Characterization of a new model of thromboembolic stroke in C57 black/6J mice

Abstract

This study characterizes a new model of thromboembolic stroke of the middle cerebral artery in C57 black/6J mice, thus offering an opportunity to use the model for studying ischemic stroke in transgenic mice. Thromboembolic stroke was induced by local injection of either 1.5 or 3.0 UI of thrombin directly into the right MCA of C57 black/6J mice. Cerebral blood flow (CBF) velocity was measured continuously by laser Doppler flowmetry, which allowed documentation of both MCA occlusion and of spontaneous recanalization. After 24 h, all animals were euthanized. Cryosections were cut at 400-μm intervals and silver stained with the high-contrast method for volumetric assessment of infarct size. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), caspase-3 and hsp 70 protein levels were investigated by immunofluorescence. Thrombin injection resulted in clot formation in all animals. Cortical infarction occurred in 63% of the mice while 37% had a spontaneous MCA recanalization during the first 20 min following thrombin injection. In cases of successful MCA occlusion with consequent infarction, the clot was stable up to 2 h after formation. Subsequently, 20% recanalized spontaneously. Infarctions were restricted to the cortex with a mean lesion volume of 36 ± 5 for 1.5 UI and 56 ± 8 for 3.0 UI thrombin. Protein levels of IL-6, TNF-α, caspase-3, and hsp 70 were significantly increased after MCAO. The results demonstrate that the mouse thromboembolic stroke model produces cortical infarctions of consistent size in C57 black/6J mice, which is dependent upon the amount of thrombin used for clot formation. Spontaneous MCA recanalization occurs after 2 h of ischemia in 20% of mice. Thus, the thromboembolic model is an applicable stroke model for C57 black/6J mice, which mimics many of the features of human stroke, including spontaneous recanalization. However, strain differences between Swiss and C57 black/6J mice must be taken into account when using the model.

Fig. 1

Illustration of the occlusion of the MCA. In situ thrombin injection in the bifurcation and subsequent clot formation. The branches of the MCA become dark red because of the obstructed blood flow and a white clot becomes visible in the bifurcation of the MCA

Fig. 2

Detailed description of the experiments and surgery outcome

Fig. 3

Flow chart of the experiments

Fig. 4

Illustration of clot stability for 1.5 units thrombin. Data were obtained by laser Doppler flowmetry and data are expressed as mean ± s.e.m. values, n = 6

Fig. 5

Size of infarct volume. Data are expressed as mean ± s.e.m.; 1.5 units thrombin n = 9, 3.0 units thrombin n = 9

Fig. 6

Representative hematoxylin–eosin–saffron (HES) staining for 1.5 units thrombin

Fig. 7

Bar graphs showing semi-quantification of fluorescence intensity for IL-6, TNF-α, caspase 3 and hsp 70 protein levels. Data are presented as the mean percentage relative to control ± s.e.m.; n = 9–13, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, significant difference between control groups and MCAO. Statistical analyses were performed using Kruskal–Wallis non-parametric test together with Dunn's post-hoc test

Fig. 8

Sections from the brain tissue showing IL-6, TNF-α, caspase 3, and hsp 70 protein expressions. The images represent the control and MCAO. There are significant increases in IL-6, TNF-α, caspase 3 and hsp 70 protein levels in the MCAO group compared to the control groups. Data were obtained with confocal microscopy