Optimising the photothrombotic model of stroke in the C57BI/6 and FVB/N strains of mouse

Abstract

The photothrombotic stroke model relies on the interaction between photosensitive-dye and light for clot formation. Interestingly, the relationship between the length of light exposure and stroke-outcome has never been examined. This model has yet to be established in the FVB/N strain, even though stroke-outcomes are strain-specific. Therefore, this study aimed to examine the effect of different lengths of light exposure in two strains of mice on photothrombotic stroke. Male FVB/N and C57Bl/6 mice were subjected to stroke using 15, 18, or 20-min light exposure. Mice underwent functional testing for up to 7 days. Infarct volume was assessed with thionin staining, and cellular responses to injury analysed via immunofluorescence at 7-days post-stroke. Blood brain barrier (BBB) breakdown was assessed using Evans blue dye at 4.5-h post-stroke. Increasing light exposure from 15 to 20-min increased infarct volume but not functional deficit. Interestingly, there were strain-specific differences in functional outcomes, with FVB/N mice having less deficit on the hanging wire test than C57BI/6 after 15-min of light exposure. The opposite was seen in the adhesive removal test. There was no difference in the number of neurons, astrocytes, microglia, macrophages, and T cells between the strains, despite FVB/N mice demonstrating greater BBB breakdown and an enlarged spleen post-stroke. Increasing light exposure systematically increases infarct volume but does not worsen functional outcomes. FVB/N and C57Bl/6 mice exhibit subtle differences in functional outcomes post stroke, which highlights the need to choose tests which are appropriate for the mouse strain being used.

Figure 1

(a) Representative images of thionin stained coronal brain sections and infarct volumes mm³ (b) from either sham operated non-stroked animals (C57Bl/6 n = 11, FVB/N n = 10), or animals with light exposures of 15 min (C57Bl/6 n = 8, FVB/N n = 16), 18 min (C57Bl/6 n = 10, FVB/N n = 14), and 20 min (C57Bl/6 n = 8, FVB/N n = 15) during stroke induction. Infarct area signified with a red line. Data are presented as mean ± SEM; *P < 0.05, ***P < 0.001 vs 15 min in respective group, two-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar represents 1 mm.

Figure 2

C57Bl/6 mice have greater motor impairment and stroke-induced weight loss compared to FVB/N mice. In contrast, FVB/N mice are more sensitive to the adhesive removal test. Effects of light exposures of 15 (C57Bl/6 n = 8, FVB/N n = 16), 18 (C57Bl/6 n = 10, FVB/N n = 14), and 20 min (C57Bl/6 n = 8, FVB/N n = 15) during stroke induction, prior to stroke, and 1, 3, and 7 days after stroke on: The hanging wire test in C57Bl/6 mice (a) or FVB/N mice (b), expressed as latency to fall (s); the adhesive removal test for C57Bl/6 (c) and FVB/N (d) mice, expressed as time to remove adhesives (s); change in body weight for the C57Bl/6 (e) and FVB/N (f) strains of mice, expressed as a change in percentage of the pre-stroke baseline weight. Data are presented as mean ± SEM, with *P < 0.05 vs pre-stroke, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs sham, ^$P < 0.05 vs 15 min, two-way RM ANOVA followed by Tukey’s multiple comparisons test.

Figure 3

The mean number of NeuN + cells per region, illustrating the number of viable neurons (A); the mean percentage of fluorescence per region illustrating the amount of GFAP + staining for reactive astrocytes (B); the mean percentage of fluorescence per region of Iba1 + staining for microglia (C) in the non-stroke hemisphere, and the infarct, peri-infarct and undamaged regions of the stroke hemisphere in C57Bl/6 and FVB/N mice subjected to 18 min of light exposure during photothrombosis, or sham operated non-stroked mice. Data are represented as mean ± SEM, with ^#P < 0.05, ^###P < 0.001 vs sham, two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 4

The total number of CD3 + cells per hemisphere of the brain in C57Bl/6 and FVB/N mice subjected to 18 min of light exposure during photothrombosis, or sham operated non-stroked mice. Data are represented as mean ± SEM, with *P < 0.05 vs non-stroke hemisphere, two-way ANOVA followed by Sidak’s multiple comparisons test.

Figure 5

FVB/N mice have greater BBB breakdown compared to C57Bl/6 mice after 18 min of photothrombosis. Representative images (a) and quantification (b) of evans blue dye extravasation in the brain following 18 min of photothrombosis or sham surgery in the C57Bl/6 and FVB/N strains of mice. ^###P < 0.001 vs sham, **P < 0.01 vs C57Bl/6, one-way ANOVA followed by Tukey’s multiple comparisons test.