Background Immune cell immigration and activation play a central role within the pathophysiology of ischemic stroke. In a recent preclinical study, we demonstrated that the nuclear receptor NR4A1 has a potential protective role within the pathophysiology of cerebral ischemia. NR4A1-deficient mice subjected to experimental stroke (middle cerebral artery occlusion, MCAO) develop an increased neutrophil influx, increased brain damage and worsened functional outcome. In an extended gain-of-function experiment, we now aim to clarify whether NR4A1-activation using the NR4A1-specific ligand Cytosporone-B results in enhanced functional recovery and reduced tissue damage in the ischemic rodent brain. Preliminary data from experiments in male mice indicate that activation of NR4A1 results in reduced numbers of intracerebral neutrophil granulocytes, improved functional recovery and reduced tissue damage. In order to fulfill fundamental quality standards for preclinical studies, these findings shall now be validated in a bicentric study. For this purpose, experiments will be repeated at the original research site (University of Muenster, Germany) in female mice and subsequently in both male and female mice at a second study-center (University Hospital Essen, Germany). Assignment to groups (placebo versus active substance) will be performed with a random number generator (GraphPad Prism - assign subjects to groups). Both, drug and placebo units will be coded in a blinded manner and provided by the Muenster site. Subsequent functional testing, evaluation of functional data and determination of infarct volumes will be performed in a blinded fashion. Unblinding as well as group-assignment will be performed after data collection. Infarct volume will be used as the primary outcome measure; functional recovery as a secondary outcome measure. In addition, histological analyses will be performed at the end of the proposed project to decipher the role of NR4A1 activation on potentially protective / regenerative neutrophils. In order to fulfill fundamental requirements of preclinical trials, the following protocol has been established: harmonization of experimental protocols among both study centers, a priori sample size calculation, randomization of the treatment groups and blinding of all investigators. Cytosporone-B treatment Cytosporone-B or the corresponding placebo (16.5% DMSO in PBS) will be applied to wildtype mice (13 mg/kg body weight, i.p.) 3h after induction of reperfusion. Middle cerebral artery occlusion Occlusion of the middle cerebral artery (MCAO) will be induced in anesthetized mice under maintenance of 37˚C body temperature during the whole procedure. After midline neck incision, the left common artery and carotid bifurcation will be exposed and the external and proximal left common arteries will be ligated subsequently. Retrograde perfusion of the left common carotid artery will be transiently interrupted using a microvascular clip. The common carotid artery will then be incised using a micro-dissecting scissors, and a silicon-coated 8–0 nylon monofilament (701956PK5Re or 702056PK5Re, Doccol Corporation, Sharon, MA) is going to be advanced into the middle cerebral artery. Following 30 minutes of MCA occlusion, the filament will be retracted to allow reperfusion of the MCA. Functional testing For comparison of sensorimotor deficits the foot fault test will be used. In the foot fault test, the animals are placed individually on an elevated 10-mm square wire mesh (total grid area of 40 cm x 40 cm) and are subsequently videotaped while walking freely for 2 minutes. The number of foot faults as well as the total number of total steps is counted. The percentage of foot faults is then calculated as number of foot faults of the affected limb / number of total steps of the affected limb*100. Inclusion criteria: - Duration of the MCAO procedure ≤ 60 minutes. - Sufficient occlusion of the middle cerebral artery for 30 minutes. - Successful application of the placebo / agent. Exclusion criteria: - Duration of the MCAO procedure > 60 minutes. - Insufficient occlusion of the middle cerebral artery - Intraoperative bleeding - Animal is circling after MCAO - Weight loss of ≥ 20% - Persistent abnormal posture Based on our results following Cytosporon-B application (3 hours after MCAO) in male mice (placebo infarctvolume 39.85±11.6 vs. Cytosporon-B infarctvolume 24.7±14.1), the following animal numbers were calculated (GPower 3.1.9.2): t tests - Means: Wilcoxon-Mann-Whitney test (two groups) Analysis: A priori: Compute required sample size Input Tail(s) = One Parent distribution = Normal Effect size d = 1.191 α err prob = 0.05 Power (1-β err prob) = 0.8 Allocation ratio N2/N1 = 1 Output: Noncentrality parameter δ = 2.60 Critical t = 1.74 Df = 17.1 Sample size group 1 = 10 Sample size group 2 = 10 Total sample size = 20 Actual power = 0.803 Group size is n=10. Date of registration: 03 January 2023 Study start date: 23 January 2023 Anticipated completion date: 31 May 2023 Country: Germany Funding: This study is funded by the Deutsche Forschungsgemeinschaft (CRC TRR332 TP B2) Prelimininary data has been published at https://www.biorxiv.org/content/10.1101/2022.02.27.482146v1 No conflicts of interest are to report. Contact details for further information: jan.strecker@ukmuenster.de