Role of neutrophil granulocytes after stroke: mechanisms of brain entry, neutrophil-induced disturbed brain recovery and remodeling

在许多情况下，已发现多形中性粒细胞（PMN）在缺血性中风的病理生理学中起重要作用，缺血性中风是工业化国家发病率和死亡率的主要原因之一。在这种情况下，我们可以通过抗体介导的阻断证明PMN使用整合素VLA-4粘附到发炎的血脑屏障并进入缺血性脑。在这里，它们被局部小胶质细胞识别，其能够吞噬细胞，从而潜在地改善脑实质内的PMN的否则严重的神经毒性作用。然而，由于缺乏合适的工具，我们还不能研究，当中性粒细胞永久缺乏VLA 4时，中风是如何发展的。此外，我们无法阐明PMN发挥其神经毒性功能的病理生理机制。为了改变这一点，我们开发了一种新的小鼠模型，现在允许PMN选择性遗传操作和荧光标记。本研究旨在阐明以下问题：（1）中性粒细胞如何影响微血管开放、重塑和血管生成？微血管的分支和树状结构是如何改变的？局部脑血流量的后果是什么？(2)侵入脑的中性粒细胞如何与脑神经元相互作用？它们如何改变神经元的分支和活力？这个过程是如何被大脑中的小胶质细胞改变的？(3)中性粒细胞胞外陷阱（NETs）在中性粒细胞相关脑损伤中的作用是什么？NET形成的抑制如何影响微血管开放/重塑、神经元变性和神经元可塑性？(4)脑内浸润性PMN的分子特征与外周血PMN有何不同？这些分子特征是否可以通过实验靶向来恢复神经元的存活？为了研究这些问题，我们将采用先进的显微镜成像，包括活体2光子和光片显微镜的清除大脑。此外，我们将利用独特而复杂的动物模型。通过这项研究，我们将对中性粒细胞介导的中风效应有一个全新的认识，这有望为急需的新治疗方案铺平道路。

Polymorphonuclear neutrophil granulocytes (PMN) have been found in many instances to play an important role in the pathophysiology of ischemic stroke, one of the major causes of morbidity and mortality in industrialized countries. In this context we could demonstrate by means of antibody mediated blockade that PMN use the integrin VLA-4 for adhesion to the inflamed blood brain barrier and entry into ischemic brain. Here they are recognized by local microglia, which is able to phagocytose the cells thereby potentially ameliorating the otherwise severely neurotoxic action of PMN within the brain parenchyma. However, due to the lack of suitable tools we could not yet study, how stroke develops when PMN are permanently lacking VLA 4. Furthermore, we could not clarify the pathophysiological mechanisms by which PMN exert their neurotoxic functions. To change this, we have developed a novel mouse model that now allows PMN-selective genetic manipulation and fluorescent labelling. Using this mouse model we aim at clarifying the following questions: (1) How do PMN influence microvascular patency, microvascular remodeling and angiogenesis? How are microvascular branching and arborisation altered? Which are the consequences for regional cerebral blood flow? (2) How do brain-invading PMN interact with brain neurons? How do they alter neuronal arborisation and viability? How is this process modified by brain-resident microglia? (3) Which is the role of neutrophil extracellular traps (NETs) in PMN-associated brain injury? How does inhibition of NET formation influence microvascular patency/remodeling, neuronal degeneration and neuronal plasticity? (4) How do the molecular signatures of brain-infiltrating PMN differ from PMN in the periphery? Can these molecular signatures be experimentally targeted in order to restore neuronal survival? To study these questions, we will employ advanced microscopical imaging including intravital 2 photon and lightsheet microscopy of cleared brains. In addition, we will make use of unique and complex animal models. With this study we will get a completely new understanding on the PMN-mediated effects of stroke which hopefully will pave the way to new treatment options that are needed urgently.