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Ccl2-ccr2 Signaling Is Critical To Macrophage Recruitment, Angiogenesis, And Regeneration Across Nerve Autograft Alternatives
Matthew D. Wood, PhD, Deng Pan, PhD, Lauren Schellhardt, B.A., Anja Fuchs, PhD, Alison K. Snyder-Warwick, MD, Susan E. Mackinnon, MD.
Washington University in St. Louis, St. Louis, MO, USA.

PURPOSE: Traumatic nerve injuries often necessitate surgical repair with nerve autografts or alternatives. Concerns when using alternatives to autografts, such as acellular nerve allograft (ANAs), still exist and include the general variability and inconsistency in outcomes achieved, as well as a known nerve gap length limit when alternatives are used to repair longer (>3cm) nerve gaps. Therefore, further knowledge regarding the biology of regeneration across alternatives is critical toward understanding these shortcomings and improving their designs. We explored the contribution of macrophages to angiogenesis and nerve regeneration following repair with ANAs.
METHODS: ANAs were generated using a chemical detergent protocol. One (1) cm ANAs were used to repair sciatic nerve gaps in CCL2KO, CCR2KO, or wild-type (WT; control) mice. As well, in select mice cabozantinib or clodronate liposomes were used to disrupt angiogenesis (via VEGFR) or deplete systemic monocytes/macrophages, respectively. Regeneration was quantified using immunofluorescence analysis.
RESULTS: First, we established a timeline of endothelial cell migration and vessel formation within ANAs. At 10 days following repair with ANAs, limited CD31+ area (endothelial cells) were present. Subsequently, the proportion of CD31+ cells increased significantly over time with the formation of elongated vessels. Similarly, based on the extent of S100+ area (Schwann cells), Schwann cell accumulation within ANAs closely followed endothelial cell accumulation and increased in proportion to the extent of vessel formation. Then, to determine the role of angiogenesis in subsequent regeneration, WT mice were treated with cabozantinib resulting in drastically reduced endothelial cells quantities within ANAs compared to untreated mice. As a result, treated ANAs also had severely reduced Schwann cell and T cell (CD3+) accumulation compared to ANAs from untreated mice. To understand how angiogenesis was promoted, macrophage were depleted through administering clodronate liposomes to WT mice, which severely reduced macrophage accumulation within the ANAs in a dose dependent manner. Regardless of clodronate liposome dose, however, endothelial cell quantities were reduced within ANAs compared to ANAs from untreated mice. In turn, Schwann cell accumulation within ANAs from clodronate liposome treated mice were also reduced compared to ANAs from untreated mice. Given these results, we then assessed if CCL2-CCR2 signaling was responsible for monocyte/macrophage recruitment within ANAs. Following nerve repair with ANAs, the accumulation of CD68 macrophages was greater within ANAs from WT mice compared to CCL2KO or CCR2KO mice. As well, ANAs from WT mice had significantly more endothelial cells and Schwann cells compared to CCL2KO or CCR2KO mice.
CONCLUSION: Macrophages are critical for angiogenesis within ANAs, where the macrophages that promote angiogenesis within ANAs are primarily hematogenous-derived macrophages, promoted via CCL2-CCR2 signaling. Efficient Schwann cell and T cell accumulation within ANAs to promote nerve regeneration only occurs following angiogenesis and vascularization of ANAs.


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