Sheep models for cerebrovascular pathology

Worldwide, 5.5 M people die from stroke each year. Currently, stroke is still the third cause of death in industrialized countries and the leading cause of permanent disability in adulthood. About 78% of the incident stroke patients suffer from cerebral ischemia by occlusion of a cerebral vessel, while hemorrhage accounts for ~17% of all strokes [1]. Both conditions are medical emergencies with limited time for treatment. Recanalization by rtPA or thrombectomy are the only available therapeutic options, both being additionally hampered by a number of exclusion criteria. Despite thorough research, mostly in rodent models, no alternative treatment option has so far been successfully translated into clinical routine. Consequently, international academic and industrial expert consortia (STAIR, STEPS, [2]) suggest preclinical research strategies including validation of novel treatment strategies in gyrencephalic animal models. As a result, we subjected adult Merino sheep to (1) stereotaxic application of autologous blood as a model for intracerebral hemorrhage (ICH) [3], or to (2) territorial infarction by surgical occlusion of the middle cerebral artery (MCAO) [4]. We show the characteristics of perilesional deficits after ICH and the evaluation of a novel nicotinic acetylcholine receptor (nAChR) tracer for the identification of inflammation after MCAO by positron-emission-tomography (PET). All experimental procedures were approved by the local authority (animal licenses TVV33/12 and TVV56/15).
The ICH model led to ipsilateral GM and WM ablation and lateralization. The ipsilateral lateral ventricle was compressed and the contralateral ventricle dilated . Voxel-based-morphometry supports the compression of the ipsilateral ventricle, while the ventricle of the olfactory bulb was dilated bilaterally. Diffusion-weighed-imaging (DWI) revealed free diffused water of the hematoma, which was surrounded by reduced diffusion. The diffusion/perfusion mismatch was
calculated with 0.56 (Figure 1). Histological examination of the perilesional zone revealed hypoxic-ischemic neurons and WM vacuolation including axonal degenerations. The plasma protein fibrinogen was detected around small arteries distant from the cavity.
The MCAO model includes the transcranial surgical occlusion of all 3 branches of the MCAO, subsequently was confirmed by MR angiography. Acute ischemic alteration at day 1 were detected by [15O]H2O brain perfusion PET, MR perfusion and DWI, while structural MRI revealed the infarction at d7 and d14.. The infarctions were delineated at day 7 and 14 by FLAIR sequence. However, prominent edema complicated segmentation procedures. PET/MRI showed decreased nAChR tracer signal in the ischemia-related brain regions on day1 and day7, but a significant signal increase in the peri-infarct region on day14. These PET findings were confirmed by ex vivo autoradiography. Preliminary histopathological evaluation attributed the PET signal increase to glial activation and macrophage infiltration.
Peri-hematomal characteristics of the ICH altered brain tissue are comparable to the findings reported in human beings and other animal models. We show preliminary evidence for a protocol to visualize post-stroke neuroinflammation by PET/MRI. These results supports a potential role for our ovine models in translational stroke research.
Fig1: Intracerebral hemorrhage (ICH) in sheep: PWI (perfusion) and DWI (diffusion) were aligned to T2w turbo-spin-echography, resliced and smoothed . Crosshairs located at [-17, 0, 8].
Reference List
(1) Heuschmann, P. U., Busse, O., Wagner, M., Endres, M., Villringer, A., Röther, J., Kolominsky-Rabas, P. L., and Berger, K. Frequency and Care of Stroke in Germany. Akt Neurol. 2010. 37(7), 333-40.
(2) STAIR-group. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999. 30(12):2752-58.
(3) Nitzsche, B., Lobsien, D., Geiger, K., Barthel, H., Zeisig, V., Boltze, J., and Dreyer, A. Large Mammals - translational stroke models in sheep. Amos, K. (Ed.). 9th International congress on vascular dementia. Ljubljana. 2015. Medimont. 2016.
(4) Nitzsche, B., Barthel, H., Lobsien, D., Boltze, J., Zeisig, V., and Dreyer, A. Focal cerebral ischemia by permanent middle cerebral artery occlusion in sheep - surgical technique, clinical imaging and histopathological results. In: Janowski, M., (Ed.) Experimental Neurosurgery in Animal Models .1 ed. Humana Press; 2016.