Long-Term Neutrophil Depletion and Flow Cytometric Analysis of Brain Neutrophils in the 5xFAD Mouse Model

Background

Alzheimer’s disease (AD) is one of the most serious neurodegenerative disorders worldwide. The pathology of AD is complex and involves multiple pathologies, including amyloid-β accumulation, tau neurofibrillary tangles, abnormal neuroinflammation, and oxidative stress. Recently, increasing evidence has suggested that innate immune cells contribute to the progression of AD. Elevated neutrophil numbers have been observed in patients with AD and in the brains of AD mouse models, including 5xFAD and 3xTg mice, compared with healthy controls [1,2]. Neutrophils may contribute to disease progression by impairing cerebral blood flow and vascular function [3]. However, the precise role of neutrophils in AD pathologies remains unclear. Experimental approaches that enable sustained manipulation of neutrophils and subsequent characterization of brain-associated neutrophils are therefore valuable for investigating their contribution to AD and other chronic neurodegenerative diseases.

Neutrophil depletion using anti-Ly6G antibodies is widely used to investigate neutrophil function in mouse models. Most published depletion protocols, however, use treatment periods of less than two to three weeks [4,5]. The commonly used rat anti-Ly6G clone 1A8 can induce anti-rat antibodies following repeated administration, potentially accelerating clearance of the injected antibody and reducing depletion efficiency during prolonged treatment [6]. The use of murinized anti-Ly6G antibodies provides an approach for overcoming this limitation. Olofsen et al. demonstrated sustained neutrophil depletion using a murinized anti-Ly6G antibody for 28 days [7]. However, chronic neurodegenerative diseases may require neutrophil manipulation over longer experimental periods, and the performance of prolonged depletion strategies in an AD model has not been established. In addition, prolonged anti-Ly6G treatment creates challenges for monitoring depletion because occupancy of Ly6G by the depleting antibody can interfere with subsequent detection of the same epitope. Alternative identification strategies, including forward scatter (FSC)/side scatter (SSC) characteristics in combination with GR-1 and CD11b expression, can therefore be used to evaluate circulating neutrophils during treatment as described before [7].

In addition to sustained depletion, investigation of neutrophil function in neurodegenerative disease requires reliable recovery and characterization of neutrophils from brain tissue. Current studies investigating neutrophils in AD have primarily relied on microscopy to identify neutrophils within the brain parenchyma [8]. Flow cytometry provides an alternative approach that enables phenotypic characterization of individual immune-cell populations, but established protocols for brain immune-cell isolation generally focus on microglia or broader myeloid populations [9–11]. Mechanical tissue dissociation and Percoll density-gradient separation are established methods for recovering leukocytes from brain tissue. Nevertheless, analysis of brain-infiltrating neutrophils remains technically challenging because neutrophils represent a small fraction of brain leukocytes and are short-lived cells that can be sensitive to tissue processing. Thus, a workflow that applies established brain leukocyte isolation approaches specifically to the recovery and flow cytometric characterization of neutrophils would facilitate their investigation in AD mouse models.

Here, we describe two independent procedures for studying neutrophils in the 5xFAD mouse model of AD. Part I and Part II can be performed independently according to the experimental objective. However, they may also be combined when both long-term neutrophil depletion and subsequent analysis of brain-associated neutrophils are required. Part I describes neutrophil depletion. We adopted the use of murine anti-Ly6G and the alternative flow cytometric identification strategy described for long-term depletion [7], while extending the experimental setting from a 28-day timeline to a 6-week treatment in a chronic neurodegenerative disease model. Part II describes the processing of brain tissue for flow cytometric analysis of brain-infiltrating neutrophils. We apply established mechanical dissociation and Percoll-based leukocyte separation procedures [11] to 5xFAD mouse brain, using gentle mechanical homogenization followed by 40%/80% Percoll density-gradient separation and flow cytometric identification of neutrophils. This workflow consistently enabled the identification of a distinct CD45⁺CD11b⁺Ly6G⁺Ly6C^med population in both wild-type and 5xFAD mouse brains. Thus, rather than introducing the individual depletion, dissociation, or density-gradient techniques themselves, the present protocol extends and integrates these approaches for prolonged neutrophil manipulation and subsequent brain neutrophil analysis in an AD mouse model.