PDF(Pubmed)
Abstract：
BACKGROUND: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed.
METHODS: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining.
RESULTS: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired.
CONCLUSIONS: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.
METHODS: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining.
RESULTS: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired.
CONCLUSIONS: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.
摘要：
背景：自1990年代以来，已经积累的证据表明,巨噬细胞促进周围神经再生,并且是在调节损伤(CL)反应中增强再生所必需的.坐骨神经损伤后，巨噬细胞在损伤部位积聚，该部位远端的神经，和轴突切除的背根神经节（DRGs）。在周围神经系统，和其他组织一样，巨噬细胞应答来自常驻巨噬细胞和募集的单核细胞源性巨噬细胞(MDMs).尚未解决的问题是：巨噬细胞在哪些部位增强神经再生，是需要的特定人群。
方法：使用Ccr2敲除（KO）和Ccr2gfp/gfp敲入/KO小鼠来防止MDM募集。在坐骨CL范例中使用这些菌株，我们研究了MDMs和居民体内CL增强再生的必要性，以及特征性损伤引起的神经炎症。CL范式变体，包括添加药理学巨噬细胞消耗方法，测试了各种巨噬细胞群体在启动或维持CL反应中的作用。体内再生，从2天后的双侧近端测试病变（TLs）测量，和巨噬细胞通过免疫荧光染色定量。
结果：在挤压和横切CLs之间，外周CL增强的再生是相当的，并且在Ccr2KO和WT小鼠中，尽管MDM耗尽，但仍持续28天。同样，在Ccr2KO小鼠中，背根中测得的中央CL反应没有变化。TL和CL的巨噬细胞，但不是在他们之间，为促再生标记染色，精氨酸酶1.TL巨噬细胞主要是CCR2依赖性MDM，在Ccr2KO和Ccr2gfp/gfpKO小鼠中几乎不存在。然而,由于常驻巨噬细胞补偿,CCR2空CLs中的Arg1+巨噬细胞仅比对照略少.将酵母聚糖注射到完整的WT坐骨神经中，募集了Arg1巨噬细胞，但并未增强再生。最后,Ccr2gfpKOCLs中注射氯膦酸盐可显着减少CL巨噬细胞。结合Ccr2gfpKO背景，耗尽MDM和TL巨噬细胞，和横切CL，物理移除远端神经环境，神经中几乎所有的巨噬细胞都被切除了,然而CL增强的再生没有受损。
结论：坐骨神经中的巨噬细胞既不需要也不足以产生CL反应。
方法：使用Ccr2敲除（KO）和Ccr2gfp/gfp敲入/KO小鼠来防止MDM募集。在坐骨CL范例中使用这些菌株，我们研究了MDMs和居民体内CL增强再生的必要性，以及特征性损伤引起的神经炎症。CL范式变体，包括添加药理学巨噬细胞消耗方法，测试了各种巨噬细胞群体在启动或维持CL反应中的作用。体内再生，从2天后的双侧近端测试病变（TLs）测量，和巨噬细胞通过免疫荧光染色定量。
结果：在挤压和横切CLs之间，外周CL增强的再生是相当的，并且在Ccr2KO和WT小鼠中，尽管MDM耗尽，但仍持续28天。同样，在Ccr2KO小鼠中，背根中测得的中央CL反应没有变化。TL和CL的巨噬细胞，但不是在他们之间，为促再生标记染色，精氨酸酶1.TL巨噬细胞主要是CCR2依赖性MDM，在Ccr2KO和Ccr2gfp/gfpKO小鼠中几乎不存在。然而,由于常驻巨噬细胞补偿,CCR2空CLs中的Arg1+巨噬细胞仅比对照略少.将酵母聚糖注射到完整的WT坐骨神经中，募集了Arg1巨噬细胞，但并未增强再生。最后,Ccr2gfpKOCLs中注射氯膦酸盐可显着减少CL巨噬细胞。结合Ccr2gfpKO背景，耗尽MDM和TL巨噬细胞，和横切CL，物理移除远端神经环境，神经中几乎所有的巨噬细胞都被切除了,然而CL增强的再生没有受损。
结论：坐骨神经中的巨噬细胞既不需要也不足以产生CL反应。
