| Objective To explore TLR4 signaling is included in Wallerian degeneration(WD)and nerve regeneration or not after peripheral nerve injury.Methods In order to observe the Wallerian degeneration,rat peripheral nerve transection model is established.Forty male Wistar rats were assigned into two groups at random:sham group(n=20)and model group(n=20).TOLL like receptor 4(TLR4)signaling pathway was regulated in rat sciatic nerve transection model.Sixty male Wistar rats were assigned into three groups at random: control group(G1)(n=20),TAK-242 group(G2)(n=20)and LPS group(G3)(n=20).The right sciatic nerves of rats in model,G1,G2 and G3 groups were cut and sutured end-to-end,while the sciatic nerves of rats in sham group were only exposed.The rats were treated with 0.15 mg/kg TAK-242 via tail vein 1 hour preoperatively and 7 days postoperatively in the G2 group,and the rats in G3 group were given microinjections of LPS(2 g/L)in a final volume of 1 μL.The rats in G1 groups were given intravenous injection of the same volume of saline,and the rats in model groups received no treatment.The sciatic nerves were taken at 1.5,24 hours,3,4,7 days after surgery.Real-time quantitative PCR(q RT-PCR)was adopted to search the dynamic expressions of TIR-domain-containing adaptor inducinginterferon-β(TRIF),interleukin-1(IL-1β)and monocyte chemoattractant protein-1(MCP-1)m RNA.Immunofluorescence(IF)staining was adopted to search the expression of CD68+ macrophages and iba1+schwann cells in sciatic nerves.Luxol Fast Blue(LFB)staining was applied to test sciatic nerves myelin,and Haematoxylin-eosin(HE)staining was conducted in sciatic nerves tissue to observe the pathological variations.Immunohistochemistry(IHC)was applied to test the form of TRIF and growth associated protein-43(GAP-43)proteins in sciatic nerve,and sciatic function index(SFI)was applied to assess the motor function’s recovery in rats.Results The following changes were observed in the rat model of sciatic nerve transaction.Using q RT-PCR,this study was researched that compared with the sham group,expressions of IL-1β m RNA and MCP-1 m RNA were significantly increased in the model group at 24 hours after surgery(both P < 0.001),and immunofluorescence showed that up-regulated expression of CD68+ cells appeared in the model group at 3 days after surgery(P < 0.01).Hematoxylin-eosin staining showed that a lot of inflammatory cells,Schwann cells and regenerated nerve fibers at the sciatic nerve stump were found in the model group at 7 days after surgery.Luxol fast blue staining revealed that demyelination at the sciatic nerve stump appeared in model groups at postoperative 7 days compared with the sham group(P < 0.001).Besides,the SFI scores of model group at each time point(10,20,30,40 and 50 d post-injury)was lower than the sham group’s SFI scores.The following changes were found when the TLR4 signaling pathway was regulated in rats’ sciatic nerve transection model.Using q RT-PCR and IHC,we found that TRIF were significantly decreased in the in TAK-242-injected rats compared with compared with controls(P < 0.001,P < 0.05).Compared with controls,q RT-PCR indicated that expressions of IL-1β m RNA and MCP-1 m RNA were significantly compromised in TAK-242-injected rats at 24 hours after surgery(both P < 0.001),while the expressions of them were significantly increased in LPS-injected rats(both P < 0.001).Immunofluorescence showed that compared with controls,down-regulated expression of Schwann cells and macrophages appeared in TAK-242-injected rats and up-regulated expression of them appeared in LPS-injected rats at 3 days post-injury(all P < 0.05).LFB staining were used to test sciatic nerves myelin clearance at 7 d post-injury,and the data showed compared with controls,myelin debris clearance in TAK-242-injected rats significantly delayed(P < 0.05)and it in LPS-injected rats significantly advanced(P <0.05).HE staining indicated a poor organization of repair site in TAK-242-injected rats and an optimal organization of repair site in LPS-injected rats in distal stump at 7 d postinjury.In addition,we use GAP-43 immunohistochemistry to assessed axonal regeneration at 4 d post-injury,and the data showed a delay on axonal regeneration inTAK-242-injected rats compared with controls(P < 0.05).Finally,compared with control group’s SFI score,the score of TAK-242 group at each time point(20,30,and 40 d postinjury)was lower(P < 0.05),and the score of LPS group at 20 d post-injury was higher(P< 0.05).Conclusion TLR4 signaling has the possibility to control the immune response to affect myelin phagocytosis and nerve regeneration during WD in rat after peripheral nerve injury. |
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