PRF-DRG improves the neuropathic pain-induced depressive behaviors in rats with SNI
First, we examined the changes in the mechanical pain threshold in rats with SNI. Compared with that in the sham group, the 50% PWT in the SNI group was significantly reduced after SNI (P < 0.05, Additional file 1: Table 1, Table 2, and Fig. 2), and reached a peak on day 7 after SNI; thereafter, it maintained a lower level until the end of the observation. The occurrence of mechanical allodynia indicated the successful establishment of the neuropathic pain model. Second, we explored whether depressive behaviors could be induced by neuropathic pain in the present study. On day 42 after SNI, the sucrose preference rate was significantly reduced (P < 0.05, Fig. 3a), whereas the forced swimming immobility time was remarkably prolonged (P < 0.05, Fig. 3b) in the SNI group compared to the sham group. However, there were no significant differences in the sucrose preference rate and forced swimming immobility time among the six groups on day 21 after SNI. These findings indicated that in the present study, SNI induced depressive-like behaviors in rats on day 42 but not on day 21 after SNI. Third, on day 42 after SNI, the 50% PWT (P < 0.05, Additional file 1: Table 1, Table 2, and Fig. 2) and sucrose preference rate were increased (P < 0.05, Fig. 3a), while the forced swimming immobility time was reduced (P < 0.05, Fig. 3b) in the PRF group compared with those in the SPRF and SNI groups. Thus, PRF-DRG could ease the depressive behaviors induced by SNI.
Table 1 Statistical table of 50% PWT at different times for each group (mean ± SD)
An additional .txt file shows this in more detail [see Additional file 1]
Table 2 50%MWT repeated measurement ANOVAs results
Source of Variation
|
SS
|
DF
|
MS
|
F value
|
P value
|
Time x Column Factor
|
801.5
|
45
|
17.81
|
23.81
|
P < 0.0001
|
Time
|
1622
|
9
|
180.2
|
240.9
|
P < 0.0001
|
Column Factor
|
4818
|
5
|
963.5
|
1534
|
P < 0.0001
|
Subject
|
33.93
|
54
|
0.6282
|
0.8399
|
P= 0.7837
|
Residual
|
363.5
|
486
|
0.748
|
|
|
SS: sum of aquares of deviation from mean; DF: degree of freedom; MS: mean aquare
As can be seen from Table 2 overall, the difference of 50% PWT in each group was statistically significant, and the difference of 50% PWT at each time point was statistically significant. At the same time, there was a cross effect between the group and time, so it could be considered that the 50% PWT was not identical at different time points, and the rate of change at different times in different groups was not identical. In contrast, we can see that sham>PRF=IRF8 siRNA>SNI=SPRF=NFW, indicating that the 50% PWT in the SNI group, PRF group, IRF8 siRNA group, SPRF group, and NFW group were significantly reduced after SNI, and reached a peak on day 7 after SNI, and 50% PWT was increased in the PRF group and IRF8 siRNA compared with those in the SPRF, SNI, and NFW groups. There were no significant differences in the 50% PWT between the PRF and IRF8 siRNA groups.
PRF-DRG therapy increases the hippocampal BDNF by inactivating the spinal IRF8 to involve in the improvement of depressive-like behaviors in rats with SNI
On day 42 after SNI, we found that the 50% PWT (P < 0.05, Additional file 1: Table 1, Table 2, and Fig. 2) and the sucrose preference rate were increased (P < 0.05, Fig. 3a), while the immobility time in the FST was reduced (P < 0.05, Fig. 3b) in the IRF8 siRNA group compared with those in the NFW and SNI groups. Thus, intrathecal injection of IRF8 siRNA could protect against depressive-like behaviors induced by SNI in rats. We next evaluated the expression of BDNF in the hippocampus and IRF8 in the spinal cord of rats by IHC (Fig.4), qRT-PCR (Fig.5), and WB (Fig.6). The levels of hippocampal BDNF were remarkably decreased, and spinal IRF8 was significantly increased in the SNI and NFW groups compared with those in the sham group on days 21 (P < 0.05) and 42 after SNI (P < 0.05). We further examined whether hippocampal BDNF expression could be influenced by spinal IRF8 activation. Biochemical analysis showed that intrathecal injection of IRF8 siRNA reversed the increased spinal IRF8 (P < 0.05) and decreased hippocampal BDNF expression on days 21 (P< 0.05) and 42 (P < 0.05) in the IRF8 siRNA group compared with those in the NFW and SNI groups. Therefore, these results indicate that spinal IRF8 negatively regulates hippocampal BDNF expression.
Similar to the IRF8 siRNA group, biochemical analysis (Fig. 4-6) showed that PRF-DRG therapy inhibited the enhanced expression of spinal IRF8 (P < 0.05) while reversing the reduction in hippocampal BDNF expression in the PRF group compared to that in the SPRF group on days 21 (P < 0.05) and 42 (P < 0.05) after SNI. There were no significant differences in the depressive-like behaviors, the levels of spinal IRF8, and hippocampal BDNF between the PRF and IRF8 siRNA groups on days 21 and 42 after SNI. Since spinal IRF8 activation could mediate the reduction of BDNF in the hippocampus, we considered that PRF-DRG therapy could mimic the IRF8 siRNA to improve depressive-like behaviors. Thus, PRF-DRG probably increased hippocampal BDNF by inactivating spinal IRF8 to improve pain and depression comorbidity.
PRF-DRG therapy also negatively regulates hippocampal IL-1β and TNF-α by inactivating spinal IRF8 to ameliorate depressive-like behaviors in rats with SNI
We also evaluated the expression levels of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) in the hippocampus of rats by ELISA (Fig. 7). On day 42 after SNI, the concentrations of hippocampal IL-1β and TNF-α were increased (P < 0.05) in the SNI, SPRF, and NFW groups compared with those in the sham group (P < 0.05). There were no significant differences in IL-1β and TNF-α among the six groups on day 21 and in IL-6 among the six groups on days 21 and 42. These data indicate that hippocampal IL-1β and TNF-α are involved in neuropathic pain-induced depression. Compared with those in the SNI, SPRF, and NFW groups, intrathecal injection of IRF8 siRNA or PRF-DRG treatment resulted in a significant reduction in hippocampal IL-1β and TNF-α concentrations on day 42 after SNI (P < 0.05, Fig. 7). There were no significant differences in IL-1β and TNF-α levels between the PRF and IRF8 siRNA groups on day 42. Hence, PRF-DRG therapy could mimic intrathecal injection of IRF8 siRNA to reduce the levels of hippocampal IL-1β and TNF-α. In summary, PRF-DRG decreased hippocampal IL-1β and TNF-α by inactivating spinal IRF8 to participate in pain and depression comorbidity improvement.