Fig. 2. L-EV treatment during the stress-treatment period blocked the stress-induced decrease in the expression of neurotrophic factors in the hippocampus and inhibited stress-induced depressive-like behaviors. (A) Experimental design (Exp. 1). Mice were treated with restraint for 2-h daily for 14 days (2h×14 d RST). Saline or L-EV (0.1 µg/kg) were i.p. injected 30 min before restraint treatment each day. Behavioral tests were performed on post-stress days 1-3 (p1~p3) and mice were sacrificed on post-stress day 7 (p7). Control mice injected with saline (CON+veh), mice treated with L-EV (CON+L-EV), mice treated with repeated restraint and injected with saline (CRST+veh), and mice treated with repeated restraint and injected with L-EV (CRST+L-EV) were prepared. (B) Body weight (g) changes of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=8~12 animals per group). (C) Expression levels of Ngf, tBdnf, Bdnf1, Bdnf4, Nt3, Nt4/5, and Trkb in the hippocampus of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=6 animals and 4~8 PCR repeats per group). Two-way ANOVA followed by Bonferroni post hoc test (Ngf, F(1,28)=2.900 and p=0.0997 for CRST main effect, F(1,28)=0.6187 and p=0.4381 for L-EV main effect, and F(1,28)=1.024 and p=0.3202 for CRST×L-EV; tBdnf, F(1,25)=0.2452 and p=0.6248 for CRST main effect, F(1,25)=15.43 and p=0.0006 for L-EV main effect, and F(1,25)=1.932 and p=0.1768 for CRST×L-EV; Bdnf1, F(1,12)=9.542 and p=0.0094 for CRST main effect, F(1,12)=15.01 and p=0.0022 for L-EV main effect, and F(1,12)=2.324 and p=0.1533 for CRST×L-EV; Bdnf4, F(1,12)=13.95 and p=0.0028 for CRST main effect, F(1,12)=9.418 and p=0.0097 for L-EV main effect, and F(1,12)=8.003 and p=0.0152 for CRST×L-EV; Nt3, F(1,28)0.9424 and p=0.3400 for CRST main effect, F(1,28)=0.1996 and p=0.6585 for L-EV main effect, and F(1,28)=1.974 and p=0.1710 for CRST×L-EV; Nt4/5, F(1,24)=14.45 and p=0.0009 for CRST main effect, F(1,24)=1.654 and p=0.2106 for L-EV main effect, and F(1,24)=8.419 and p=0.0078 for CRST×L-EV; Trkb, F(1,14)=0.0650 and p=0.8025 for CRST main effect, F(1,14)=1.768 and p=0.2049 for L-EV main effect, and F(1,14)=2.969 and p=0.1069 for CRST×L-EV). (D) Western blot data showing the expression level of proBDNF in the hippocampus of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=6 animals and 4 WB repeats per group). Two-way ANOVA followed by Bonferroni post hoc test (F(1,12)=12.29 and p=0.0043 for CRST main effect, F(1,12)=1.697 and p=0.2172 for L-EV main effect, and F(1,12)=8.952 and p=0.0112 for CRST×L-EV). (E) Expression levels of Sirt1, Hdac2, and Creb1 in the hippocampus of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=6 animals and 6–10 PCR repeats per group). Two-way ANOVA followed by Bonferroni post hoc test (Sirt1, F(1,23)=6.483 and p=0.0181 for CRST main effect, F(1,23)=12.17 and p=0.0020 for L-EV main effect, and F(1,23)=2.365 and p=0.1377 for CRST×L-EV; Hdac2, F(1,25)=10.31 and p=0.0036 for CRST main effect, F(1,25)=5.110 and p=0.0327 for L-EV main effect, and F(1,25)=1.340 and p=0.2280 for CRST×L-EV; Creb1, F(1,22)=0.2456 and p=0.6251 for CRST main effect, F(1,22)=2.829 and p=0.1067 for L-EV main effect, and F(1,22)=1.358 and p=0.2564 for CRST×L-EV). (F, G) Immobility time in the tail suspension test (TST; F) and forced swim test (FST; G) of CON+veh, CON+L-EV, CRST+veh, and CRST+L-EV (n=7~12 animals per group). Two-way ANOVA followed by Bonferroni post hoc test (TST, F(1,33)=11.90 and p=0.0016 for CRST main effect, F(1,33)=6.235 and p=0.0177 for L-EV main effect, and F(1,33)=1.665 and p=0.2059 for CRST×L-EV; FST, F(1,36)=23.84 and p<0.0001 for CRST main effect, F(1,36)=3.265 and p=0.0792 for L-EV main effect, and F(1,36)=12.17 and p=0.0013 for CRST×L-EV). Data are presented as mean±SEM. *p<0.05; **p<0.01.
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