Department of Physiology, Spinal Cord and Brain Injury Research Center, University of Kentucky College of Medicine, Lexington, KY, 40536, USA.
Science Honors Program of Georgetown College, Georgetown, KY, 40324, USA.
J Neuroinflammation. 2021 May 13;18(1):113. doi: 10.1186/s12974-021-02161-8.
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.
性别和年龄是影响脊髓损伤 (SCI) 恢复的重要变量。尽管在 SCI 发生时，年龄逐渐趋于老龄化，但性别或年龄对炎症的影响仍有待阐明。本研究旨在确定 SCI 后急性期内性别和年龄对固有免疫反应的影响。
4 月龄和 14 月龄的雄性和雌性小鼠接受 T9 挫伤 SCI，分别于损伤后 3 天和 7 天（DPI）通过流式细胞术测定损伤周围小胶质细胞、单核细胞衍生的巨噬细胞（MDM）和中性粒细胞的比例。在 3-DPI 时通过免疫组织化学获得小胶质细胞和 MDM 的细胞计数以验证流式细胞术结果。在 3 天 DPI 时通过荧光激活细胞分选（FACS）分别分离小胶质细胞和 MDM，以评估与激活、氧化还原和碎片代谢/清除相关的 27 个基因的 RNA 表达。
流式细胞术显示，受伤时为雌性且年龄较大显著增加了 MDM 相对于其他吞噬细胞的比例，特别是在 3-DPI 时增加了 MDM 与小胶质细胞的比值。免疫组织化学细胞计数显示，雄性小鼠 SCI 损伤内的总小胶质细胞更多，这可以解释 MDM/小胶质细胞比值较低的原因。通过 NanoString 对 27 个基因进行分析，仅在 MDM 中发现 1 个基因在性别间存在差异表达；具体而言，补体蛋白 C1qa 在雄性中增加。年龄对 MDM 中无基因产生影响。在控制假发现率后，仅在性别间有 2 个基因在小胶质细胞中存在差异调节，具体为作为活性氧 (ROS) 相关标志物的 CYBB（NOX2）以及与修复表型相关的基因 MRC1（CD206）。这两个基因在雌性小胶质细胞中均增加。年龄对小胶质细胞中无基因存在差异调节。在分析的 27 个基因中，26 个基因在性别间存在差异，除三个例外，所有基因均在 MDM 中表达更高。具体而言，C1qa、cPLA2 和 CD86 在小胶质细胞中表达更高。
这些发现表明，SCI 后的炎症反应在细胞募集和基因表达水平上均具有性别依赖性。
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