Gut Dysbiosis and Cerebral Small Vessel Disease

脑部小血管疾病(CSVD)是NINDS确定的一个公认的优先事项，是 老年人的认知障碍。在这个提议中，我们提出了一个新的想法，即抗原起源于 肠道微生物区系构成了CSVD潜在炎症的基本来源。我们建议你的直觉 生物失调使细菌和细菌成分移位肠道上皮屏障，并 最终进入大脑，在那里它们启动并维持必要的炎症 慢性阻塞性肺疾病的发展。这一假说得到了强有力的初步数据的支持：(1)肠道生物失调发生在 CSVD的相关模型--自发性高血压卒中易感大鼠(SHRSP)与其亲本的比较 (2)改变WKY大鼠的肠道微生物群，使其类似于SHRSP，伴随着 SHRSP的病理改变；(3)SHRSP的肠上皮屏障受损；(4)SHRSP的脑组织 SHRSP含有比WKY大鼠多52%的细菌DNA；(5)革兰氏阴性菌代表着一种实质性的 与WKY大鼠相比，SHRSP大鼠脑内细菌DNA的总比例更高；以及(6) 内毒素是由革兰氏阴性菌产生的细菌内毒素，在很大程度上 与WKY大鼠相比，SHRSP大鼠脑内含量增加。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区系而产生或防止的。如果肠道菌群失调是导致 那么我们应该能够通过以下方式在对照WKY大鼠(SHRSP的亲本株)中诱导CSVD 导致生物失调。或者，我们应该能够通过以下方式消除或推迟SHRSP大鼠CSVD的发病 防止生态失调。在特定目标2中，我们将识别肠道和脑细菌以及细菌成分 参与了CSVD的启动。(一)我们会分析肠道和大脑的细菌DNA组成 从粪便、盲肠内容物和脑中测定细菌16S rRNA基因的序列。这一分析可以确定 细菌到属的分类水平。我们将进一步鉴定细菌到物种水平，并有针对性地 QPCR.(B)我们会使用RT-PCR和原位荧光技术来确定大脑中是否存在完整的细菌 SHRSP和WKY大鼠的杂交。(C)我们将尝试从SHRSP和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，a 这个过程被称为移位，从肠道到大脑。首先，我们将确定荧光标记的内毒素和 当给SHRSP和WKY大鼠灌胃时，肽多糖可以追踪到大脑。第二，我们将聘用 我们和记者一起设计了一种细菌，从肠道到大脑追踪细菌，并确定革兰氏杆菌- 在患有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)会影响大脑的健康。建立胆识 微生物群作为CSVD相关炎症的来源可能会极大地将我们的注意力重新集中在GI上 作为脑部病理的潜在原因，也为治疗干预提供了靶点。

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.