Recombinant Slit2 Reduces Surgical Brain Injury Induced Blood Brain Barrier Disruption via Robo4 Dependent Rac1 Activation in a Rodent Model

Abstract

Brain tissue surrounding surgical resection site can be injured inadvertently due to procedures such as incision, retractor stretch, and electrocauterization when performing neurosurgical procedures, which is termed as surgical brain injury (SBI). Blood brain barrier (BBB) disruption due to SBI can exacerbate brain edema in the post-operative period. Previous studies showed that Slit2 exhibited vascular anti-permeability effects outside the brain. However, BBB protective effects of Slit2 following SBI has not been evaluated. The objective of this study was to evaluate whether recombinant Slit2 via its receptor roundabout4 (Robo4) and the adaptor protein, Paxillin were involved in reducing BBB permeability in SBI rat model. Our results showed that endogenous Slit2 increased in the surrounding peri-resection brain tissue post-SBI, Robo4 remained unchanged and Paxillin showed a decreasing trend. Recombinant Slit2 administered 1 h before injury increased BBB junction proteins, reduced BBB permeability, and decreased neurodeficits 24 h post-SBI. Furthermore, recombinant Slit2 administration increased Rac1 activity which was reversed by Robo4 and Paxillin siRNA. Our findings suggest that recombinant Slit2 reduced SBI-induced BBB permeability, possibly by stabilizing BBB tight junction via Robo4 mediated Rac1 activation. Slit2 may be beneficial for BBB protection during elective neurosurgeries.

Figure 1

Temporal expression of endogenous Slit2, Robo4 and Paxillin in the right frontal perisurgical site after SBI. (A) Representative western blot showed that the expression of endogenous Slit2 increased in the right frontal perisurgical region at various time points after SBI. Data are expressed as mean ± SEM. ANOVA, SNK. N = 4/group. *p < 0.05 compared to Sham, ^&p < 0.05 compared to SBI 6 h. (B) The expression of Robo4 did not change at the right frontal perisurgical site up to 72 h after SBI. Data are expressed as mean ± SEM. ANOVA, SNK. N = 4/group. (C) Western blot showed a trend in reduced expression of Paxillin at 24 and 72 h after SBI at the right frontal perisurgical site. Data are expressed as mean ± SEM. ANOVA, SNK. N = 4/group. The full length blots for western blot pictures shown in panels A, B, and C are presented in Supplementary Figure S1.

Figure 2

Localization of Robo4 in the brain at the right frontal perisurgical site 24 h after SBI. (A) Representative microphotographs of immunofluorescence staining showed co-localization of Robo4 (FITC/green) with the endothelial marker von Willibrand Factor (vWF) (Rhodamine Red/red) and DAPI (blue). (B) Immunofluorescence staining showed Robo4 (FITC/green) was expressed by neurons but did not co-localize with the astrocyte marker glial fibrillary acidic protein (GFAP) (Rhodamine Red/red). (C) Immunofluorescence staining showed Robo4 (FITC/green) co-localized with neuronal marker NeuN (Rhodamine Red/red) and DAPI (blue) at the perisurgical site 24 h after SBI. Arrows and arrowheads indicate cells with positive staining. Scale bar = 50 μm.

Figure 3

Localization of Paxillin in the brain at the right frontal perisurgical site 24 h after SBI. (A) Representative microphotographs of immunofluorescence staining showing co-localization of Paxillin (FITC/green) with von Willibrand Factor (vWF) (Rhodamine Red/red), and DAPI (blue). (B) The pictures show co-localization of Paxillin (FITC/green) with GFAP (Rhodamine Red/red), and DAPI (blue). (C) Immunofluorescence staining showed Paxillin (FITC/green) did not co-localize with neuronal marker NeuN (Rhodamine Red/red). Arrows indicate cells with positive staining. Scale bar = 50 μm.

Figure 4

Effect of recombinant Slit2 on BBB junction protein expression 24 h after SBI. (A) Representative western blot analysis showed recombinant Slit2 (10 μg/Kg) administration significantly increased the expression of occludin after SBI. (B) The expression of claudin 3 showed a trend to increase after SBI with recombinant Slit2 (10 µg/Kg). (C) The expression of VE cadherin did not change after SBI. Data are expressed as mean ± SEM. ANOVA, SNK. N = 4–6/group. *p < 0.05 compared to Sham, ^†p < 0.05 compared to SBI + Vehicle. The full length blots for western blot pictures shown in panels A, B, and C are presented in Supplementary Figure S2.

Figure 5

Effect of recombinant Slit2 on BBB permeability and neurological function 24 h after SBI. (A) Evans blue dye extravasation in the right frontal perisurgical site was reduced by recombinant Slit2 (10 μg/Kg). (B) Modified Garcia test showed recombinant Slit2 (10 µg/Kg) administration improved neurological score 24 h after SBI. Data are expressed as mean ± SEM. ANOVA, SNK. N = 6–7/group. *p < 0.05 compared to Sham, ^†p < 0.05 compared to SBI + Vehicle.

Figure 6

Role of Robo4-Paxillin pathway in Slit2 mediated protection after SBI. (A) Representative western blot analysis at 24 h after SBI to show the efficacy of Robo4 knockdown using siRNA. The expression of Robo4 did not change after SBI or with recombinant Slit2 (10 μg/Kg) administration compared to Sham. The expression of Robo4 was significantly reduced with Robo4 siRNA but not with Paxillin siRNA or scramble siRNA. (B) Paxillin siRNA but not scramble siRNA significantly reduced Paxillin expression. (C) Rac1 activity assay showed recombinant Slit2 (10 μg/Kg) administration increased Rac1 activity 24 h after SBI. Robo4 siRNA and Paxillin siRNA reversed this effect but not scramble siRNA. Data are expressed as mean ± SEM. ANOVA, SNK. N = 4/group. *p < 0.05 compared to Sham, ^†p < 0.05 compared to SBI + Vehicle, ^@p < 0.05 compared to SBI + Slit2 10 μg/Kg, ^#p < 0.05 compared to SBI + Slit2 + Robo4 siRNA, ^$p < 0.05 compared to SBI + Slit2 + Paxillin siRNA. The full length blots for western blot pictures shown in panels A and B, and Rac1 activity assay shown in panel C are presented in Supplementary Figure S4.