New tools to study leukocyte infiltration into the CNS

描述(申请人提供)：脑和脊髓浸润性炎症单核细胞和中性粒细胞有助于多种神经系统疾病的发病机制、损伤和修复/再生，包括中风、癫痫、脱髓鞘疾病、阿尔茨海默病、肌萎缩侧索硬化症、癌症、疼痛、脑外伤、脊髓损伤和感染。虽然存在一些表面标记可以提供不同的单核单核细胞群的不同和重叠的分辨率，但这些工具在识别、跟踪和定量目标组织(如脑)中的单核细胞和中性粒细胞方面并不是最理想的。GRAF建立的LysM-EGFP小鼠，只有骨髓单核细胞系的细胞表达GFP，为单核细胞和中性粒细胞的跟踪提供了更复杂的工具。与此同时，一个迅速发展但相互矛盾的文献表明，中性粒细胞和单核细胞募集到中枢神经系统是相当复杂的，依赖于各种趋化因子和趋化因子受体的相互作用。例如，一般情况下，中性粒细胞的运输可能依赖于通过CXCR2和CXCR3轴的信号，而单核细胞的运输可能依赖于CCR2和CCR5受体。基于这一概念，并在努力剖析特定的趋化因子受体在粒单核细胞向中枢神经系统运输中的作用，我们在这个小额拨款提案中的第一个目标是 将LysM-EGFP小鼠与CCR2、CCR5、CXCR2或CXCR3纯合缺陷小鼠杂交。我们的第二个目标是描述感染了泰勒氏小鼠脑脊髓炎病毒的小鼠脑内粒单核细胞渗入的动力学和空间分布。为了实现这一目标，我们打算使用基于光纤的荧光内窥镜来获取活体动物中GFP阳性中性粒细胞和炎性单核细胞运输的深层组织图像，并确定趋化因子受体缺陷是否改变了细胞的运输。我们的长期目标是利用这四条线路 确定导致白细胞渗入中枢神经系统的因素，并评估颞叶 粒单核细胞之间的相互关系，通过阻止一个群体的渗透(例如，由于CXCR2缺乏而导致的中性粒细胞)和量化其他群体的渗透(例如，单核细胞)。我们打算使用这些小鼠模型来确定在我们特定的病毒模型中白细胞进入大脑的基本方面，并使这些线可用于其他研究中风、脑外伤、脊髓损伤等的研究人员。该项目具有创新性，因为它将产生新的小鼠模型，用于更仔细地研究中性粒细胞和炎性单核细胞向中枢神经系统的转移，还因为它使用光纤显微镜来观察这些细胞在活体动物脑深部结构中的转移。我们提议的项目意义重大，因为预计它将提供工具，解决有关白细胞向中枢神经系统运输机制的一些相互冲突的概念。通过扩大我们对中性粒细胞和单核细胞转运机制的了解，这些工具有可能极大地影响改善人类疾病的治疗策略的发展。

DESCRIPTION (provided by applicant): Brain-and spinal cord-infiltrating inflammatory monocytes and neutrophils contribute to pathogenesis, injury, and repair/regeneration in a wide array of neurologic diseases, including stroke, epilepsy, demyelinating disease, Alzheimer disease, ALS, cancer, pain, TBI, spinal cord injury, and infection. While a number of surface markers exist that provide variable and overlapping resolution of different myelomonocytic populations, these tools are suboptimal for identifying, tracking, and quantitating monocytes and neutrophils in target tissues such as the brain. The development of the LysM-eGFP mouse by Graf, in which only cells of myelomonocytic lineage express GFP, has provided a far more sophisticated tool for following monocytes and neutrophils. At the same time, a burgeoning but conflicted literature indicates that neutrophil and monocyte recruitment to the CNS is quite complex and dependent upon a variety of chemokines and chemokine receptor interactions. For example, in general, neutrophil trafficking may depend upon signaling through the CXCR2 and CXCR3 axis, while monocyte trafficking may depend upon CCR2 and CCR5 receptors. Based on this concept, and in an effort to dissect the role of specific chemokine receptors in the trafficking of myelomonocytic cells to the CNS, our first objective in this small grant proposal is to cross LysM-eGFP mice with mice that are homozygously deficient in CCR2, CCR5, CXCR2, or CXCR3. Our second objective is to characterize the kinetics and spatial distribution of myelomonocytic cells infiltrating the brain in mice infected with the Theiler's murine encephalomyelitis virus. To accomplish this objective, we intend to use a fiber optic-based fluorescence endoscope to acquire deep tissue images of GFP-positive neutrophil and inflammatory monocyte trafficking in live animals and determine whether chemokine receptor deficiency alters the trafficking of the cells. Our long- term goals are to use these four lines to identify the factors responsible for leukocyte infiltration into the CNS and to assess the temporal inter-relation between myelomonocytic cells by thwarting infiltration of one population (for example neutrophils via CXCR2 deficiency) and quantifying the infiltration of other populations (for example monocytes). We intend to use these mouse models to determine basic aspects of leukocyte trafficking into the brain in our specific virus model and to make these lines available to other investigators studying stroke, TBI, spinal cord injury, etc. This project is innovative because it will generate new mouse models for more carefully studying neutrophil and inflammatory monocyte trafficking into the CNS and because it employs a fiber optic microscope to observe the trafficking of these cells within deep brain structures in living animals. Our proposed project is significant because it is expected to provide tools that will resolve a number of conflicting concepts regarding the mechanisms of leukocyte trafficking to the CNS. By extending our knowledge of neutrophil and monocyte trafficking mechanisms, these tools have the potential to greatly impact the development of therapeutic strategies for ameliorating human disease.