Division of Vascular Surgery, Northwestern University, Chicago, IL, United States of America.
Department of Surgery and Animal Resources Center, University of Chicago, Chicago, IL, United States of America.
PLoS One. 2018 Dec 6;13(12):e0208426. doi: 10.1371/journal.pone.0208426. eCollection 2018.
The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice.
Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods.
All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-γ-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response.
GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.
微生物组在许多炎症过程和疾病状态中具有功能作用。虽然内膜增生的发展与炎症有关，但微生物组在内膜增生中的直接作用尚未确定。无菌（GF）小鼠是研究共生生物与宿主之间因果关系的一种非常宝贵的模型。我们假设与常规饲养（CONV-R）小鼠相比，GF 小鼠在颈结扎后会出现不同的内膜增生。
20 周龄雄性 C57BL/6 GF 小鼠在无菌条件下进行左颈动脉结扎。通过粪便培养和 16S rRNA qPCR 评估无菌状态。28 天后，通过动脉切片的形态计量学和组织学分析评估内膜增生。五天时通过 Ki67 和炎症细胞标志物的免疫荧光评估局部动脉细胞增殖和炎症。通过血清的多重免疫测定评估全身炎症。以相同方式处理的 CONV-R 小鼠作为对照队列。使用标准统计方法比较 GF 和 CONV-R 小鼠。
整个研究期间，所有 GF 小鼠均保持无菌。颈结扎后 28 天，CONV-R 小鼠的内膜增生发展明显多于 GF 小鼠，如内膜面积、中膜面积、内膜+中膜面积和内膜面积/（内膜+中膜）面积。Masson 三色染色显示，新生内膜病变的胶原含量在质量上相似。结扎后 5 天，GF 颈动脉中层和外膜的 Ki67 免疫反应明显减少。与 CONV-R 动脉相比，GF 小鼠动脉中抗炎 M2 巨噬细胞的浸润明显增加，成熟中性粒细胞的比例降低。GF 小鼠颈结扎后 5 天血清 IFN-γ诱导蛋白（IP）-10 和 MIP-2 明显减少，提示全身炎症反应减轻。
与 CONV-R 小鼠相比，GF 小鼠的内膜增生发展减弱，这可能与伤口愈合和急性炎症的动力学改变有关。认识到共生体在动脉重塑调节中的作用将深入了解再狭窄的病理生理学，并支持通过操纵微生物群来治疗或降低再狭窄风险的策略。
