Neuroinflammation in Wallerian Degeneration and Regeneration: Neutrophils Play a Primary Role as Phagocytes

了解神经退行性变的过程是重要的，以了解 神经系统对损伤和疾病的反应。这类研究中常用的模型是坐骨神经的远端节段。 神经切断或挤压后的神经。在这样的损害之后，发生沃勒变性，其中远端 神经碎片和变性的片段，以及由此产生的轴突和髓鞘碎片被清除。 有趣的是，虽然这一过程在外周神经系统(PNS)中迅速发生，但在 中枢神经系统(CNS)，部分原因是，再生在大脑中通常是无效的 和脊髓。目前普遍认为炎性巨噬细胞(mφS)来源于血源性单核细胞。 是吞噬轴突和髓鞘碎片所必需的。免疫细胞进入受损组织以回应 趋化性细胞因子或趋化因子。术后大量单核细胞渗入远端神经。 对趋化因子CCL2的反应，趋化因子通过趋化因子受体CCR2作用于单核细胞。在……里面 在CCR2基因被敲除的小鼠中，CCR2+单核细胞不会进入神经。因此，它是， 当我们发现在CCR2基因敲除中髓鞘和轴突蛋白的清除都是正常的时，非常令人惊讶 老鼠。我们随后发现，正常清除的一个重要原因似乎是吞噬作用 中性粒细胞是一种免疫细胞，以前与沃勒变性无关。事实上，尽管中性粒细胞 中枢神经系统的活动开始被研究，例如在多发性硬化症的模型中，几乎有 没有关于他们在PNS中的行为的研究。我们的初步证据是使用中性粒细胞耗竭抗体抗- Ly6G建议，但不能证明，中性粒细胞直接吞噬和代谢髓鞘。我们会 通过检测蛋白质印迹前后髓鞘蛋白的清除来验证这一假说。 中性粒细胞耗竭和油红O联合标记组织，髓鞘代谢物和细胞类型 特定的中性粒细胞抗体。我们已经证明，在轴突切断后，两个吸引趋化因子的中性粒细胞 在坐骨神经、CXCL1和CXCL2中诱导。为了确定这些趋化因子及其受体， CXCR2，都参与了中性粒细胞向神经的渗透，我们将使用中和抗体、药理学 拮抗者和基因敲除老鼠。我们还将调查中性粒细胞是否参与了沃勒氏病 退化对于随后的再生很重要。“沃勒式”退变发生在几个 脱髓鞘神经病，我们将检查中性粒细胞是否在这一现象中发挥作用。我们会 根据我们最近的发现，中性粒细胞进入坐骨神经，建立格林-巴利综合征的小鼠模型 在这个模型中有胆量。CCR2基因敲除中髓鞘清除正常的意外发现 小鼠，我们的实验将检测中性粒细胞在沃勒变性和脱髓鞘中的作用 疾病。鉴于已知的沃勒变性对神经再生的重要性，这是由于移除 髓鞘蛋白，我们的研究将提出促进三叉神经节再生的方法，可能还包括中枢神经系统。

Understanding the process of neural degeneration is important in order to understand the responses of the nervous system to injury and disease. A common model used in such studies is the distal segment of the sciatic nerve after nerve transection or crush. Following such a lesion, Wallerian degeneration occurs in which the distal segment of the nerve fragments and degenerates, and the resulting axonal and myelin debris are cleared away. Interestingly, while this process occurs rapidly in the peripheral nervous system (PNS), it is extremely slow in the central nervous system (CNS), and, partly because of this, regeneration is generally ineffective in the brain and spinal cord. It is widely believed that inflammatory macrophages (mφs) derived from blood-borne monocytes are required for the phagocytosis of axonal and myelin debris. Immune cells enter injured tissue in response to chemotactic cytokines or chemokines. A major population of monocytes infiltrates into the distal nerve after axotomy in response to the chemokine CCL2, which acts on monocytes via the chemokine receptor CCR2. In mice in which the gene for CCR2 is knocked out, CCR2+ monocytes do not enter the nerve. It was, therefore, very surprising when we found that the clearance of both myelin and axonal protein is normal in Ccr2 knockout mice. We subsequently found that an important reason for the normal clearance appears to be phagocytosis by neutrophils, an immune cell not previously implicated in Wallerian degeneration. In fact, although neutrophil actions in the CNS are beginning to be examined, for example in models of multiple sclerosis, there are almost no studies on their actions in the PNS. Our preliminary evidence using the neutrophil-depleting antibody anti- Ly6G suggests, but does not prove, that neutrophils directly phagocytose and metabolize myelin. We will examine this hypothesis by examining the clearance of myelin proteins by western blotting before and after neutrophil depletion and by co-labeling tissue with Oil Red O, a stain for myelin metabolites, and with cell type specific neutrophils antibodies. We have shown that after axotomy two neutrophil attracting chemokines are induced in the sciatic nerve, CXCL1 and CXCL2. To determine whether these chemokines and their receptor, CXCR2, are involved in neutrophil infiltration into the nerve, we will use neutralizing antibodies, pharmacological antagonists, and knockout mice. We will investigate also whether the involvement of neutrophils in Wallerian degeneration is important for subsequent regeneration. “Wallerian-like” degeneration occurs in several demyelinating neuropathies, and we will examine whether neutrophils play a role in this phenomenon. We will use a mouse model for Guillain Barré syndrome based on our recent finding that neutrophils enter into the sciatic nerve in this model. Following up on our unexpected findings of normal clearance of myelin in Ccr2 knockout mice, our experiments will examine the role of neutrophils in Wallerian degeneration and in a demyelinating disease. Given the known importance of Wallerian degeneration for nerve regeneration due to the removal of myelin proteins, our studies will suggest ways of improving regeneration in the PNS and perhaps in the CNS.