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OMIP‐041: Optimized multicolor immunofluorescence panel rat microglial staining protocol

2017; Wiley; Volume: 93; Issue: 2 Linguagem: Inglês

10.1002/cyto.a.23267

1552-4930

Naama E. Toledano Furman, Karthik S. Prabhakara, Supinder S. Bedi, Charles S. Cox, Scott D. Olson,

Immune Response and Inflammation

The common usage of animal models in a variety of preclinical studies is supported by appropriate species-specific antibodies to be utilized in immunohistochemistry (IHC), western blotting, and flow cytometry (FC) assays. Other than the technical advantages (sophisticated surgical manipulations due to their size), modest cost (relative to larger animals), and the standardized results, the similarities in metabolic activity and physiology of neurological disorders to humans make rats appropriate for neurological disease or disorders models 1, 2. However, rat-based assays are not as comprehensive or standardized as mouse or human based assays are, partially because there is a shortage in rat-specific antibodies. Rat-specific antibodies are now becoming commercially available, which allows us to set standardize criteria for rat origin cells of interest. As our research focus is in neurotherapy, we are interested specifically in microglial cells, which are the innate immune cells in the brain 3 and spinal cord 4. Microglial cells play a critical role in traumatic brain injuries (TBI) and spinal cord injuries (SCI), and their presence, activation, and effect are highly investigated in those models 4-6. Microglial characterization via FC would save many hours of work as a substitute for IHC analysis, yield unbiased statistics, and overall help research move at a faster pace 7. Here we present multicolor phenotyping panels for assessing microglia derived from rat brain or spinal cord for their activation states, polarization, and number (see Table 1). The microglial cells used are immediately isolated from fresh brain or spinal cord tissues, using a Neural Tissue Dissociation kit, followed by myelin removal and purification using anti-rat CD11b/c microbeads. The Optimized Multicolor Immunofluorescence Panel (OMIP) was designed to phenotypically characterize rat-derived microglial cells isolated from brain or spinal cord, in TBI or SCI rat models. Central nervous system (CNS) injury immediately results in a robust inflammatory cell-mediated response. The key players in the response to the injury are microglial cells, which are a major cellular component of the CNS innate immune system. In normal situations, microglia maintains a dynamic and ramified phenotype, while an injury results in a dramatic amoeboid phenotype change. CNS injuries also lead to infiltration of macrophages to the injury site and both microglia and macrophages share the expression of surface markers such as CD45, CD11b/c, and CX3CR1. The common way of identifying microglia using FC is as CD11b/c+ CD45low 8, 9. In this study, we developed a new multiparametric flow cytometric analysis that not only enables differentiation between macrophages and resident microglia but also defines phenotypic changes in microglia in rat model. Recently, microglial specific markers such as transmembrane protein 119 (Tmem119) and the p2y12 receptor were found to distinguish between resident microglia and infiltrating macrophages which originate from the same myeloid lineage 10. The p2y12 receptor is an APD-responsive G protein-coupled receptor. In the CNS, its expression is restricted to microglia. It is now known that the p2y12 receptor is selectively expressed in nonactivated microglia and mediates process motility during early injury responses 11. Both anti-p2y12 and anti-Tmem119 antibodies were produced and studied well in mouse and human 8, but less so in rat models. As far as we know, only one anti-rat p2y12 antibody (Alomone Labs, Jerusalem, Israel) is currently available for FC purposes. Microglial activation is not one defined path of phenotypic changes, but a heterogeneous phenotype expression profile that is unique to the cells, particular conditions/diseases, and variable over time. Similar to the M1-M2 polarization process of macrophages in non-neuronal tissues, microglia present multiple activation phenotypes. However, the markers for each polarization state were defined using in vitro models, and as such, this paradigm are oversimplified. Nonetheless, given the limited data on the role microglia after TBI or SCI, this classification establishes a solid base to investigate the role of microglia and evaluate new treatment strategies. The classical M1 activation of microglia, in response to TNF-α and IFN-γ, involves phagocytosis, ability to kill pathogens and ROS release 12. As microglia are antigen presenting cells and communicate with T cells, activated microglia will upregulate their cell surface markers such as MHC-II and CD86 13. M2 classification branch out to 3 major alternative pathways. While the phenotypic profile of the M2 cells may diverse, they are united in their purpose to downregulate, repair or protect the body from inflammation 14. Microglia can be stimulated using IL4 or IL13 in an alternative pathway that includes immunity against parasites, Th2 cell recruitment, and tissue repair. Under these conditions, cells are defined as M2a-polarized. The M2c phenotype, also called "acquired deactivation," is in response to IL10, glucocorticoids, or uptake of apoptotic cells where microglia are involved in tissue remodeling processes. In M2c, cells will overexpress transforming growth factor (TGF) beta, sphingosine kinase (SPHK1), and CD163, the membrane-bound scavenger receptor for haptoglobin/hemoglobin complexes 15-17. The M2b phenotype has characteristics of both M1 and M2 and is associated with memory immune response 18. Microglia are being evaluated via FC, however, most data published are presenting human or mouse microglia with only few publications showing microglia of rat origin. Moreover, no uniform, validated, or standardized panel to evaluate rat microglia is currently available. Our method allows the evaluation of rat microglia originating from CNS-related tissues following an injury, as well as be useful in variety of applications other than one suggested here. The procedure is optimized to sample a single brain hemisphere or a 1 inch long section of spinal cord tissue (see Supporting Information Fig. S4 for microglia isolation process illustration). We developed this panel (Figure 1) based on an existing mouse microglia panel that was previously developed and utilized in our lab 7. The rationale was to first identify the main microglia population using CD45, CD11b/c, and p2y12 antibodies. Next, we chose markers that are available for rat that could indicate the polarization state a cell is in, see Table 2. Antibodies for intracellular markers were omitted from this protocol (such as Iba1 and CD68), as the permeabilization conditions have been shown to critically affect the cells 28. Additional benefits of limiting the protocol to surface staining are the retained ability to sort live cells for functional experiments and the simplicity of the procedure. Gating strategy for rat microglia. After isolation, each cell sample was divided into 2 tubes. The pro- and anti- inflammatory paths were evaluated separately in tube 1 (M1) and tube 2 (M2), respectively. (A) The main cellular population was gated to exclude doublets, and viable cells were gated under the single cells gate. (B) Identification of microglia using the triplet anchor CD45, CD11b/c and p2y12. Microglia are positive for all 3 markers. Cells that are positive for CD45+ and CD11b/c but negative for p2y12 (noted with *) are considered all other myeloid cells (such as monocytes and macrophages). (C) The M1 markers acquired were CD32 and CD86, under the microglia gating. (D) M2 polarized microglia were detected by the M2 markers CD200R, RT1B, and CD163. The level of expression of all the markers in the panels may vary with the activation levels of the cells. Therefore, during experiments biological controls as sham or naïve rat derived cells are recommended for the comparison and precise analysis of cells derived from injured tissues. The panel was designed with consideration of the brightness index of the fluorophores that were matched to each marker. Finally, optimization was performed to validate the phenotypic changes of differentiated cells using antibodies titrations and fluorescence minus one controls. Male Sprague Dawley Rats (225–250 g, Harlan Labs) were the source of CNS tissue. The usage of the animals was approved by the Animal welfare committee at University of Texas Health Science Center at Houston, Texas, protocol: AWC16–0046 and AWC14–0023. Animals were handled in accordance with the standards of the American Association for the Accreditation of Laboratory Animal Care (AAALAC). This work will provide researchers with a practical tool to evaluate the polarization state of the microglial cells following brain or spinal cord injuries in rat models. This surface staining is a simple and relatively quick procedure to evaluate microglia, which are freshly harvested from brain or spinal cord. We would like to acknowledge the contributions of Deepa Bhattarai, Dr. Amit Srivastava, and Dr. Katherine Ruppert for their support and assistance in providing primary tissue for this study and final editing of the manuscript. Dr. Naama Toledano-Furman performed the final isolation of primary cells, staining, flow cytometry, analysis, and was the primary author of the manuscript. Karthik S. Prabhakara was responsible for the majority of the surgery and primary isolation of tissue and cells used in this manuscript. Dr. Supinder Bedi assisted in the design and marker selection for the panels and performed some surgical manipulations. Drs. Olson and Cox provided guidance, assistance with writing and editing the manuscript, and financial and logistical support of the work. Additional Supporting Information may be found in the online version of this article. Supporting MIFlowCyt Supporting Figure 1A Supporting Figure 1B Supporting Figure 2A Supporting Figure 2B Supporting Figure 3 Supporting Figure 4 Supporting Figure 5 Supporting OMIP Online Material Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.