American Society for Peripheral Nerve

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Knockout Of Toll-Like Receptors Delay The Nerve Regeneration In Sciatic Nerve Crushing Injury Of Mice
Ching-Hua Hsieh, MD, PhD; Lu Tsu-Hsiang, BA
Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan

Purpose: Toll-like receptors (TLRs) can recognize specific endogenous molecules associated to danger signals that are released from damaged cells or tissues after injury and trigger an innate immune response. TLRs had been suggested to be crucial in Wallerian degeneration after peripheral nerve injury. This study was designed to explore the effect of TLRs knockout on the nerve regeneration in a mice sciatic nerve crushing injury model.

Materials and Methods: The right sciatic nerve at the mid-thigh level was crushed by a No.5 Jeweler forcep for 30 seconds in C57BL/6, Tlr2-/-, Tlr3-/-, Tlr4-/-, Tlr5-/-, and Tlr7-/- mice. At 1, 3, 7, 14 d after nerve crush injury, one cm nerve segment distal to the crush site were harvested and detected by PCRarray to detect the change mRNAs regarding to demyelination and remyelination factors including myelination protein (Mal, Mbp, Plp, Pmp22), myelination promoting factors (Bdnf, Brn2, Gdnf, Igf, Krox20, Nrg1, Oct6, Sox10, Hdac1, Ndrg1), and myelination inhibiting factors (Jun, Notch1, Ntf3, Pax3, Sox2, Tgfb). Four mice in each group at indicated postoperative days were harvested for Western blotting of the myelin protein MPZ as well as re-myelination transcription factors Oct6 and Sox10. Sciatic nerve segments 5 mm distal to the crushed site were harvested at postoperative day 10 (n=6) and fixed in 3% EM grade glutaraldehyde at 4oC and stained with 1% toluidine blue dye for a quantitative assessment of the regenerated nerve fibers with the Leco IA32 Image Analysis System.

Results: Analysis of the histomorphometric image of the nerve specimens from these Tlr-/- mice presented prominent debris and less percentage nerve tissue with less fiber count, fiber width, fiber area, total fiber area, myelin area, axon area and axon width than those from the C57BL/6 mice. PCRarray experiments demonstrate that TLR knockout changed the expression profile of the myelination-promoting and -inhibiting factors from the nerve segment after the crush injury. In addition, delayed re-myelination of the distal nerve stump in Tlr2-/- and Tlr4-/- mice with an average 4-day delayed expression of the myelin protein MPZ was observed with delayed occurrence of Oct6 and Sox10 for 4 and 7 days, respectively.

Conclusion: This study using a mouse model of sciatic nerve crushing injury demonstrated that the knockout of toll-like receptors deter the nerve regeneration with change of the balance between the expression of myelination promoting and inhibiting factors in the distal nerve segment. 1

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