Critical role of EphA4 in early brain injury after subarachnoid hemorrhage in rat

Abstract

Early brain injury (EBI) is reported as a primary cause of mortality in subarachnoid hemorrhage (SAH) patients. Eph receptor A4 (EphA4) has been associated with blood-brain barrier integrity and pro-apoptosis. We aimed to investigate a role of EphA4 in EBI after SAH. One hundred and seventy-nine male adult Sprague-Dawley rats were randomly divided into sham versus endovascular perforation model of SAH groups. SAH grade, neurological score, Evans blue dye extravasation, brain water content, mortality, Fluoro-Jade staining, immunofluorescence staining, and western blot experiments were performed after SAH. Small interfering RNA (siRNA) for EphA4, recombinant Ephexin-1 (rEphx-1), and Fasudil, a potent ROCK2 inhibitor, were used for intervention to study a role of EphA4 on EBI after SAH. The expression of EphA4, Ephexin-1, RhoA, and ROCK2 significantly increased after SAH. Knockdown of EphA4 using EphA4 siRNA injection intracerebroventricularly (i.c.v) reduced Evans blue extravasation, decreased brain water content, and alleviated neurobehavioral dysfunction after SAH. Additionally, the expression of Ephexin-1, RhoA, ROCK2 and cleaved caspase-3 were decreased. Tight junction proteins increased, and apoptotic neuron death decreased. The effects of EphA4 siRNA were abolished by rEphx-1. In contrast, Fasudil abolished the effects of rEphx-1. These results suggest that EphA4, a novel and promising target for treatment, exacerbates EBI through an Ephexin-1/ROCK2 pathway after SAH.

Keywords: Blood-brain barrier; EBI; EphA4; Neuronal apoptosis; Subarachnoid hemorrhage.

Fig. 1.

Expression changes of EphA4, Ephexin-1, RhoA, and ROCK2 after SAH. A, representative western blot images. B, C, D, E, quantitative analyses of time-dependent expression of EphA4, Ephexin-1, RhoA, and ROCK2 in the left hemisphere at 24 h after SAH. Relative densities of each protein have been normalized against the Sham group. n = 6 per group. *P < 0.05 vs. Sham.

Fig. 2.

Cellular localization of EphA4 after SAH. Representative images of immunofluorescent staining of EphA4 (red) and neurons (NeuN, green) (A), EphA4 (green) and astrocytes (GFAP, red) (B), and EphA4 (red) and microglia (Iba-1, green) (C) in Sham and SAH rats after 24 h surgery. Nuclei are stained with DAPI (blue). Top panel indicates the location of staining (small black box). Arrows indicate colocalization of EphA4/NeuN, EphA4/GFAP, and EphA4/Iba-1. n = 3 per group. Bar = 30 μm.

Fig. 3.

The effect of Ephrin A4 (EphA4) knockdown on neurological deficits and BBB disruption after subarachnoid hemorrhage (SAH). In rats receiving intracerebraventricular (i.c.v.) administration of small interfering ribonucleic acid (siRNA) for EphA4, the modified Garcia (A) and beam balance score (B) were significantly higher, and brain water content (C) and Evans blue extravasation (D) were lower at 24 h after SAH. n = 6 per group. Vehicle, phosphate-buffered saline; *P < 0.05 vs. Sham; ^@P < 0.05 vs. SAH + Vehicle; ^#P < 0.05 vs. SAH + Scramble siRNA.

Fig. 4.

Ephrin receptor A4 (EphA4) knockdown upregulates the expression of tight junction proteins, and inhibits cleaved caspase 3 (CC-3) from the left hemisphere at 24 h after SAH. Silencing EphA4 by using small interfering ribonucleic acid (siRNA) significantly decreases the expression of EphA4 (A). Representative western blot images and quantitative analysis of Ephexin-1 (B), ROCK2 (C), zonula occludens-1 (ZO-1) (D), Claudin-5 (E), and CC-3 (F) from the left hemisphere at 24 h after SAH. n = 6 per group. rEphx-1, recombinant Ephexin-1; Vehicle, phosphate-buffered saline; *P < 0.05 vs. Sham; ^@P < 0.05 vs. Vehicle; ^#P < 0.05 vs. Scramble siRNA; ^&P < 0.05 vs. EphA4 siRNA; ^$P < 0.05 vs. EphA4 siRNA + rEphx-1.

Fig. 5.

Effect of Ephrin receptor A4 (EphA4) knockdown on apoptotic neuron death after SAH. (A) Representative images of Fluoro-Jade staining at 24 h hours after SAH. (B) Quantitative analyses of mean apoptotic cell counts from 8 fields of left hemisphere at 24 h hours after SAH (NO/mm²). n = 3 per group. Vehicle, phosphate-buffered saline; siRNA, small interfering ribonucleic acid; rEphx-1, recombinant Ephexin-1. Top panel indicates the location of staining (small black box). ^*P < 0.05 vs. Sham; ^@P < 0.05 vs. Vehicle; ^#P < 0.05 vs. Scramble siRNA; ^&P < 0.05 vs. EphA4 siRNA; ^$P < 0.05 vs. EphA4 siRNA + rEphx-1. Bar = 50 μm.