Abstract
BackgroundPlenty 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.MethodsImmunohistochemistry, 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.Results1. 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.ConclusionsOur 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.
Highlights
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, creating a conducive microenvironment for nerve regeneration
Identification of macrophage‐specific knockout of RhoA in Lyz2Cre+/−RhoAflox/flox mice To investigate the roles of RhoA in macrophages after peripheral nerve injury (PNI), we firstly generated mice with macrophage-specific RhoA knockout under control Lyz2Cre+RhoA+/+ (Cre)-loxp system
Mice carrying floxed RhoA alleles were crossed with mice expressing Cre recombinase under the controlled by Lyz2 regulatory sequences
Summary
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, creating a conducive microenvironment for nerve regeneration. Drugs targeting the RhoA pathway have been widely used to promote peripheral axonal regeneration. The role of RhoA in macrophage during Wallerian degeneration and nerve regeneration after peripheral nerve injury is still unknown. 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. Considering that the drugs used to treat the injured nerve may not just affect neurons and impact on other cells, and a certain molecule might play diverse roles in different cell types [8,9,10]. Our team has found several surprising effects of RhoA on Schwann cells, such as RhoA knockdown depressing Schwann cell’s proliferation, migration, and myelination, and the typical downstream molecule ROCK does not contribute to some effects of RhoA on Schwann cells. RhoA regulates Schwann cell’s differentiation through JNK pathway, while influencing its proliferation by regulating AKT pathway [8, 10]
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