH effectively preserved all cell types, although 33℃ was superior. Longer cooling durations overcame moderate delays to cooling initiation. In contrast, TH interfered with astrocyte paracrine protection of neurons in a temperature-dependent manner. These findings suggest that longer TH is needed to overcome delays to TH onset, but shorter TH durations may be superior to longer, perhaps due to suppression of astrocytic paracrine support of neurons during injury. We propose a scheme for optimizing TH after cerebral injury to stimulate further studies of cardiac arrest and stroke.

Therapeutic hypothermia is the most potent neuroprotectant for experimental cerebral ischemia, illustrated in a 2007 meta-analysis published in this journal. To address recent therapeutic nihilism, we systematically reviewed recent experimental literature. Quality scoring showed considerable improvement in study design. Using several outcome measures in a variety of models and species, therapeutic hypothermia was protective compared with normothermia, with powerful and statistically significant normalized treatment effect sizes, in 60 papers comprising 216 comparisons. In the past 5 years, preclinical studies of ischemic stroke re-emphasize that therapeutic hypothermia is potently effective, justifying further development in larger human clinical trials.

Protease activated receptors (PARs) populate neurons and astrocytes in the brain. The serine protease thrombin, which activates PAR-1 during the first hours after stroke, appears to be associated with the cytotoxicity. Thrombin antagonists and PAR-1 inhibitors have been correlated with reduced cell death and behavioral protection after stroke, but no data yet supports a mechanistic link between PAR-1 action and benefit. We sought to establish the essential role of PAR-1 in mediating ischemic damage. Using a short hairpin mRNA packaged with green fluorescent protein in a lentivirus vector, we knocked downPAR-1 in the medial caudate nucleus prior to rat middle cerebral artery occlusion (MCAo) and in rat neurons prior to oxygen-glucose deprivation. We also compared aged PAR-1 knockout mice with aged PAR-3, PAR-4 mice and young wild-type mice in a standard MCAo model. Silencing PAR-1 significantly reduced neurological deficits, reduced endothelial barrier leakage, and decreased neuronal degeneration in vivo during MCAo. PAR-1 knock-down in the ischemic medial caudate allowed cells to survive the ischemic injury; infected cells were negative for TUNEL and c-Fos injury markers. Primary cultured neurons infected with PAR-1 shRNA showed increased neuroprotection during hypoxic/aglycemic conditions with or without added thrombin. The aged PAR-1 knockout mice showed decreased infarction and vascular disruption compared to aged controls or young wild types. We demonstrated an essential role for PAR-1 during ischemia. Silencing or removing PAR-1 significantly protected neurons and astrocytes. Further development of agents that act at PAR-1or its downstream pathways could yield powerful stroke therapy.

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.

Background and Purpose We showed previously robust neuroprotection with the thrombin inhibitor argatroban, and now sought additional support for its neuroprotective potential. Methods We used behavioral and histological endpoints; rigorously blinded the study groups; extended the treatment window to 3 hours following ischemia onset; and used 2 separate models. First, 2-h filament MCAo in 64 male Sprague-Dawley rats was followed by learning and memory testing and quantitative histomporphometry. Randomly assigned treatment was 0.45mg argatroban, saline, or 0.4U thrombin. Second, we used the quantal bioassay (n=272) after 2-hour MCAo to detect the longest time delay after which therapy failed. Results Argatroban powerfully and significantly reversed learning and memory deficits due to focal ischemia compared to saline or thrombin (p<0.03, ANOVA). Argatroban was significantly (p<0.05, t-test with Bonferroni) protective when given immediately or after 1, 2, 3 but not 4 hours delay. Conclusions We obtained supportive evidence for argatroban protection of the neurovascular unit using behavioral and histological measurements at realistic therapeutic time windows.