GA attenuates the infiltration of inflammatory cells and the expression of cytokines and chemokines after SCI
Since GA prevented BSCB disruption after SCI, we examined the effect of GA treatment on the infiltration of blood cells by immunohistochemistry using antibodies against MPO and ED-1 and Western blot analysis for ED-1. Fig. 6A shows a schematic drawing depicting infiltrated neutrophils and macrophages that were observed both rostral and caudal to the lesion area at 1 d and 5 d after injury. Examination of the distribution of MPO-positive neutrophils at 1 d and ED-1-positive macrophages at 5 d cells along the length of the lesion site showed that these cells were mainly observed in the dorsal column of injured spinal cord. Furthermore, infiltrated neutrophils and macrophages were observed from the lesion site to 2500 µm, and the farther away from the lesion site, the smaller the number of cells. However, GA treatment significantly alleviated the infiltration of neutrophils and macrophages into any location when compared with vehicle control. The relative fluorescence intensity analysis shows that GA treatment significantly suppressed the infiltration of neutrophils and macrophages compared with that of the vehicle control (Fig. 6B and C). Western blot analysis also revealed that the SCI-induced increase in ED-1 was significantly reduced by GA treatment at 5 d after injury compared with that of the vehicle control (Fig. 6D).
Next we determined the effect of GA on the expression of inflammatory mediators and chemokines after SCI by RT-PCR and Western blot. The results show that the increases in Tnf-α , IL-1β (at 2 h),IL-6, Cox-2 and iNos (at 6 h) mRNA levels after SCI were significantly inhibited by GA treatment (Fig. 6E and F). In addition, GA significantly inhibited the increases in mRNA levels ofMcp-1, Mip-1α, Mip-1β, Gro-α (at 2 h) and Mip-2α (at 6 h) after injury (Fig. 6G and H). The protein levels of COX-2 and iNOS at 1 d after injury were also significantly alleviated in GA-treated group compared with the vehicle control group (Fig. 6I).