V. Pamulapati2, T. L. Smith1, L. Xiong1, S. Swaminathan4, C. M. Cuda4, K. J. Ho1 1Feinberg School Of Medicine – Northwestern University, Division Of Vascular Surgery, Department Of Surgery, Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University, Department Of Surgery, Chicago, IL, USA 4Feinberg School Of Medicine – Northwestern University, Division Of Rheumatology, Department Of Medicine, Chicago, IL, USA
Introduction:
Murine femoral artery wire injury (FAWI) is a commonly used model of neointimal hyperplasia, an inflammatory process resulting in restenosis and failure of surgery for arterial occlusive disease. High-parameter flow cytometry allows high-resolution classification, quantification, and sorting of cell populations from a single tissue sample, but has not previously been used to study the dynamics of post-operative inflammatory cellular infiltration following FAWI.
Methods:
We designed an 18-fluorochrome flow cytometry panel for leukocyte identification in digested femoral artery (FA) samples. C57BL/6 mice underwent FAWI followed by euthanasia at 1, 3, 7, and 14 days post-injury. Injured left femoral arteries and uninjured right femoral arteries were enzymatically digested into single cell suspensions that were stained and analyzed on a BD FACSymphony A5 flow cytometer and FlowJo v10 software. Immunofluorescence (IF) staining of femoral artery frozen sections from the same time points with CD45 primary antibodies was used to validate leukocyte quantification from flow cytometry.
Results:
Both IF cell counts and flow cytometry quantification suggest that infiltrating CD45+ leukocytes plateau in quantity by 7 days post-injury, then begin to decline by 14 days post-injury (Figure, panel A). As expected, injured femoral artery samples demonstrated significantly higher numbers of live leukocytes (Live/Dead–CD45+) than uninjured femoral arteries at the 1-day (54,883 vs. 777 cells, p=0.05), 3-day (32085 vs. 534 cells, p=0.004), and 7-day (49,885 vs. 970 cells, p=0.02) post-injury time points. Using our high-parameter flow cytometry panel, we were able to identify and quantify live leukocyte (Live/Dead–CD45+), CD4 and CD8 T-lymphocyte (CD3+CD4+/CD8+), B-lymphocyte (CD3+CD19+), neutrophil (CD45+Ly6G+), NK cell (CD11b–NK1.1+), eosinophil (SiglecF+), dendritic cell (CD64–MHCII+), classical/intermediate/non-classical monocyte (CD11b+CD64–Tim4–), and multiple macrophage (CD64dim/+Tim4–/+) sub-populations and quantify them across a 14-day post-injury time course (Figure, panel B). Leukocyte infiltration at the 1-day post-injury time point was dominated by neutrophils, however, by 7-days post-injury, macrophage sub-populations composed the majority of infiltrating leukocytes.
Conclusion:
Our high-parameter flow cytometry method allows for broad phenotyping of highly defined leukocyte sub-populations infiltrating in the arterial wall following FAWI with a single staining panel. This methodology has numerous downstream applications, including time-series analyses, cell sorting for gene expression analyses, identification of inflammatory surrogate markers (in conjunction with cytokine analysis), and correlation with vascular lesion formation.
