Background and Objectives
Animal models relevant to man are indispensable in investigating the efficacy of new therapeutic modalities. The importance of both white matter and grey matter damage, in experimental stroke research is also being increasingly recognized. In accordance with the demands for experimental stroke models to include species with gyrencephalic brains, we have focused on the miniature pig. This paper provides information on both the surgical technique for middle cerebral artery occlusion (MCAO) and the methods of quantitation of ischemic damage to both grey and white matter using line diagrams of the miniature pig brain.
Methods
Sixteen miniature pigs weighing 14–24 kg were randomly divided into three groups: permanent MCAO (n=5), permanent internal carotid artery occlusion (ICAO) (n=6) and a sham operated group (n=5). The operation was performed under general anesthesia via a frontotemporal approach. MCAO and ICAO was induced by electrocoagulation of the MCA from a point proximal to the origin of the lenticulostriate artery to a distal point where it crosses the olfactory tract and the ICA at a point proximal to the posterior communicating artery, respectively. At 24 hours after surgery the volume of ischemic damage was evaluated by MRI and then the animal was sacrificed, a coronal brain sections stained with haematoxylin and eosin (grey matter damage) or amyloid precursor protein (APP) immunohistochemistry (white matter damage). Quantitative histopathology was used to map the infarct onto 16 pre-selected line diagrams.
Results
Histological examination and MRI findings demonstrated no areas of ischemia in the cerebral hemispheres of any of the sham-operated animals. By contrast, in the MCAO group, all animals had ischemic damage in basal ganglia and frontal cortex. The volume of the infarct in the MCAO and the ICAO measured directly from MRI scans was 16.2 ± 1.1 and 1.5 ± 0.5 cm3 (mean ± SD), respectively. There was a good correlation between the histopathological and MRI findings (r2= 0.86, p < 0.0001) (Figure 1) although the infarct volume measured by quantitative histopathology was smaller than that measured by MRI, since mapping onto line diagrams reduces the impact of cerebral edema and brain swelling on measurement of infarct volume. For quantification of axonal damage immunohistochemical study using APP antibody was used and provided total APP score of 43.8 ± 3.5 (mean ± SD) in the MCAO.
Conclusions
This paper describes a simple and reproducible method for the induction of white and grey matter damage in focal cerebral ischemia with minimum invasiveness. This miniature pig stroke model has utility for studying the pathophysiology of ischemia in gyrencephalic brain and for the assessing the therapeutic efficacy of drugs prior to initiating human clinical trials.