| Objectives:1.To explore whether TLR4 and TNF-α in hippocampus are involved in the occurrence and maintenance of neuropathic pain in CCI rats.2.To explore whether intra-hippocampal CA1 injection of minocycline or miR-181 c agomir may produce an analgesic effect through down-regulating the expression of TLR4 and TNF-α in hippocampus of CCI rats.Methods:We randomly selected SD male rats with 5~6 weeks old.Two guide cannulaes were inserted into the hippocampus of all rats.1.All rats were randomized into three groups(n=8):(1)sham group(sham operation);(2)vehicle-treated CCI group(intra-hippocampal CA1 injection of 0.5 μL PBS);(3)Minocycline-treated group(intra-hippocampal CA1 injection of 0.5 μL minocycline).The animal in sham group and the CCI+PBS group received CCI surgery and injection of0.01 MPBS.In minocycline-treated group,minocycline(0.5 μL)was injected twice a day for consecutive 7 days(2 μg/μl,twice a day).The MWT(mechanical withdraw threshold)was evaluated at 1 h after drug administration before the surgery and on 1,3,5 and 7days after surgery.The expression of Iba-1,TLR2,TLR4,IL-1β,TNF-α and i NOS at the m RNA level in hippocampus were assessed by using RTFQ-PCR(real-time fluorescence quantitative PCR)at 7 post-operative days.The content of IL-1β and TNF-α in hippocampus were examined by ELISA at 7 post-operative days.2.All rats were randomized into four groups(n=8):(1)sham group(sham operation),(2)vehicle-treated CCI group,(3)negative control-treated CCI group(intra-hippocampal CA1 injection of 1 n M negative control),(4)miR-181 c agomir-treated group(intra-hippocampal CA1 injection of 1 n M miR-181 c agomir).Since the third day after operation,different drugs were applied.The sham group and vehicle-treated CCI group received normal saline(1 μL)for consecutive 4 days.The negative control-treated group received negative control(1 μL)for consecutive 4 days.The miR-181 c agomir-treated group received 1n M miR-181 c agomir(1 μL,once a day)for consecutive 4 days.The MWT was evaluated before the surgery and on 1,3,5 and 7 days after surgery.The expression of miR-181 c,Iba-1,TLR4,IL-1β,TNF-α and i NOS in hippocampus were assessed by using RTFQ-PCR at 7 post-operative days.The content of IL-1β and TNF-αin hippocampus were examined by ELISA at 7 post-operative days.Results:1.Compared with sham group,CCI rats displayed significantly decreased MWT on day1 after nerve injury(P<0.01),and the allodynia was sustained for 7 days.Repeated administrated of minocycline markedly suppressed this decreased in MWT after nerve injury(compared with PBS-treated CCI group,P<0.05).2.The RTFQ-PCR results indicated that,compared with sham group,the expression of Iba-1,TLR2,TLR4,IL-1β,TNF-α and i NOS m RNA in hippocampus were significantly increased in CCI rat(P<0.01).Intra-hippocampal CA1 injection of minocycline obviously suppressed the increased expression of Iba-1,TLR2,TLR4 and TNF-α m RNA(compared with CCI group,P<0.01;compared with sham group,P>0.05)and partially blocked the increased expression of IL-1β and i NOS m RNA(compared with CCI group,P<0.01;compared with sham group,P<0.01).3.The ELISA results indicated that,compared with sham group,the content of IL-1β and TNF-α in hippocampus were significantly increased in hippocampus of CCI rats(P<0.01).Intra-hippocampal CA1 injection of minocycline partially blocked the increased content of IL-1β and obviously suppressed the increased content of TNF-α(IL-1β: compared with CCI group,P<0.05;compared with sham group,P<0.05;TNF-α :compared with CCI group,P<0.01;compared with sham group,P>0.05).4.Compared with sham group,intra-hippocampal CA1 injection of miR-181 c agomir from 3 days to 6 days could significantly improve CCI-induced mechanical hyperalgesia(P<0.05).There was no significant difference between vehicle-treated CCI group and negative control-treated CCI group.5.The RTFQ-PCR results indicated that,compared with sham group,the expression of miR-181 c m RNA was significantly decreased(P<0.05).At the same time,Iba-1,TLR4,IL-1β,TNF-α and i NOS m RNA in hippocampus were significantly increased in CCI rats(P<0.01).Intra-hippocampal CA1 injection of miR-181 c agomir obviously suppressed the increased expression of Iba-1,TLR4,IL-1β,TNF-α and i NOS m RNA(compared with CCI group,P<0.01;Compared with sham group,P>0.05).There was no significant difference between vehicle-treated CCI group and negative control-treated CCI group.6.The ELISA results indicated that,compared with sham group,the content of IL-1β and TNF-α in hippocampus were significantly increased in CCI rat(P<0.01).Intra-hippocampal CA1 injection of miR-181 c agomir obviously suppressed the increased content of IL-1β and TNF-α(compared with CCI group,P<0.01;compared with sham group,P>0.05).There was no significant difference between vehicle-treated CCI group and negative control-treated CCI group.Conclusions:1.These observations suggest that TLR4 and TNF-α in hippocampus contribute to occurrence and maintenance of neuropathic pain in CCI rats.2.Intra-hippocampal CA1 injection of minocycline or miR-181 c agomir can alleviate pain response by inhibiting the elevated expression of TLR4,TNF-α and other inflammatory factors in hippocampus of CCI rats. |
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