Sex and age are emerging as influential variables that affect spinal cord injury (SCI) recovery. Despite a changing demographic towards older age at the time of SCI, the effects of sex or age on inflammation remain to be elucidated. This study determined the sex- and age-dependency of the innate immune response acutely after SCI.
Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the lesion were determined at 3- and 7-day post-injury (DPI) using flow cytometry. Cell counts of microglia and MDMs were obtained using immunohistochemistry to verify flow cytometry results at 3-DPI. Microglia and MDMs were separately isolated using fluorescence-activated cell sorting (FACS) at 3-day post-injury (DPI) to assess RNA expression of 27 genes associated with activation, redox, and debris metabolism/clearance.
Flow cytometry revealed that being female and older at the time of injury significantly increased MDMs relative to other phagocytes, specifically increasing the ratio of MDMs to microglia at 3-DPI. Cell counts using immunohistochemistry revealed that male mice have more total microglia within SCI lesions that can account for a lower MDM/microglia ratio. With NanoString analyses of 27 genes, only 1 was differentially expressed between sexes in MDMs; specifically, complement protein C1qa was increased in males. No genes were affected by age in MDMs. Only 2 genes were differentially regulated in microglia between sexes after controlling for false discovery rate, specifically CYBB (NOX2) as a reactive oxygen species (ROS)-associated marker as well as MRC1 (CD206), a gene associated with reparative phenotypes. Both genes were increased in female microglia. No microglial genes were differentially regulated between ages. Differences between microglia and MDMs were found in 26 of 27 genes analyzed, all expressed higher in MDMs with three exceptions. Specifically, C1qa, cPLA2, and CD86 were expressed higher in microglia.
These findings indicate that inflammatory responses to SCI are sex-dependent at both the level of cellular recruitment and gene expression.
Digital Object Identifier (DOI)
Funding support provided by the Craig H. Neilson Foundation under award #465079, the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH) under Awards: R01NS091582 and F32NS111241, as well as, the National Institute on Alcohol Abuse and Alcoholism training grant T32AA027488 and National Cancer Institute grant P30CA177558.
Data generated in this study is available upon request and will be deposited in the Open Data Commons for Spinal Cord Injury upon publication (ODC-SCI; https://scicrunch.org/odc-sci).
Supplementary Figure 1. (available for download as an additional file listed at the end of this record) Annotation markup for MDM and microglial cell counts. Unbiased sampling procedures were used to count MDMs and microglia throughout the lesion. a Lesions were traced (blue trace) and partitioned into 100 μm2, randomly placed, boxes that were evenly spaced at 100 μm apart (green boxes). b Within each sampling area, MDMs (brown cells; red circles) were counted as long as cresyl violet nuclei were contained within, or touching, the right or top margins of the box. Microglia (black cells; blue measuring bar; blue arrow) were counted that met pre-defined criterion of presenting with a soma and at least 5 μm in length or diameter, and were contained within, or touching, the right or top margins of the box. Cells with nuclei touching the bottom or left margins (yellow arrow) were excluded from cell counts.
Stewart, Andrew N.; Lowe, John L.; Glaser, Ethan P.; Mott, Caitlin A.; Shahidehpour, Ryan K.; McFarlane, Katelyn E.; Bailey, William M.; Zhang, Bei; and Gensel, John C., "Acute Inflammatory Profiles Differ with Sex and Age After Spinal Cord Injury" (2021). Physiology Faculty Publications. 171.