Flow cytometric identification of dynamic immune cell populations in a rat model of post-traumatic elbow contracture

Post-traumatic joint contracture (PTJC) commonly occurs after elbow injury. Previous findings implied immune system activation in capsules of contracted joints; however, quantifying immune cell populations in rodent joint tissues is challenging due to small size and low cellularity. Here, we used flow cytometry to investigate the temporal immune response and enumerate cell populations in the rat elbow capsule after traumatic injury. After inducing PTJC, capsules were harvested from injured, sham, and control rat elbows at multiple time points, stained with surface markers for immune cells, and quantified via flow cytometry. Results were compared to previously published mechanics and histology data from the same model. Another injured group was treated with celecoxib to determine if changes due to anti-inflammatory treatment could be detected. Compared to control, injured animals displayed elevated leukocytes, T cells, and natural killer cells. CD45+ cells exhibited similar temporal changes as mechanics, which increased and then decreased after injury. CD3+, CD4+, and CD8a+ T cells followed a similar pattern as histology scores, which increased and remained elevated. Treating injured animals with celecoxib increased leukocytes but decreased several immune subpopulations. A method for flow cytometry on rat elbow capsule was established and used to quantify immune cell populations, which changed in response to injury and anti-inflammatory treatment. Comparisons between flow cytometry and previously published mechanics and histology revealed additional insights about temporal patterns in cell-, tissue-, and joint-level changes. Future work will investigate whether changes in immune cells attenuate PTJC symptoms.

创伤后关节挛缩（Post-traumatic joint contracture, PTJC）常继发于肘部损伤。既往研究表明，挛缩关节的滑膜囊存在免疫系统激活现象，但由于啮齿类动物关节组织体积微小且细胞丰度较低，对其免疫细胞群体进行定量分析存在较大挑战。本研究采用流式细胞术（flow cytometry），探究创伤后大鼠肘部滑膜囊的时序性免疫应答并计数细胞群体。在成功构建PTJC模型后，本研究于多个时间点从损伤组、假手术组及对照组大鼠肘部采集滑膜囊组织，采用免疫细胞表面标志物进行染色后，通过流式细胞术完成定量检测。将检测结果与该模型已发表的力学及组织学数据进行对比分析。另设一组损伤大鼠给予塞来昔布（celecoxib）干预，以明确能否检测到抗炎治疗引发的细胞变化。与对照组相比，损伤组大鼠的白细胞、T细胞及自然杀伤细胞（natural killer cells）水平均显著升高。CD45+细胞的时序变化趋势与力学指标一致，即损伤后先升高后降低。CD3+、CD4+及CD8a+ T细胞的变化模式则与组织学评分高度契合，即损伤后升高并维持在较高水平。对损伤大鼠给予塞来昔布干预后，其白细胞水平有所升高，但部分免疫亚群的数量显著降低。本研究建立了大鼠肘部滑膜囊流式细胞术检测方法，并利用该方法定量分析了免疫细胞群体的变化，这些变化可响应损伤及抗炎治疗干预。通过将流式细胞术结果与已发表的力学及组织学数据进行对比，本研究进一步揭示了细胞、组织及关节层面变化的时序模式。未来研究将探究免疫细胞的变化是否可缓解PTJC相关症状。