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BACKGROUND

A stable and reproducible experimental bacterial pneumonia model postintracerebral hemorrhage (ICH) is necessary to help investigating the pathogenesis and novel treatments of Stroke-associated pneumonia (SAP).

AIM

To establish a Gram-negative bacterial pneumonia-complicating ICH rat model and an acute lung injury (ALI)-complicating ICH rat model.

METHODS

We established two standardized models of post-ICH pneumonia by nasal inoculation with () or intratracheal inoculation with lipopolysaccharide (LPS). Survival and neurological scores were monitored. Magnetic resonance imaging was performed to evaluate hematoma volume. Abdominal aortic blood was collected for leukocyte counting, serum was isolated to determine concentrations of S100β and proinflammatory cytokines using ELISAs. Histopathological changes of brain, lung and gut were assessed using hematoxylin-eosin staining. Lung was isolated for immunofluorescence staining for myeloperoxidase (MPO). Bronchoalveolar lavage fluid was collected for leukocyte counting, and supernatant was prepared to measure MPO activity. Ileum was isolated for immunofluorescence staining for tight junction proteins ZO-1 and γδ TCRs/IL-17A and for Alcian blue-nuclear fast red staining of acidic mucins. Feces were collected, 16S rRNA sequencing, untargeted metabolomics and Spearman's correlation analyses were performed to explore changes of gut microbiota, metabolites and their interactions.

RESULTS

In -induced bacterial pneumonia-complicating ICH rats, we demonstrated that challenge caused more severe neurological deficits, brain damage, neuroinflammation, and aggravated pneumonia and lung injury. Disruptions of the intestinal structure and gut barrier and the reductions of the protective intestinal IL-17A-producing γδT cells were also observed. challenge exacerbated the gut microbiota dysbiosis and fecal metabolic profile disorders, which were characterized by abnormal sphingolipid metabolism especially elevated ceramide levels; increased levels of neurotoxic quinolinic acid and an upregulation of tryptophan (Trp)-serotonin-melatonin pathway. Spearman's correlation analyses further revealed that the reduction or depletion of some beneficial bacteria, such as and , and the blooming of some opportunistic pathogens, such as , , and in -induced SAP rats were associated with the disordered sphingolipid and Trp metabolism. Using an LPS-induced ALI complicating ICH model, we also characterized SAP-induced brain, lung and gut histopathology injuries; peripheral immune disorders and intense pulmonary inflammatory responses.

CONCLUSIONS

These two models may be highly useful for investigating the pathogenesis and screening and optimizing potential treatments for SAP. Moreover, the differential genera and sphingolipid or Trp metabolites identified above seem to be promising therapeutic targets.

背景

脑出血（ICH）后稳定且可重复的实验性细菌性肺炎模型对于研究卒中相关性肺炎（SAP）的发病机制和新治疗方法是必要的。

目的

建立革兰阴性菌肺炎并发ICH大鼠模型和急性肺损伤（ALI）并发ICH大鼠模型。

方法

我们通过鼻内接种（）或气管内接种脂多糖（LPS）建立了两种标准化的ICH后肺炎模型。监测生存率和神经学评分。进行磁共振成像以评估血肿体积。采集腹主动脉血进行白细胞计数，分离血清以使用酶联免疫吸附测定法测定S100β和促炎细胞因子的浓度。使用苏木精-伊红染色评估脑、肺和肠道的组织病理学变化。分离肺组织进行髓过氧化物酶（MPO）免疫荧光染色。收集支气管肺泡灌洗液进行白细胞计数，并制备上清液以测量MPO活性。分离回肠进行紧密连接蛋白ZO-1和γδT细胞受体/IL-17A的免疫荧光染色以及酸性粘蛋白的阿尔辛蓝-核固红染色。收集粪便，进行16S rRNA测序、非靶向代谢组学和斯皮尔曼相关性分析，以探索肠道微生物群、代谢物及其相互作用的变化。

结果

在诱导的细菌性肺炎并发ICH大鼠中，我们证明攻击导致更严重的神经功能缺损、脑损伤、神经炎症，并加重肺炎和肺损伤。还观察到肠道结构和肠道屏障的破坏以及产生保护性肠道IL-17A的γδT细胞的减少。攻击加剧了肠道微生物群失调和粪便代谢谱紊乱，其特征是鞘脂代谢异常，尤其是神经酰胺水平升高；神经毒性喹啉酸水平升高以及色氨酸（Trp）-血清素-褪黑素途径上调。斯皮尔曼相关性分析进一步表明，诱导的SAP大鼠中一些有益细菌（如和）的减少或耗竭以及一些机会性病原体（如、、和）的大量繁殖与鞘脂和Trp代谢紊乱有关。使用LPS诱导的ALI并发ICH模型，我们还表征了SAP诱导的脑、肺和肠道组织病理学损伤；外周免疫紊乱和强烈的肺部炎症反应。