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).