Blood–Brain Barrier Disruption in Preclinical Mouse Models of Stroke Can Be an Experimental Artifact Caused by Craniectomy

Craniectomy in mice unilaterally increased BBB permeability to EBD and fluorescein. A1–C3, Mice were subjected to craniectomy above the right motor cortex and intravenously injected with EBD (A1–A3), fluorescein (B1–B3), or 10 kDa dextran-FITC (C1–C3) 24 h later. Extravasation of the circulating tracers into the left (contralateral) hemisphere, right (ipsilateral) hemisphere, and cerebellum was used as a measure of BBB permeability, and extravasation of the tracers into the liver and the concentrations of the tracers remaining in the blood were used as measures of tracer stability in the circulation. n = 8 mice/group for EBD (A1–A3) and n = 5 mice/time point for fluorescein (B1–B3) and 10 kDa dextran-FITC (C1–C3). The concentrations of tracers in the brain were compared by two-way repeated-measures ANOVA (matching brain regions from the same mouse) followed by Holm–Sidak multiple-comparisons test (A1, B1, and C1). The concentrations of tracers in the liver (A2, B2, and C2) and blood (A3, B3, and C3) were compared with unpaired t test. **p < 0.01, ***p < 0.001, compared with the control. n.s., No significant difference.

Craniectomy contributed to BBB disruption but not to cerebral infarction and neurodegeneration in a preclinical mouse model of stroke. A, Mice were subjected to focal cerebral ischemia induced by dMCAO or craniectomy above the right motor cortex and intravenously injected with EBD 24 h later. Extravasation of the circulating tracers into the left (contralateral, nonischemic) hemisphere and right (ipsilateral, ischemic) hemisphere was used as a measure of BBB permeability (n = 10 mice/group). The concentrations of tracers in the brain were compared by two-way repeated-measures ANOVA (matching brain regions from the same mouse) followed by Holm–Sidak multiple-comparisons test. *p < 0.05; ***p < 0.001, compared with the control or for dMCAO versus craniectomy. n.s., No significant difference. B, C, Mice were subjected to focal cerebral ischemia induced by dMCAO or craniectomy above the right motor cortex and killed 24 h later to prepare coronal brain sections, which were stained with TTC or Fluoro-Jade to identify infarcted brain regions (B) and assess neurodegeneration (C), respectively. Each image in B and C is representative of n = 3 mice/group; there were no noticeable differences between individual mice from the same group.

dMCAO unilaterally increased cerebral extravasation of endogenous albumin in mice. A, Mice were subjected to focal cerebral ischemia of the right motor cortex and perfused to remove circulating albumin from the blood 3, 6, or 12 h later, and extravasation of albumin into the left (contralateral, nonischemic) and right (ipsilateral, ischemic) hemispheres was assessed by Western blotting. Control mice exhibited no cerebral ischemia. B, Summarized results for A. C, Mice were subjected to focal cerebral ischemia of the right motor cortex and perfused to remove circulating albumin from the blood 12, 24, 48, or 72 h later, and extravasation of albumin into the left (contralateral, nonischemic) and right (ipsilateral, ischemic) hemispheres was evaluated by Western blotting. D, Summarized result for C. In B and D, albumin extravasation was compared by two-way repeated-measures ANOVA (matching brain regions from the same mouse) followed by Holm–Sidak multiple-comparisons test. **p < 0.01, ***p < 0.001. n.s., No significant difference.

Craniectomy contributed to increased cerebral extravasation of endogenous albumin in a preclinical stroke model independent of dural tearing. A, Mice were subjected to craniectomy above the right motor cortex and perfused to remove circulating albumin from the blood 3 or 24 h later, and extravasation of albumin into the left (contralateral) and right (ipsilateral) hemispheres was assessed by Western blotting. B, Summarized result for A. C, Mice were subjected to focal cerebral ischemia induced by dMCAO to or craniectomy above the right motor cortex and perfused to remove circulating albumin from the blood 24 h later, and extravasation of albumin into the left (contralateral, nonischemic) and right (ipsilateral, ischemic) hemispheres was assessed by Western blotting. D, Summarized data for C. E, To determine the effect of dural tearing, rats were injected with Evans blue dye to facilitate identification of the dura mater during craniectomy and were perfused to remove circulating albumin from the blood 24 h later; and extravasation of albumin into the left (contralateral, nonischemic) and right (ipsilateral, ischemic) hemispheres was evaluated by Western blotting. F, Summarized data for E. In B, D, and F, albumin extravasation was compared by two-way repeated-measures ANOVA (matching brain regions from the same mouse) followed by Holm–Sidak multiple-comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., No significant difference.

BBB disruption caused by craniectomy was region specific. A–F, Mice were subjected to craniectomy above the right motor cortex and perfused to remove circulating albumin from the blood 24 h later, and extravasation of albumin into the left (contralateral) and right (ipsilateral) prefrontal cortex (A, B), motor cortex (C, D), and striatum (E, F) was assessed by Western blotting. B, Summarized result for A. D, Summarized result for C. F, Summarized result for E. In B, D, and F, albumin extravasation was compared by two-way repeated-measures ANOVA (matching brain regions from the same mouse) followed by Holm–Sidak multiple-comparisons test. ***p < 0.001. n.s., no significant difference.