Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice

Abstract

Background Plenty of macrophages are recruited to the injured nerve to play key roles in the immunoreaction and engulf the debris of degenerated axons and myelin during Wallerian degeneration, thus creating a conducive microenvironment for nerve regeneration. Recently, drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. However, the role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. Herein, we come up with the hypothesis that RhoA might influence Wallerian degeneration and nerve regeneration by affecting the migration and phagocytosis of macrophages after peripheral nerve injury. Methods Immunohistochemistry, Western blotting, H&E staining, and electrophysiology were performed to access the Wallerian degeneration and axonal regeneration after sciatic nerve transection and crush injury in the LyzCre+/−; RhoAflox/flox (cKO) mice or Lyz2Cre+/− (Cre) mice, regardless of sex. Macrophages’ migration and phagocytosis were detected in the injured nerves and the cultured macrophages. Moreover, the expression and potential roles of ROCK and MLCK were also evaluated in the cultured macrophages. Results 1. RhoA was specifically knocked out in macrophages of the cKO mice; 2. The segmentation of axons and myelin, the axonal regeneration, and nerve conduction in the injured nerve were significantly impeded while the myoatrophy was more severe in the cKO mice compared with those in Cre mice; 3. RhoA knockout attenuated the migration and phagocytosis of macrophages in vivo and in vitro; 4. ROCK and MLCK were downregulated in the cKO macrophages while inhibition of ROCK and MLCK could weaken the migration and phagocytosis of macrophages. Conclusions Our findings suggest that RhoA depletion in macrophages exerts a detrimental effect on Wallerian degeneration and nerve regeneration, which is most likely due to the impaired migration and phagocytosis of macrophages resulted from disrupted RhoA/ROCK/MLCK pathway. Since previous research has proved RhoA inhibition in neurons was favoring for axonal regeneration, the present study reminds us of that the cellular specificity of RhoA-targeted drugs is needed to be considered in the future application for treating peripheral nerve injury.

miR-301a Deficiency Attenuates the Macrophage Migration and Phagocytosis through YY1/CXCR4 Pathway

“…Recently, we reported that RhoA-conditional knockout in macrophages resulted in fewer macrophages migrating into the injured sciatic nerve and weaker capability of phagocytosis for removing the debris of degenerated axons and myelin during the Wallerian degeneration [ 25 ]. We believe the sciatic nerve injury can also be used as a credible animal model to test the effects of miR-301a KO on macrophage migration and phagocytosis.…”

“…For nerve injury, the right sciatic nerve was bluntly exposed. A sciatic nerve transection injury model was established through a transection at 0.3 cm distal to the sciatic notch [ 25 , 26 ]. For the sham operation, the left sciatic nerve was only exposed without transection.…”

“…Myelin debris were produced using mouse brains and spinal cords as described elsewhere [ 25 ]. In brief, brains and spinal cords were isolated from wild type mice aged 8–10 weeks after euthanasia by cervical dislocation and then shattered into tiny particles by sonication.…”

“…The migration of the cultured macrophages was assessed by a transwell assay using 6.5 mm transwell chambers (8 µm pores, Corning Costar, New York, NY, USA, 3422) as described previously [ 25 , 29 ]. After the chambers were pretreated with 0.1 mg/mL Poly-L-lysine hydrobromide (PLL, Sigma-Aldrich, St. Louis, MO, USA, P1274) solution or culture medium, 1 × 10 5 macrophages in 100 µL of DMEM/F12 containing 1% FBS were seeded into the upper chamber, and the lower chamber was filled with 600 µL DMEM/F12 containing 10% FBS and 1 mg/mL myelin debris without cells.…”

Re-Analysis of Single-Nucleus Transcriptomics Reveals Diverse Dorsal Root Ganglia Macrophage Responses Following Peripheral Nerve Injury

“…The depletion of macrophages in the DRG has been shown to prevent the development of nerve injury-induced mechanical hypersensitivity [ 3 ], suggesting that pro-nociceptive neuronal and non-neuronal cellular interactions also occur in the DRG and may contribute to and be required for neuropathic pain initiation and maintenance. DRG macrophages express both pro-inflammatory and regenerative genes in a temporal manner after a nerve injury [ 4 ], and the infiltration of the phagocytic macrophages to the site of a nerve injury is crucial for the regeneration of the nerve especially after a sciatic crush injury [ 5 , 6 ]. These studies suggest that while a PNI induces macrophage responses in different peripheral and central tissues, the consequences of such effects are highly dynamic, location dependent, and specific to the type of nerve injury.…”