Role of Macrophages in CL-Enhanced Regeneration After Peripheral Nerve Injury

Neurology Life Sciences Neurosurgery Immunology Medicine Cell Biology
Macrophages Peripheral nerve regeneration CL response Neuroinflammation CCR2

Key Research on the Role of Macrophages in Peripheral Nerve Regeneration

Background and Research Objectives

Since the 1990s, research has shown that macrophages play a promoting role in peripheral nerve regeneration, and are particularly important in the conditioning lesion (CL) response. After sciatic nerve injury, macrophages accumulate at the injury site, the distal nerve, and the removed dorsal root ganglia (DRG). The macrophage response comes from both resident macrophages and recruited monocyte-derived macrophages (MDMs). However, the specific locations and macrophage populations that play a critical role in nerve regeneration remain to be elucidated. Therefore, the authors conducted a series of experiments to investigate the roles of different types of macrophages in CL-enhanced nerve regeneration.

Authors and Affiliations

The authors of this paper are Aaron D. Talsma, Jon P. Niemi, and Richard E. Zigmond, all affiliated with Case Western Reserve University. This paper was published in the Journal of Neuroinflammation in 2024.

Research Process

Experimental Design

The authors used CCR2 knockout (Ko) and CCR2gfp/gfp knock-in/knock-out (Ki/Ko) mice to prevent the recruitment of MDMs. In the sciatic nerve CL model, they examined the necessity of MDMs and resident macrophages in CL-enhanced regeneration and characterized injury-induced neuroinflammation.

Experimental Procedures and Treatment Methods

  1. Macrophage Isolation and Injection:

    • Experiments were conducted using mice aged 8 to 20 weeks, with no significant sex differences observed in the CL response, so equal numbers of males and females were used.
    • Unilateral sciatic nerve transection was performed by exposing and cutting the sciatic nerve through microsurgery, creating a CL model.

  2. Regeneration Measurement and Immunofluorescence Staining:

    • In vivo regeneration was measured at 2 days after bilateral test lesions (TLs), and macrophages were quantified using immunofluorescence staining.
    • For analysis, Luxol Fast Blue staining and ImageJ software were used for image analysis and quantification.

  3. Specific Methods and Tools:

    • Innovatively used nanoliposome carrier injection solutions (e.g., Clodronate) to deplete macrophages, combined with lentiviral transduction for specific macrophage labeling and tracking.
    • Employed ImageJ and image analysis algorithms for quantification and processing of nerve regeneration data.

Data Analysis and Results

Regeneration Results and Macrophage Population Analysis

  1. Regeneration in CCR2 Knockout Mice:

    • The study found that CCR2 knockout mice maintained normal CL-enhanced peripheral nerve regeneration and central CL response in the spinal dorsal root. This indicates that MDMs and the DN environment are not necessary for regeneration.

  2. Macrophages at the CL Site:

    • Macrophages were distributed at both TL and CL sites, but not significantly different between them. TL macrophages were primarily CCR2-dependent and nearly absent in CCR2 knockout mice, while Arg1+ macrophages at the CL site were compensated.
    • Inducing inflammation with Zymosan injected into intact WT sciatic nerves resulted in the recruitment of Arg1+ macrophages but did not enhance nerve regeneration.

  3. Effects of Clodronate Treatment:

    • Injecting Clodronate into the CL site of CCR2gfp KO mice significantly reduced the number of macrophages at the CL site, but regeneration was unaffected.

Conclusions and Significance

The experiments demonstrated that macrophages in the sciatic nerve are neither necessary nor sufficient to produce the CL response. Injury-induced macrophages in the DN environment cannot promote regeneration, indicating that the role of macrophages in post-injury nerve regeneration needs to be reevaluated.

Research Highlights

  1. Application of Innovative Techniques: The study utilized CCR2 knockout models and nanoliposome CL experiments to precisely block and observe macrophage changes at different locations and time points.
  2. Data Quantification and Image Processing: Luxol Fast Blue staining and ImageJ software were used for accurate data quantification and rigorous image processing, ensuring the reliability of the experiments.
  3. In-depth Understanding of Regeneration Mechanisms: The experiments systematically excluded the necessity of MDMs and the DN environment in nerve regeneration, providing new perspectives and insights into the regeneration mechanisms.

These findings provide important theoretical support and practical guidance for the future treatment and research of peripheral nerve injury, potentially influencing the direction of future clinical and basic research.

This study not only achieved innovation in technical approaches but also provided important insights into scientific theory, making a significant contribution to advancing the field of regenerative medicine.