LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats

Abstract

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250-280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

Figure 1. Effect of LXW7 on P-Flk (A, C) and vascular endothelial growth factor (VEGF) expression (B, D) in rats with middle cerebral artery occlusion (MCAO) treated with LXW7 (LXW7 group), PBS (ischemia group) and sham-operated rats (sham group) at 24 h. Arrowheads indicate positive cells in the brain tissue. Scale bar: 50 μm. Data are reported as means±SD (n=6 for each group). ^#P<0.05 compared to sham group, *P<0.05 compared to ischemia group (ANOVA, followed by Fisher's post hoc test).

Figure 2. Effect of LXW7 on brain water content (A), neurological deficits (B), and brain infarct volume (C, D) in rats with middle cerebral artery occlusion (MCAO) treated with LXW7 (LXW7 group), PBS (ischemia group) and sham-operated rats (sham group) at 24 h. Data reported as means±SD (n=6 for each group). ^#P<0.05 compared to sham group, *P<0.05 compared to ischemia group (ANOVA, followed by Fisher's post hoc test).

Figure 3. Protein levels for proinflammatory cytokines TNF-α, IL-1β, and marker of microglia Iba1 in the peri-ischemic brain tissue 24 h after middle cerebral artery occlusion in rats treated with LXW7 (LXW7 group), PBS (ischemia group) and sham-operated rats (sham group). Expression of TNF-α is shown in panel A, and IL-1β in panel B, by ELISA analysis. Representative western blots are shown in panels C, D, and E. Data are reported as means±SD (n=6 for each group) and corrected by β-actin. ^#P<0.05 compared to sham group, *P<0.05 compared to ischemia group (ANOVA, followed by Fisher's post hoc test).

Figure 4. Treatment of middle cerebral artery occlusion rats with LXW7 (LXW7 group) resulted in the reduction of TNF-α, and IL-1β expression in activated microglia. Confocal images showing the expression of TNF-α (A, D, G, green), IL-1β (J, M, P, green) in microglia (B, E, H, K, N, Q, red), in peri-ischemic brain tissue 24 h after MCAO in rats (D-F, M-O) and following treatment with LXW7 (A-C, J-L) (n=6 for each group). Expression of TNF-α and IL-1β in microglia cells (arrows) is markedly enhanced following MCAO, but a noticeable reduction in TNF-α and IL-1β expression can be observed in the activated microglia in the LXW7-treated rat brain. Activated microglia were reduced with LXW7 treatment (B, K). Scale bars: 50 µm.