Limited Nerve Regeneration across Acellular Nerve Allografts (ANAs) Coincides with Changes in Blood Vessel Morphology and the Development of a Pro-Inflammatory Microenvironment.

The use of acellular nerve allografts (ANAs) to reconstruct long nerve gaps (>3 cm) is associated with limited axon regeneration. To understand why ANA length might limit regeneration, we focused on identifying differences in the regenerative and vascular microenvironment that develop within ANAs based on their length. A rat sciatic nerve gap model was repaired with either short (2 cm) or long (4 cm) ANAs, and histomorphometry was used to measure myelinated axon regeneration and blood vessel morphology at various timepoints (2-, 4- and 8-weeks). Both groups demonstrated robust axonal regeneration within the proximal graft region, which continued across the mid-distal graft of short ANAs as time progressed. By 8 weeks, long ANAs had limited regeneration across the ANA and into the distal nerve (98 vs. 7583 axons in short ANAs). Interestingly, blood vessels within the mid-distal graft of long ANAs underwent morphological changes characteristic of an inflammatory pathology by 8 weeks post surgery. Gene expression analysis revealed an increased expression of pro-inflammatory cytokines within the mid-distal graft region of long vs. short ANAs, which coincided with pathological changes in blood vessels. Our data show evidence of limited axonal regeneration and the development of a pro-inflammatory environment within long ANAs.

Loss of Gata1 decreased eosinophils, macrophages, and type 2 cytokines in regenerating nerve and delayed axon regeneration after a segmental nerve injury.

The immune system has garnered attention for its role in peripheral nerve regeneration, particularly as it pertains to regeneration across segmental injuries. Previous work demonstrated that eosinophils are recruited to regenerating nerve and express interleukin-4, amongst potential cytokines. These results suggest a direct role for eosinophils in promoting nerve regeneration. Therefore, we further considered eosinophils roles in nerve regeneration using a segmental nerve injury and Gata1 knockout (KO) mice, which are severely eosinophil deficient, compared to wild-type BALB/c mice (WT). Mice receiving a sciatic nerve gap injury demonstrated distinct cytokine expression and leukocytes within regenerating nerve. Compared to controls, Gata1 KO regenerated nerves contained decreased expression of type 2 cytokines, including Il-5 and Il-13, and decreased recruitment of eosinophils and macrophages. At this early time point during ongoing regeneration, the macrophages within Gata1 KO nerves also demonstrated significantly less M2 polarization compared to controls. Subsequently, motor and sensory axon regeneration across the gap injury was decreased in Gata1 KO compared to WT during ongoing nerve regeneration. Over longer observation to allow for more complete nerve regeneration, behavioral recovery measured by grid-walk assessment was not different comparing groups but modestly delayed in Gata1 KO compared to WT. The extent of final axon regeneration was not different amongst groups. Our data provide additional evidence suggesting eosinophils contribute to nerve regeneration across a nerve gap injury, but are not essential to regeneration in this context. Our evidence also suggests eosinophils may regulate cytokines that promote distinct macrophage phenotypes and axon regeneration.

IL-4 expressing cells are recruited to nerve after injury and promote regeneration.

Interleukin-4 (IL-4) has garnered interest as a cytokine that mediates regeneration across multiple tissues including peripheral nerve. Within nerve, we previously showed endogenous IL-4 was critical to regeneration across nerve gaps. Here, we determined a generalizable role of IL-4 in nerve injury and regeneration. In wild-type (WT) mice receiving a sciatic nerve crush, IL-4 expressing cells preferentially accumulated within the injured nerve compared to affected sites proximal, such as dorsal root ganglia (DRGs), or distal muscle. Immunohistochemistry and flow cytometry confirmed that eosinophils (CD45+, CD11b+, CD64-, Siglec-F+) were sources of IL-4 expression. Examination of targets for IL-4 within nerve revealed macrophages, as well as subsets of neurons expressed IL-4R, while Schwann cells expressed limited IL-4R. Dorsal root ganglia cultures were exposed to IL-4 and demonstrated an increased proportion of neurons that extended axons compared to cultures without IL-4 (control), as well as longer myelinated axons compared to cultures without IL-4. The role of endogenous IL-4 during nerve injury and regeneration in vivo was assessed following a sciatic nerve crush using IL-4 knockout (KO) mice. Loss of IL-4 affected macrophage accumulation within injured nerve compared to WT mice, as well as shifted macrophage phenotype towards a CD206- phenotype with altered gene expression. Furthermore, this loss of IL-4 delayed initial axon regeneration from the injury crush site and subsequently delayed functional recovery and re-innervation of neuromuscular junctions compared to wild-type mice. Given the role of endogenous IL-4 in nerve regeneration, exogenous IL-4 was administered daily to WT mice following a nerve crush to examine regeneration. Daily IL-4 administration increased early axonal extension and CD206+ macrophage accumulation but did not alter functional recovery compared to untreated mice. Our data demonstrate IL-4 promotes nerve regeneration and recovery after injury.

Liposomes embedded within fibrin gels facilitate localized macrophage manipulations within nerve.

BACKGROUND: Understanding the role of macrophages at discrete spatial locations during nerve regeneration after injury is important. But, methodologies that systemically manipulate macrophages can obscure their roles within discrete spatial locations within nerve. NEW METHOD: Liposomes were embedded within fibrin gels to construct a delivery system that facilitated macrophage-specific manipulations at a sole spatial region, as macrophages accumulated within the fibrin. Clodronate liposomes were characterized for their toxicity to specific cells composing nerve in vitro, then tested for macrophage-specific depletion in vivo. This delivery system using clodronate liposomes was used to repair a mouse sciatic nerve gap to evaluate its efficacy and effects. RESULT: Clodronate liposomes showed specific toxicity to macrophages without affecting dorsal root ganglia (DRG)-derived neurons, endothelial cells, or Schwann cells in culture. The delivery system demonstrated sustained release of liposomes for more than 7 days while still retaining liposomes within the fibrin. In vivo, the delivery system demonstrated macrophages were targeted by liposomes, and the use of clodronate liposomes minimized macrophage accumulation within fibrin, while not affecting macrophage accumulation within DRG. Nerve regeneration across the nerve gap repaired using this delivery system was associated with decreased angiogenesis, Schwann cell accumulation, axon growth, and reinnervation of affected muscle. COMPARISON WITH EXISTING METHODS: This delivery system allowed specific perturbation of macrophages locally in nerve. This method could be applicable across species without the need for genetic manipulations or systemic pharmaceuticals. CONCLUSION: Liposomes embedded within fibrin gels locally target macrophages at the site of nerve injury, which enables greater precision in conclusions regarding their roles in nerve.

Brief Electrical Stimulation Accelerates Axon Regeneration and Promotes Recovery Following Nerve Transection and Repair in Mice.

BACKGROUND: Clinical outcomes following nerve injury repair can be inadequate. Pulsed-current electrical stimulation (ES) is a therapeutic method that facilitates functional recovery by accelerating axon regeneration. However, current clinical ES protocols involve the application of ES for 60 minutes during surgery, which can increase operative complexity and time. Shorter ES protocols could be a strategy to facilitate broader clinical adoption. The purpose of the present study was to determine if a 10-minute ES protocol could improve outcomes. METHODS: C57BL/6J mice were randomized to 3 groups: no ES, 10 minutes of ES, and 60 minutes of ES. In all groups, the sciatic nerve was transected and repaired, and, in the latter 2 groups, ES was applied after repair. Postoperatively, changes to gene expression from dorsal root ganglia were measured after 24 hours. The number of motoneurons regenerating axons was determined by retrograde labeling at 7 days. Histomorphological analyses of the nerve were performed at 14 days. Function was evaluated serially with use of behavioral tests up to 56 days postoperatively, and relative muscle weight was evaluated. RESULTS: Compared with the no-ES group, both ES groups demonstrated increased regeneration-associated gene expression within dorsal root ganglia. The 10-minute and 60-minute ES groups demonstrated accelerated axon regeneration compared with the no-ES group based on increased numbers of labeled motoneurons regenerating axons (mean difference, 202.0 [95% confidence interval (CI), 17.5 to 386.5] and 219.4 [95% CI, 34.9 to 403.9], respectively) and myelinated axon counts (mean difference, 559.3 [95% CI, 241.1 to 877.5] and 339.4 [95% CI, 21.2 to 657.6], respectively). The 10-minute and 60-minute ES groups had improved behavioral recovery, including on grid-walking analysis, compared with the no-ES group (mean difference, 11.9% [95% CI, 3.8% to 20.0%] and 10.9% [95% CI, 2.9% to 19.0%], respectively). There was no difference between the ES groups in measured outcomes. CONCLUSIONS: A 10-minute ES protocol accelerated axon regeneration and facilitated functional recovery. CLINICAL RELEVANCE: The brief (10-minute) ES protocol provided similar benefits to the 60-minute protocol in an acute sciatic nerve transection/repair mice model and merits further studies.

The CCL2/CCR2 axis is critical to recruiting macrophages into acellular nerve allograft bridging a nerve gap to promote angiogenesis and regeneration.

Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps.