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Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice
Jiawei Xu1,2, Jinkun Wen1,3, Lanya Fu1,2
1Department of Histology and Embryology, School of Basic Medical Sciences, Southern Medical University, Guangzhou Ave North 1838, Guangzhou, 510515, China.
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.
Insights
RhoA depletion in macrophages impairs nerve regeneration by hindering macrophage migration and phagocytosis, impacting Wallerian degeneration. Cellular specificity is crucial for future nerve injury treatments.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Macrophages are vital for clearing debris during Wallerian degeneration and promoting nerve regeneration.
- The RhoA pathway is a target for promoting peripheral nerve regeneration, but its role in macrophages is unclear.
Purpose of the Study:
- To investigate the role of RhoA in macrophage function during Wallerian degeneration and nerve regeneration.
- To test the hypothesis that RhoA affects macrophage migration and phagocytosis after peripheral nerve injury.
Main Methods:
- Used conditional knockout mice (Lyz-Cre; RhoA-flox) to specifically delete RhoA in macrophages.
- Assessed Wallerian degeneration, axonal regeneration, and nerve function via histology, electrophysiology, and macrophage assays in vivo and in vitro.
- Evaluated the expression and role of ROCK and MLCK in macrophages.
Main Results:
- RhoA was successfully knocked out in macrophages of cKO mice.
- Axonal regeneration, nerve conduction, and Wallerian degeneration were impaired in cKO mice, with increased myoatrophy.
- Macrophage migration and phagocytosis were reduced in cKO mice, both in injured nerves and in cultured cells.
- ROCK and MLCK were downregulated in cKO macrophages, and their inhibition impaired macrophage function.
Conclusions:
- RhoA in macrophages is essential for effective Wallerian degeneration and nerve regeneration.
- Impaired macrophage migration and phagocytosis due to disrupted RhoA/ROCK/MLCK signaling underlie the detrimental effects.
- Future therapies targeting RhoA for peripheral nerve injury must consider cell-specific effects.
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