Gut Dysbiosis and Cerebral Small Vessel Disease

脑小血管疾病（CSVD）是NINDS确定的公认优先事项，是脑血管病的主要原因。 老年人的认知障碍。在这个建议中，我们提出了新的想法，即抗原来自于 肠道微生物群构成了CSVD基础炎症的基本来源。我们认为肠道 生态失调允许细菌和细菌组分移位肠上皮屏障， 最终进入大脑，在那里它们启动并维持炎症， CSVD的发展。这一假设得到了强有力的初步数据的支持：（1）肠道生态失调发生在 自发性高血压卒中易感大鼠（SHRSP），CSVD的相关模型，与其母体相比 应变，WKY大鼠;（2）改变WKY大鼠的肠道微生物组，以类似于SHRSP，伴随着 SHRSP中发生的病理变化;（3）SHRSP中肠上皮屏障受损;（4） SHRSP比WKY大鼠含有多52%的细菌DNA;（5）革兰氏阴性菌代表了SHRSP的基本特征。 与WKY大鼠相比，SHRSP脑中总细菌DNA的比例更大;以及（6） 脂多糖（LPS），来源于革兰氏阴性菌的细菌内毒素，基本上是 与WKY大鼠相比，SHRSP的脑中增加。在具体目标1中，我们将确定CSVD是否可以 通过操纵肠道微生物群来产生或预防。如果肠道生态失调是导致 那么我们应该能够在对照WKY大鼠（SHRSP的亲本品系）中诱导CSVD， 导致生态失调或者，我们应该能够消除或延迟SHRSP大鼠CSVD的发作， 防止生态失调。在具体目标2中，我们将识别肠道和大脑细菌以及细菌成分 参与了CSVD的启动。(a)我们将分析肠道和大脑的细菌DNA组成， 对粪便、盲肠内容物和大脑中的细菌16 s rRNA基因进行测序。通过分析可以发现 细菌的分类水平属。我们将进一步确定细菌的物种水平与目标 qPCR。(b)我们将使用RT-PCR和荧光原位技术来确定完整的细菌是否存在于大脑中 SHRSP与WKY大鼠杂交。(c)我们将尝试从SHRSP的大脑中培养细菌， WKY大鼠。在具体目标3中，我们建议跟踪细菌和细菌成分的运动， 这个过程被称为易位，从肠道到大脑。首先，我们将确定荧光标记的LPS和 当强饲到SHRSP和WKY大鼠的肠道中时，肽聚糖追踪到脑。第二，我们将采用 我们用报告基因6改造了细菌，以追踪从肠道到大脑的细菌，并确定克- 在发生CSVD的宿主中，阴性细菌能够更有效地从肠道转移到大脑。如果 我们的假设是有效的，那么胃肠道（GI）可以影响大脑的健康。建立肠道 微生物组作为与CSVD相关的炎症来源可能会极大地重新关注GI 以及提供治疗干预靶点。

Cerebral small vessel disease (CSVD), a recognized priority established by the NINDS, is a major cause of cognitive impairment in the elderly. In this proposal, we present the novel idea that antigens derived from the gut microbiota constitute a fundamental source for the inflammation underlying CSVD. We propose that gut dysbiosis allows bacteria and bacterial components to translocate the gut epithelial barrier and ultimately gain access to the brain where they initiate and maintain inflammation necessary for the development of CSVD. The hypothesis is supported with strong preliminary data: (1) gut dysbiosis occurs in spontaneously hypertensive stroke-prone rats (SHRSP), a relevant model for CSVD, compared to its parent strain, WKY rats; (2) altering the gut microbiome in WKY rats to resemble that of SHRSP is accompanied by pathological changes occurring in SHRSP; (3) the gut epithelial barrier is compromised in SHRSP; (4) brains of SHRSP contain 52% more bacterial DNA than WKY rats; (5) gram negative bacteria represent a substantially greater proportion of the overall bacterial DNA in brain of SHRSP compared to WKY rats; and (6) Lipopolysaccharides (LPS), bacterial endotoxin derived from gram negative bacteria, are substantially increased in brains of SHRSP compared to WKY rats. In Specific Aim 1 we will determine if CSVD can be produced or prevented through manipulation of the gut microbiota. If gut dysbiosis is an underlying cause of CSVD then we should be able to induce CSVD in control WKY rats, the parent strain for the SHRSP, by inducing dysbiosis. Alternatively, we should be able to abolish or delay the onset of CSVD in SHRSP rats by preventing dysbiosis. In Specific Aim 2 we will identify gut and brain bacteria and bacterial components involved with the initiation of CSVD. (a) We will analyze the bacterial DNA composition of the gut and brain by sequencing the bacterial 16s rRNA gene from feces, cecal content, and brain. This analysis can identify bacteria to the taxonomic level of genus. We will further identify bacteria to the species level with targeted qPCR. (b) We will determine if intact bacteria are resident in the brain using RT-PCR and fluorescence in situ hybridization of SHRSP and WKY rats. (c) We will attempt to culture bacteria from the brains of SHRSP and WKY rats. In Specific Aim 3 we propose to track the movement of bacteria and bacterial components, a process termed translocation, from the gut to the brain. First, we will determine if fluorescently labeled LPS and peptidoglycan track to the brain when gavaged into the gut of SHRSP and WKY rats. Second, we will employ bacteria that we have engineered with reporters 6 to track bacteria from gut to brain and determine if gram- negative bacteria are capable of translocating from gut to brain more efficiently in hosts developing CSVD. If our hypothesis is valid, then the gastrointestinal tract (GI) can affect the health of the brain. Establishing the gut microbiome as a source for inflammation related to CSVD could dramatically refocus our attention on the GI tract as a potential cause for brain pathologies as well as provide a target for therapeutic intervention.