Inhibiting 12/15-lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis models

Abstract

12/15-Lipoxygenase (12/15-LOX) contributes to the brain damage after middle cerebral artery occlusion (MCAO) in the acute phase of stroke. The aim of this study was to investigate the effects of a 12/15-LOX inhibitor, LOXBlock-1(LB1), in mice using a FeCl₃-induced permanent distal MCAO model and FeCl₃-induced ischemia/thrombolysis with tPA. In order to induce permanent distal MCAO, 30% FeCl₃ was used in C57BL6 mice. LB1 or DMSO treatments were applied intraperitoneally 2 h following MCAO. For FeCl₃-induced ischemia/thrombolysis experiments, 10% FeCl₃ was preferred so as to obtain reperfusion with tPA in CD1 mice. 4 h following ischemia either LB1 or DMSO and iv tPA was administered. Outcomes were NSS, weight loss, infarct volume, hemorrhage area and reperfusion rate. FeCl₃-induced distal MCAO caused an increase in 12/15-LOX signal in the ischemic cortex with an increase in MDA2 and AIF immunoreactivity. LB1 treatment, applied 2 h after ischemia, significantly decreased the infarct volume at 24 h of permanent distal MCAO. Weight loss was also significantly reduced in LB1 treated group. Distal MCAO and tPA application with LB1 or DMSO showed that treatment significantly decreased the infarct volume and the hemorrhage area. The reperfusion rate in the LB1-treated group was surprisingly higher than in the DMSO group and NSS results were significantly improved. These data suggest that LB1 can be used as an adjuvant agent to tPA. This study not only shows the effects of LB1 treatment in distal MCAO but also confirms that FeCl₃-induced MCAO model can be a useful tool to screen novel treatment options in stroke.

Keywords: 12/15-LOX; FeCl(3); Reperfusion; Stroke models; tPA.

Fig. 1

LOX, MDA2 and AIF immunoreactivity after experimental stroke model. A. 30% FeCl₃ induced distal MCAO caused LOX immunoreactivity increased (arrows) in the ischemic cortex (especially in peri-infarct area) (A, J) compared to contralateral intact cortex (D, M) and sham operated brains (G, P). This LOX immunopositivity was colocalised with MDA2 immunoreactivity (B, arrows) and AIF immunoreactivity (K, arrows) which were not positive in contralateral cortex (E, N) and sham operated cortex (H, Q). Merged images of LOX, MDA2 or AIF and ToPro3 are shown in C, F, I, L, O, R. Scale bars= 20 um).

Fig. 2

30% FeCl₃ induced MCAO experiments. Systemic LB1 administration, 2 hours after ischemia, significantly decreased infarct volume at 24 hours of stroke (A). A representative infarct picture with TTC staining of LB1 treated brain (inset). NSS were in favor of LB1 treated group (B). Weight loss of treatment group was significantly lower compared to vehicle injected group (C).

Fig. 3

Distal MCAO induced by 10 % FeCl₃ and thrombolysis with tPA plus LB1 or vehicle treatment results. Systemic LB1 treatment plus tPA showed decreased infarct volume (A) along with hemorrhage area (B) at 4 hours after ischemia (p<0.05). LB1 administration also significantly reduced NSS compared to vehicle group (C). Weight loss in the treatment group was lower than in the vehicle group, but the difference was not statistically significant (D).