IntroductionMulticentre preclinical randomised controlled trials (pRCT) are emerging as a necessary step to confirm efficacy and improve translation into the clinic. The aim of this project is to perform two multicentre pRCTs (one in rats and one in mice) to investigate the efficacy of remote ischaemic conditioning (RIC) in an experimental model of severe ischaemic stroke.Methods and analysisSeven research laboratories within the Italian Stroke Organization (ISO) Basic Science network will participate in the study. Transient endovascular occlusion of the proximal right middle cerebral artery will be performed in two species (rats and mice) and in both sexes. Animals will be randomised to receive RIC by transient surgical occlusion of the right femoral artery, or sham surgery, after reperfusion. Blinded outcome assessment will be performed for dichotomised functional neuroscore (primary endpoint) and infarct volume (secondary endpoint) at 48 hours. A sample size of 80 animals per species will yield 82% power to detect a significant difference of 30% in the primary outcome in both pRCTs. Analyses will be performed in a blind status and according to an intention-to-treat paradigm. The results of this study will provide robust, translationally oriented, high-quality evidence on the efficacy of RIC in multiple species of rodents with large ischaemic stroke.Ethics and disseminationThis is approved by the Animal Welfare Regulatory Body of the University of Milano Bicocca, under project license from the Italian Ministry of Health. Trial results will be subject to publication according to the definition of the outcome presented in this protocol.Trial registration numberPCTE0000177.

Background and purposeHead down tilt 15° (HDT15°), applied before recanalization, increases collateral flow and improves outcome in experimental ischemic stroke. For its simplicity and low cost, HDT15° holds considerable potential to be developed as an emergency treatment of acute stroke in the prehospital setting, where hemorrhagic stroke is the major mimic of ischemic stroke. In this study, we assessed safety of HDT15° in the acute phase of experimental intracerebral hemorrhage.MethodsIntracerebral hemorrhage was produced by stereotaxic injection of collagenase in Wistar rats. A randomized noninferiority trial design was used to assign rats to HDT15° or flat position (n = 64). HDT15° was applied for 1 h during the time window of hematoma expansion. The primary outcome was hematoma volume at 24 h. Secondary outcomes were mass effect, mortality, and functional deficit in the main study and acute changes of intracranial pressure, hematoma growth, and cardiorespiratory parameters in separate sets of randomized animals (n = 32).ResultsHDT15° achieved the specified criteria of noninferiority for hematoma volume at 24 h. Mass effect, mortality, and functional deficit at 24 h showed no difference in the two groups. HDT15° induced a mild increase in intracranial pressure with respect to the pretreatment values (+2.91 ± 1.76 mmHg). HDT15° had a neutral effect on MRI‐based analysis of hematoma growth and cardiorespiratory parameters.ConclusionsApplication of HDT15° in the hyperacute phase of experimental intracerebral hemorrhage does not worsen early outcome. Further research is needed to implement HDT15° as an emergency collateral therapeutic for acute stroke.

Neurological outcome after ischemic stroke depends on residual salvageable brain tissue at the time of recanalization. Head down tilt 15 (HDT15) was proven effective in reducing infarct size and improving functional outcome in rats with transient middle cerebral artery occlusion (t-MCAO) by increasing cerebral perfusion within the ischemic penumbra. In this pooled analysis, individual animal-level data from three experimental series were combined in a study population of 104 t-MCAO rats (45 in HDT15 group and 59 in flat position group). Co-primary outcomes were infarct size and functional outcome at 24 h in both groups. The secondary outcome was hemodynamic change induced by HDT15 in ischemic and non-ischemic hemispheres in a subgroup of animals. Infarct size at 24 h was smaller in HDT15 group than in flat position group (absolute mean difference 31.69 mm 3 , 95% CI 9.1-54.2, Cohen's d 0.56, p = 0.006). Functional outcome at 24 h was better in HDT15 group than in flat position group (median [IQR]: 13[10-16] vs. 11), with a shift in the distribution of the neurobehavioural scores in favour of HDT15. Mean cerebral perfusion in the ischemic hemisphere was higher during HDT15 than before

Hypothermia is a promising therapeutic strategy for severe vasospasm and other types of non-thrombotic cerebral ischemia, but its clinical application is limited by significant systemic side effects. We aimed to develop an intraventricular device for the controlled cooling of the cerebrospinal fluid, to produce a targeted hypothermia in the affected cerebral hemisphere with a minimal effect on systemic temperature. An intraventricular cooling device (acronym: V-COOL) was developed by in silico modelling, in vitro testing, and in vivo proof-of-concept application in healthy Wistar rats (n = 42). Cerebral cortical temperature, rectal temperature, and intracranial pressure were monitored at increasing flow rate (0.2 to 0.8 mL/min) and duration of application (10 to 60 min). Survival, neurological outcome, and MRI volumetric analysis of the ventricular system were assessed during the first 24 h. The V-COOL prototyping was designed to minimize extra-cranial heat transfer and intra-cranial pressure load. In vivo application of the V-COOL device produced a flow rate-dependent decrease in cerebral cortical temperature, without affecting systemic temperature. The target degree of cerebral cooling (− 3.0 °C) was obtained in 4.48 min at the flow rate of 0.4 mL/min, without significant changes in intracranial pressure. Survival and neurological outcome at 24 h showed no significant difference compared to sham-treated rats. MRI study showed a transient dilation of the ventricular system (+ 38%) in a subset of animals. The V-COOL technology provides an effective, rapid, selective, and safe cerebral cooling to a clinically relevant degree of − 3.0 °C.