背景:了解脓毒症相关性脑病(SAE)背后的机制仍然是一项艰巨的任务。这项研究试图揭示在SAE小鼠模型的大脑中发生的复杂的细胞和分子改变,最终解开这种情况的潜在机制。
方法:我们在野生型和Anxa1-/-小鼠中使用腹腔注射脂多糖(LPS)建立了小鼠模型,并在0小时收集脑组织进行分析,12小时,24小时,注射后72小时。利用先进的技术,如单核RNA测序(snRNA-seq)和Stereo-seq,我们对大脑内的细胞反应和分子模式进行了全面的表征.
结果:我们的研究揭示了Anxa1-/-(膜联蛋白A1基因敲除)和野生型小鼠对LPS攻击反应的显著时间差异,特别是在注射后的12小时和24小时时间点。我们观察到这些小鼠中Astro-2和Micro-2细胞的比例显着增加。这些细胞表现出与血管亚型Vas-1的共定位模式,形成称为V1A2M2的独特区域,其中Astro-2和Micro-2细胞包围Vas-1。此外,通过进一步的分析,我们发现配体和受体如Timp1-Cd63,Timp1-Itgb1,Timp1-Lrp1以及Ccl2-Ackr1和Cxcl2-Ackr1在该区域内显著上调。此外,我们观察到在富含Micro-2细胞的区域中Cd14-Itgb1,Cd14-Tlr2和Cd14-C3ar1的表达显着增加。此外,Cxcl10-Sdc4在包含Micro-2和Astro-2细胞的大脑区域中显示出广泛的上调。值得注意的是,在LPS挑战时,在小鼠脑中观察到Anxa1表达增加。此外,我们的研究显示,Anxa1基因敲除后死亡率显著增加.然而,我们没有观察到类型的实质性差异,数字,或其他脑细胞在Anxa1-/-和野生型小鼠之间随时间的分布。然而,当比较LPS注射后24小时的时间点时,我们观察到Anxa1-/-小鼠血管附近Micro-2和Astro-2细胞的比例和分布显着降低。此外,我们注意到几种配体-受体对的表达水平降低,包括Cd14-Tlr2,Cd14-C3ar1,Cd14-Itgb1,Cxcl10-Sdc4,Ccl2-Ackr1和Cxcl2-Ackr1.
结论:通过结合snRNA-seq和立体-seq技术,我们的研究成功地确定了一种独特的细胞共定位,被称为特殊的病理生态位,包含Astro-2、Micro-2和Vas-1细胞。此外,我们观察到该生态位内的配体-受体对的上调。这些发现表明,这种细胞排列与导致SAE或在Anxa1敲低小鼠中观察到的死亡率增加的潜在机制之间存在潜在关联。
BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition.
METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain.
RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1.
CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.