Name: non-invasive model of neuropathogenic escherichia coli infection in the neonatal rat
Uploaded: 2014-10-29T13:00:00.000+00:00
Duration: 8 min 27 s
Description: Watch this Scientific Journal Video about Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat at JoVE.com

这项工作是由研究经费从医学研究委员会G0400268和MR / K018396 / 1的支持，并通过行动医学研究。进一步支持由国家卫生研究院的研究伦敦大学学院医院的生物医学研究中心提供。

在这项研究中进行的所有动物实验符合国家和欧洲立法和批准了药剂的UCL学院和伦理委员会的英国内政部（HO）。在何项目进行的所有动物上岗证PPL二千二百四十三分之八十〇和PPL七千七百七十三分之七十〇。 1.鼠准备<ol><li>开展所有在体内使用无病原体的Wistar新生大鼠的实验。 </li><li>保留所有的鼠窝（每个群体12例新生儿）在单独的笼子与哺乳期的母亲（9 - 10周龄雌性），在最佳条件（19岁 - 21°C，45 - 55％的湿度，15 - 20换气/小时12小时光照/黑暗周期）。 </li></ol> 2.细菌细胞的制备<ol><li>商店E.大肠杆菌 K1的库存量的20％（体积/体积）甘油在-80℃下。对于每个实验，板材缺货细菌到穆勒，寒春（MH）琼脂平板上，并在37°CO / N。 </li><li>喂奶前，inocu一天迟10毫升MH肉汤与单个大肠杆菌大肠杆菌 K1的殖民地，文化O / N在37℃，200转。作为介质污染的控制，制备10毫升未接种MH肉汤的孵育在37℃下以200rpm。 </li><li>转移100μl的O / N的细菌悬浮液的进9.9 MH肉汤中的溶液（1:100的体积/体积），并孵育在37℃，200rpm下，直到指数中期阶段达到时，对应于0.6的光学密度在600nm（OD 600）的波长进行测定。 </li></ol> 3.喂养新生鼠的E.大肠杆菌 K1 <ol><li>轻轻地从颈背垂直放置在一只手的动物，允许嘴巴得以打开。慢慢插入无菌移液管尖端插入动物的嘴，以及移液管将20μl接种物（〜37℃）到动物的口，在30秒的时间周期。喂奶后立即返回了动物母亲。 </li><li>验证4 - 8×10 6细菌已被送入吨o分别小狗通过连续稀释在PBS中的接种物，并点样到MH琼脂平板上。 </li></ol>由E. 4.评估殖民化大肠杆菌 K1 <ol><li>轻轻地从颈背握住动物在一只手中。浸湿无菌棉签在无菌PBS，然后轻轻擦拭肛周区域。擦拭后立即返回动物的母亲。 </li><li>将拭子末端到含有300微升无菌PBS的管并储存在冰上。 </li><li>通过在PBS中的系列稀释确定的存活细菌的数量和电镀到麦康凯琼脂平板上。 </li><li>确定可行的E.大肠杆菌 K1由噬菌体药敏试验。挑选使用无菌微生物环状单菌落，浸入200微升无菌PBS，然后进行涡旋混合。 </li><li>在一条直线上用无菌微生物环状物，亚培养至MH琼脂上。让板干燥30秒，然后吸取10微升脊梁的teriophage K1E（10 9噬斑形成单位/ ml（PFU / ml）的）上线的中心。孵育板O / N在37℃。 </li><li>第二天，检查板噬菌体介导的裂解。 </li></ol>疾病严重程度的评估5 <ol><li>使用表1中所列的7分制每日5次-评估各动物4的疾病的严重程度。 </li><li>如果动物分数≥3出7，立即宰杀的动物，以减少痛苦。记录动物的死亡。 </li></ol> 6.安乐死新生大鼠和采血车<ol><li>轻轻握住动物在一方面，露出的头部和颈部。用酒精棉签擦拭消毒动物的脖子上。消毒大对使用70％的乙醇剪刀，清洗无菌PBS去除微量乙醇之前。 </li><li>轻轻直接持有的上述动物的皮氏培养皿。杀头使用锋利的surgi新生儿卡尔剪刀，确保血液滴入培养皿。避免与磁头，以防止血液污染皮肤菌群接触。 </li><li>立即使用无菌移液管收集血液，并与肝素的钠盐混合，在20的浓度 - 在0.5 ml离心管50单位/ ml。 </li><li>通过在PBS中的系列稀释确定的存活细菌的数量和电镀到麦康凯琼脂平板上。确定可行的大肠杆菌的数量大肠杆菌 K1通过检测活细菌的敏感性噬菌体K1E。 </li></ol> 7.解剖<ol><li>继新生大鼠断头，用无菌条件下切除和收集感兴趣的组织。洗在70％乙醇和无菌PBS中的所有仪器（ 图1中示出）。 </li><li>清洁夹层板并用70％的乙醇完全润湿腹部。放置在它的后面的尸体和通过（i）固定在解剖板上钉的左后腿来用无菌针的动作表，（ⅱ）拉伸尸体和钉扎的右前肢的动作表，（ⅲ）钉扎的右后腿和左前腿的动作表。 </li><li>拉沿使用镊子下腹部的左手侧的皮肤上，然后沿着从下腹部到使用小解剖剪胸骨尸体的左手侧切断。 </li><li>延伸的切除穿过胸骨，然后向下从胸骨到使用镊子和小解剖剪小腹尸体的右手侧，以确保无下层结构的损坏。 </li><li>最后，利用虹膜弯钳解剖暴露腹膜轻轻拉下皮瓣从胸骨到小腹。 </li><li>轻轻抬起用镊子将腹膜和垂直切开，露出内脏，以确保没有潜在的器官受损。 </li></ol> 8.收集胃肠道<ol><li>识别胃，小肠，盲肠，结肠和肠系膜淋巴系统。 </li><li>轻轻横切在两侧取出胃，再放入含有1.6毫升无菌PBS用无菌镊子一个小巧玲珑。 </li><li>样结肠的直肠。小心地将整个肠道大众使用解剖镊和地点在无菌培养皿中的尸体。确保做到这一点轻轻地使肠道结构和肠系膜淋巴结构不分开。 </li></ol> 9.分离的胃肠道和肠系膜淋巴系统（图2） <ol><li>倾30毫升无菌PBS放入培养皿中，以确保在胃肠道被完全淹没。 </li><li>确定肠系膜淋巴系统的核心质量，并掐使用精细解剖钳。找出小肠最近端和捏用细镊子剥离。 </li><li>慢慢地将CENTRA肠系膜淋巴系统和远端小肠中沿相反的方向，直到两个组织的升质量都完全彼此分离。小心执行此过程，以确保小肠未拉伸的，因为这可以防止近侧，中间和远侧区的重现性集合。将肠系膜淋巴系统中的300 - 500微升无菌PBS，并放置在冰上。 </li></ol> 10.切片胃肠道<ol><li>横切GI道在盲肠从结肠分离小肠。 </li><li>将在300结肠 - 500微升无菌PBS，并置于冰上。 </li><li>收集来自小肠的近端，中间和远端区域的代表性组织。对齐从中点小肠。 </li><li>然后，（ⅰ）收集的最后2厘米之前盲肠作为远端小肠组织的，（ⅱ）收集由5所述组织 - 6厘米以上的中点作为近端小肠，并（ⅲ）收集来自3所述组织 - 下方5厘米的中点作为中间小肠。 </li></ol> 11.收集肝脏<ol><li>切除的胃之后识别大鼠肝脏的三波瓣内。 </li><li>叶拉离用细镊子解剖腹腔。 </li><li>切割用细剪刀任何附加韧带取出肝脏。将肝脏在300 - 500微升无菌PBS，并置于冰上。 </li></ol> 12.收集脾<ol><li>确定脾脏。 </li><li>脾拉离胃和用细剪刀剪开胃脾韧带。 </li><li>将脾脏在300 - 500微升无菌PBS，并置于冰上。 </li></ol> 13.收集肾脏<ol><li>鉴定肾脏，将在腹腔中取出时对胃肠道和其他器官的背面变得可见。 </li><li>切输尿管及任何附韧带ü唱精细解剖剪刀，用细镊子取出肾脏。删除肾上腺和用细镊子和剪刀剥离任何脂肪物质（如果还连着）。 </li><li>将两个肾脏在300-500微升无菌PBS，并放置在冰上。 </li></ol> 14.收集脑<ol><li>断头后，按住头用弯钳垂直位置。掐皮肤上的头部纵向使用另一套弯钳的顶部，并用细解剖剪一个切口。切颈部附近剩余的皮肤，再纵向与精细解剖剪刀。 </li><li>采用两侧细镊子揭示头骨和删除使用精细解剖剪刀皮瓣拉扯皮肤向外的方向。将精细解剖剪刀脊柱开幕，并沿走向主席台颅骨切开。 </li><li>用细镊子的方向向外拉头骨的两个部分。切去除颅骨段。 </li><l我&gt;广泛使用镊子，使用从朝主席台脊柱开幕向上运动舀取出大脑。洗了脑在PBS两次，以去除任何血液斩首的过程。 </li></ol> 15.组织处理和均质化<ol><li>对于需要生存能力计数或DNA提取，放置组织成含有无菌PBS试管实验。立即通过在PBS中系列稀释，并镀上麦康凯琼脂平板上，或存储在-20℃下用20％的甘油用于短期测定存活菌数。对于DNA的提取，存储组织于-20℃下。 </li><li>对于需要的RNA提取，放置组织成的RNAlater溶液适当体积（每1毫克组织10微升）的实验中，然后立即储存在-20℃下短期或在-80℃下长期贮存。 </li><li>对于实验需要成像，地方的组织在10ml Methacarn溶液（60％甲醇，30％的氯仿和10％乙酸），那么存储吨室温（20 - 25℃）达3周。 </li><li>按重量计管的前和加入组织后的比较计算组织重量。均质使用匀浆组织。在无菌PBS中的各样品的均质化之间洗一次均化器在70％乙醇和两次。 注:Methacarn固定是期望的肠样品的固定，以保持粘蛋白相关的粘膜防御结构;福尔马林固定可用于全身组织。 </li></ol>

<ol>
	<li>Harvey, D., Holt, D. E., Bedford, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+meningitis+in+the+newborn:+a+prospective+study+of+mortality+and+morbidity.">Bacterial meningitis in the newborn: a prospective study of mortality and morbidity.</a> 23 (5), 218-225 (1999).</li><li>Brouwer, M. C., Tunkel, A. R., van de Beek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+diagnosis,+and+antimicrobial+treatment+of+acute+bacterial+meningitis.">Epidemiology diagnosis, and antimicrobial treatment of acute bacterial meningitis.</a> Clin. Microbiol. Rev. 23 (3), (2010).</li><li>Silva, L. P. A., Cavalheiro, L. G., Queir&#243;s, F., Nova, C. V., Lucena, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+newborn+bacterial+meningitis+and+sepsis+during+the+pregnancy+period+for+public+health+care+system+participants+in+Salvador,+Bahia,+Brazil.">Prevalence of newborn bacterial meningitis and sepsis during the pregnancy period for public health care system participants in Salvador, Bahia, Brazil.</a> J. Infect. Dis. 11 (2), 272-276 (2007).</li><li>Levy, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+of+the+newborn:+basic+mechanisms+and+clinical+correlates.">Innate immunity of the newborn: basic mechanisms and clinical correlates.</a> Nat. Rev. Immunol. 7 (5), 379-390 (2007).</li><li>Robbins, J. B., et al., Med, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+K1+capsular+polysaccharide+associated+with+neonatal+meningitis.">Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis.</a> N. Eng. J. 290 (22), 1216-1220 (1974).</li><li>Korhonen, T. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hemolysin+production,+and+receptor+recognition+of+Escherichia+coli+strains+associated+with+neonatal+sepsis+and+meningitis.">hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis.</a> Infect. Immun. 48 (2), 486-491 (1985).</li><li>Rutishauser, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysialic+acid+in+the+plasticity+of+the+developing+and+adult+vertebrate+nervous+system.">Polysialic acid in the plasticity of the developing and adult vertebrate nervous system.</a> Nat. Rev. Neurosci. 9 (1), 26-35 (2008).</li><li>Koedel, U., Pfister, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+experimental+bacterial+meningitis.">Models of experimental bacterial meningitis.</a> Infect. Dis. Clin. North Am. 13 (3), 549-577 (1999).</li><li>Tunkel, A. R., Scheld, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+pathophysiology+of+bacterial+meningiits.">Pathogenesis and pathophysiology of bacterial meningiits.</a> Clin. Microbiol. Rev. 6 (2), 118-136 (1993).</li><li>Kim, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+K1+capsule+is+the+critical+determinant+in+the+development+of+Escherichia+coli+meningitis+in+the+rat.">The K1 capsule is the critical determinant in the development of Escherichia coli meningitis in the rat.</a> J. Clin. Invest. 90 (3), 897-905 (1992).</li><li>Obata-Yasuoka, M., Ba-Thein, W., Tsukamoto, T., Yoshikawa, H., Hayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaginal+Escherichia+coli+share+common+virulence+factor+profiles,+serotypes+and+phylogeny+with+other+extraintestinal+E.+coli.">Vaginal Escherichia coli share common virulence factor profiles, serotypes and phylogeny with other extraintestinal E. coli.</a> Microbiology. 148 (9), 2745-2752 (2002).</li><li>Sarff, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Escherichia+coli+K1+in+healthy+and+diseased+newborns.">Epidemiology of Escherichia coli K1 in healthy and diseased newborns.</a> Lancet. 305 (7916), 1099-1104 (1975).</li><li>Schiffer, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review:+relation+between+invasiveness+and+the+K1+capsular+polysaccharide+of+Escherichia+coli.">A review: relation between invasiveness and the K1 capsular polysaccharide of Escherichia coli.</a> Pediatr. Res. 10 (2), 82-87 (1976).</li><li>Palmer, C., Bik, E. M., DiGuilio, D. B., Relman, D. A., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+human+infant+intestinal+microbiota.">Development of the human infant intestinal microbiota.</a> PLoS Biol. 5 (7), 1556-1573 (2007).</li><li>Nassif, X., Bourdoulous, S., Eug&#232;ne, E., Couraud, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+extracellular+pathogens+cross+the+blood-brain+barrier.">How do extracellular pathogens cross the blood-brain barrier.</a> Trends Microbiol. 10 (5), 227-232 (2002).</li><li>Moxon, E. R., Glode, M. P., Sutton, A., Robbins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infant+rat+as+a+model+of+bacterial+meningitis.">The infant rat as a model of bacterial meningitis.</a> J. Infect. Dis. 136, 186-190 (1977).</li><li>Birchenough, G. M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+innate+defenses+in+the+neonatal+gastrointestinal+tract+in+response+to+colonization+by+neuropathogenic+Escherichia+coli.">Altered innate defenses in the neonatal gastrointestinal tract in response to colonization by neuropathogenic Escherichia coli.</a> Infect. Immun. 81 (9), 3264-3275 (2013).</li><li>Ganz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensins:+antimicrobial+peptides+of+innate+immunity.">Defensins: antimicrobial peptides of innate immunity.</a> Nat. Rev. Immunol. 3 (9), 710-720 (2003).</li><li>Bevins, C. L., Salzman, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paneth+cells,+antimicrobial+peptides+and+maintenance+of+intestinal+homeostasis.">Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.</a> Nat. Rev. Microbiol. 9 (5), 356-368 (2011).</li><li>Moxon, E. R., Smith, A. L., Averill, D. R., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haemophilus+influenzae+meningitis+in+infant+rats+after+intranasal+inoculation.">Haemophilus influenzae meningitis in infant rats after intranasal inoculation.</a> J. Infect. Dis. 129 (2), 154-162 (1974).</li><li>Bortolussi, R., Ferrieri, P., Bj&#246;rkst&#233;n, B., Quie, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsular+K1+polysaccharide+of+Escherichia+coli:+relationship+to+virulence+in+newborn+rats+and+resistance+to+phagocytosis.">Capsular K1 polysaccharide of Escherichia coli: relationship to virulence in newborn rats and resistance to phagocytosis.</a> Infect. Immun. 25 (1), 293-298 (1979).</li><li>Glode, M. P., Sutton, A., Moxon, E. R., Robbins, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+neonatal+Escherichia+coli+meningitis:+induction+of+bacteremia+and+meningitis+in+infant+rats+fed+E.+coli+K1.">Pathogenesis of neonatal Escherichia coli meningitis: induction of bacteremia and meningitis in infant rats fed E. coli K1.</a> Infect. Immun. 16 (1), 75-80 (1977).</li><li>Pluschke, G., Mercer, A., Kuse&#263;ek, B., Pohl, A., Achtman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+bacteremia+in+newborn+rats+by+Escherichia+coli+K1+is+correlated+with+only+certain+O+(lipopolysaccharide)+antigen+types.">Induction of bacteremia in newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) antigen types.</a> Infect. Immun. 39 (2), 599-608 (1983).</li><li>Zelmer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+capsule-selective+endosialidase+E+minimizes+changes+in+organ+gene+expression+induced+by+experimental+systemic+infection+with+Escherichia+coli+K1.">Administration of capsule-selective endosialidase E minimizes changes in organ gene expression induced by experimental systemic infection with Escherichia coli K1.</a> Microbiology. 156 (7), 2205-2215 (2010).</li><li>Zelmer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+polysialyl+capsule+during+blood-to-brain+transit+of+neuropathogenic+Escherichia+coli+K1.">Differential expression of the polysialyl capsule during blood-to-brain transit of neuropathogenic Escherichia coli K1.</a> Microbiology. 154 (8), 2522-2532 (2008).</li><li>Mushtaq, N., Redpath, M. B., Luzio, J. P., Taylor, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+and+cure+of+systemic+Escherichia+coli+K1+infection+by+modification+of+the+bacterial+phenotype.">Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype.</a> Antimicrob. Agents Chemother. 48 (1), 1503-1508 (2004).</li><li>Achtman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+widespread+bacterial+clones+among+Escherichia+coli+K1+isolates.">Six widespread bacterial clones among Escherichia coli K1 isolates.</a> Infect. Immun. 39 (1), 315-335 (1983).</li><li>Mushtaq, N., Redpath, M. B., Luzio, J. P., Taylor, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+experimental+Escherichia+coli.+infection+with+recombinant+bacteriophage-derived+capsule+depolymerase.">Treatment of experimental Escherichia coli. infection with recombinant bacteriophage-derived capsule depolymerase.</a> J. Antimicrob. Chemother. 56 (5), 160-165 (2005).</li><li>Gross, R. J., Cheasty, T., Rowe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+bacteriophages+specific+for+the+K1+polysaccharide+antigen+of+Escherichia+coli.">Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli.</a> J. Clin. Microbiol. 6 (6), 548-550 (1977).</li></ol>

作者宣称，他们有没有竞争的财务权益。

此处所描述的动物模型基础上，任何旨在再现天然存在的感染在人中的显着特征以前的工作。新生大鼠最初用于研究由于H.婴儿脑膜炎b型流感的品种满足主要标准感染稳健的模型。因此，相关病原体的进入门应该反映人类的自然感染的和可重复给予上升到足够的时间相似病理以允许治疗性干预。该技术中使用的，不应限制本过程的适用性，并且不应向疾病结果20。 H的模型性脑膜炎由莫克森和他的同事开发出幼鼠满足这些标准的20;上呼吸道和这一重要特征的粘膜的定植用非创伤复制在幼鼠后发生的自然感染细菌的抽动滴注到鼻腔的膜。重要的是，感染的年龄依赖性的性质被复制在模型中。 同组也是率先开发E的一种非侵入性的模型大肠杆菌 K1 NBM在新生大鼠22。无病原体的Sprague-Dawley幼鼠，通过口服胃管喂食10月8日至10月10 日的细菌定植;因此接种物比由我们采用的相当高的。殖与三个K1株检查，C94（O7:K1:H-）EC3（O1:K1:H-）和LH（Ø75为:K1:H 3），发生在一个相对高的比例（48-74％） K1喂养的动物，但菌血症，脑膜炎和死亡的发生率是可变的，比定植率显著降低。 大肠杆菌的克隆性质大肠杆菌 K1的实验性感染以后被建立的23和现在是明显的，只有O18:K1和在较小程度上，O7:K1的血清型能始终如一地引起全身感染。出于这个原因，neuropathogenic 大肠杆菌的发病机制的这些调查大肠杆菌 K1是基于使用毒力增强的O18:K1株A192PP。 E.的比较通过胃管新生大鼠，如采用由Achtman的组23为用于使用前一方法，几乎可以肯定，由于损坏的死亡由Glode和同事22，和一个液滴输送方法揭示的数目过多粘膜的表面的大肠杆菌 K1馈送胃管。作为定植率与这两种方法可比较的，则建议使用供给使用移液管，用无菌尖端的细菌的较少侵入性方法，如在此所描述的通信。 在我们的研究中使用的大肠杆菌菌株A192PP是O18:K1。它是临床菌株大肠杆菌的毒性更强衍生物杆菌 A192最初回收从病人败血症27 </SUP&gt;。通过连续传代通过新生大鼠26得到的菌株的毒力增强。应变诱发年龄相关疾病的严重程度，采用100％的菌血症和死亡率当给药至2天大的动物28。与此相反，第9天龄的动物对疾病的完全抗性。 K1的特异性裂解的噬菌体可以用于区分E。大肠杆菌 K1其他E.大肠杆菌菌株29。在这项研究中，存活的细菌的敏感性噬菌体K1E应当用于（ⅰ）检查E的纯度大肠杆菌 K1的混悬剂制备要被馈送到所述动物，和（ii）区分大肠杆菌从其他大肠菌群大肠杆菌 K1，以便计算在肛周拭子，血液和组织样品的可行性。如果菌落E.大肠杆菌 K1，这将是容易的噬菌体K1E裂解，和细菌的生长会在噬菌体接种部位被抑制。如果菌落不是E.大肠杆菌 K1，将BË耐KIE噬菌体裂解，并且应该有细菌生长的噬菌体接种部位的区域。它应该牢记的是，动物模型不能反映自然发生的疾病的所有功能。当前模型可以被修改来检查neuropathogenic细菌比E.其它的毒力特征大肠杆菌 A192PP和变型中的定植接种物的大小可被容纳。该技术的未来应用可能包括急需的药品评价治疗的条件，并揭示宿主对殖民和组织侵犯的细节。 这里所描述的方法是简单而有效的。 10个单胎 - 12幼崽被用作试验组和对照组，这在-垃圾的方法确保重复性和统计有效性的高度。当务之急是对幼仔的任何procedur后尽快回到他们的自然母亲e和窝不应该因此包括动物进行不同的干预措施。重要的是，所供给的接种物被温热否则幼仔将拒绝提供培养。幼仔快速开发复杂的微生物区系，并在出生后两天胃肠道是定植从确定为在婴儿和成人消化道中最丰富的微生物的门类广泛的细菌。还没有被馈送大肠杆菌幼仔大肠杆菌 A192PP不携带E.大肠杆菌 K1在胃肠道17等确定的定植率相对来说比较简单。然而，NEUS的基于定量PCR的检测方法，定植E的大肠杆菌 K1更为敏感，传统的培养方法，并强烈推荐17。

全身性细菌感染是一个主要的威胁福祉和新生儿的存活;早产儿是特别脆弱的。新生儿细菌性脑膜炎（NBM），常与细菌性败血症，仍然是发病率和死亡率的一个显著源在最初的几个星期的生活，问题是阻力的不断发展，以一线抗菌药物1,2加剧。 NBM的情况下，是一种医疗紧急情况具有很高的医疗，社会和经济负担3;因此，目前迫切需要新的治疗剂，特别是新的预防策略，以减少感染的负担。 NBM的一些特征是不寻常的:在发达国家， 大肠杆菌和B组链球菌是负责对大多数情况下与这些菌株引发的NBM的容量几乎总是用保护多糖加利福尼亚州的存在相关联psule，使病原体逃避免疫识别处理4。 neuroinvasive E的- （85％80）的比例非常高大肠杆菌表达的K1的胶囊5,6，α-2,8连接的多聚羧酸系聚合物是结构相同的主机神经元可塑性7的调节剂。 新疗法和避孕药的NBM和相关的菌血症和败血症的评价将明显受益感染强大的动物模型，模拟疾病在人类新生儿的主要特征，尤其是强烈的年龄依赖性和感染的自然途径。广泛用于革兰氏阳性和革兰氏阴性细菌性脑膜炎型号可供选择8,9和这些都大大扩大了我们在这些感染的发病机制，病理生理学和治疗方案的知识。因此，实验性感染的大鼠，小鼠，兔和猴已被用于研究脑膜炎在两个n个eonate和成年人。然而，许多这些车型采用直接脑池内或皮下注射细菌感染引发的，创建一个人造的发病机制绕过传播的自然过程，从定植的网站。在某些情况下，接种的这些方法导致了在病理显著变化;例如， 大肠杆菌的皮下给药大肠杆菌 K1株废止与自然感染相关的年龄依赖性，产生菌血症和侵入中枢神经系统（CNS）中的两个新生儿和成人10。倾向E.大肠杆菌 NBM关键取决于病原体的垂直传播，从母亲到婴儿出生时或出生后11不久。母系来源E.大肠杆菌 K1的细菌定殖于新生儿胃肠（GI）道11-13，这是无菌的，出生但迅速获得一个复杂的微生物区14。在殖民新生儿，E.大肠杆菌K1的细菌有从肠腔穿过血-脑或血-脑脊液屏障9,15进入中枢神经系统之前易位进入全身循环的能力。实验感染的强劲机型的设计应考虑这些细节的考虑。 虽然小鼠中已广泛用于某些形式的细菌性脑膜炎8人的研究，它们不适合于新生儿感染的研究:它们是由全身性感染淹没并没有显示出人类婴儿16的强年龄依赖性特性。此外，α防御素，胃肠道由E.提供保护，防止系统入侵的肽键大肠杆菌 K 1 17，高表达于帕内特细胞和嗜中性粒细胞在人类和大鼠，但不是在小鼠18。有一个在其他的没有发现显着程度的​​重复，冗余性和异质性在小鼠防御及相关cryptidin基因imals 19。新生大鼠最初使用莫克森和他的同事20鼻内接种，调查流感嗜血杆菌性脑膜炎的发病机理，复制这个新生儿病原菌定植的自然景观在人，随后改编为年龄依赖性E.大肠杆菌 K1菌血症和脑膜炎。 Bortolussi 等人 21就业腹腔注射的细菌的接种物以引发感染但Glode的关键研究和同事22使用的口服胃饲胃肠道定植后平行感染的天然途径。作为胃管可能损伤粘膜表面，该过程被进一步完善，该接种物馈送到新生儿23。这里，该方法用于胃肠道定植和用于跟踪描述在易感大鼠幼仔的感染程序;另外，该模型的治疗性和预防性应用进行了讨论。

<table><tbody><tr><td>Pathogen-free Wistar rats (12 x neonate, 1 x lactating mother)</td><td><a href="http://www.harlan.com" target="_blank"><img src="/img/new_window.png" /> Harlan, UK</a></td><td></td><td></td></tr><tr><td>E. coli K1 A192PP</td><td><a href="http://www.ucl.ac.uk" target="_blank"><img src="/img/new_window.png" /> Taylor lab</a></td><td></td><td>Mushtaq &lt;em&gt;et al.&lt;/em&gt; 2004</td></tr><tr><td>Bacteriophage K1E</td><td><a href="http://www.ucl.ac.uk" target="_blank"><img src="/img/new_window.png" /> Taylor lab</a></td><td></td><td>Mushtaq &lt;em&gt;et al.&lt;/em&gt; 2004</td></tr><tr><td>Glycerol</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>G5516</td><td></td></tr><tr><td>Mueller-Hinton Agar</td><td><a href="http://www.oxoid.com" target="_blank"><img src="/img/new_window.png" /> Oxoid, UK</a></td><td>CM0037</td><td></td></tr><tr><td>Mueller-Hinton Broth</td><td><a href="http://www.oxoid.com" target="_blank"><img src="/img/new_window.png" /> Oxoid, UK</a></td><td>CM0405</td><td></td></tr><tr><td>MacConkey Agar</td><td><a href="http://www.oxoid.com" target="_blank"><img src="/img/new_window.png" /> Oxoid, UK</a></td><td>CM0007</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>P4417</td><td></td></tr><tr><td>Ethanol 100%</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>E7023</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>84020</td><td>Prepare 20 - 50 units/ml</td></tr><tr><td>RNAlater Solution</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>R0901</td><td>10 &amp;mu;l/mg tissue</td></tr><tr><td>Acetic Acid</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>320099</td><td></td></tr><tr><td>Chloroform</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>C2432</td><td></td></tr><tr><td>Methanol</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>322415</td><td></td></tr><tr><td>Cotton-tipped swabs</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>11542483</td><td></td></tr><tr><td>Alcotip Swabs</td><td><a href="http://www.scientificlabs.co.uk" target="_blank"><img src="/img/new_window.png" /> Scientific Laboratory Supplies, UK</a></td><td>SWA1000</td><td></td></tr><tr><td>Petri dishes</td><td><a href="http://www.sigmaaldrich.com" target="_blank"><img src="/img/new_window.png" /> Sigma, UK</a></td><td>P5856</td><td></td></tr><tr><td>30 ml Universal Tube</td><td><a href="http://www.alphalabs.co.uk" target="_blank"><img src="/img/new_window.png" /> AlphaLaboratories, UK</a></td><td>CW3890</td><td></td></tr><tr><td>0.5 ml microcentrifuge tubes</td><td><a href="http://www.starlab.co.uk" target="_blank"><img src="/img/new_window.png" /> StarLab, UK</a></td><td>I1405-1500</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td><a href="http://www.starlab.co.uk" target="_blank"><img src="/img/new_window.png" /> StarLab, UK</a></td><td>I1415-1000</td><td></td></tr><tr><td>0.1 &amp;mu;l calibrated loops</td><td><a href="http://www.starlab.co.uk" target="_blank"><img src="/img/new_window.png" /> StarLab, UK</a></td><td>E1412-0112</td><td></td></tr><tr><td>L-shaped spreaders</td><td><a href="http://www.starlab.co.uk" target="_blank"><img src="/img/new_window.png" /> StarLab, UK</a></td><td>E1412-1005</td><td></td></tr><tr><td>Cuvettes</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>10594175</td><td></td></tr><tr><td>Forceps straight with fine points</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>12780036</td><td></td></tr><tr><td>Forceps straight with blunt tips</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>12391369</td><td></td></tr><tr><td>Forceps watchmaker&#039;s curved with very fine points</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>12740926</td><td></td></tr><tr><td>Scissors straight with very fine points</td><td><a href="http://www.fisher.co.uk" target="_blank"><img src="/img/new_window.png" /> Fisher Scientific, UK</a></td><td>12972055</td><td></td></tr><tr><td>Laboratory Scissors</td><td><a href="http://www.vwr.com" target="_blank"><img src="/img/new_window.png" /> VWR, UK</a></td><td>USBE4251</td><td></td></tr><tr><td>25 G Syringe Needles</td><td><a href="http://www.greinerbioone.com" target="_blank"><img src="/img/new_window.png" /> Greiner Bio-One Ltd</a></td><td>N2525</td><td></td></tr><tr><td>LAMBDA 25 UV/Vis Spectrophotometers</td><td><a href="http://www.perkinelmer.co.uk" target="_blank"><img src="/img/new_window.png" /> PerkinElmer, UK</a></td><td>L60000BB</td><td></td></tr><tr><td>Unitemp Incubator</td><td>B&amp;amp;T, UK</td><td>OP958</td><td></td></tr><tr><td>Multitron shaking incubator</td><td><a href="http://www.infors-ht.com" target="_blank"><img src="/img/new_window.png" /> INFORS HT, UK</a></td><td>AJ118</td><td></td></tr><tr><td>Ultra-Turrax T-10 homogenizer</td><td><a href="http://www.ika.com" target="_blank"><img src="/img/new_window.png" /> IKA Werke</a></td><td>0003737000</td><td></td></tr></tbody></table>

non-invasive model of neuropathogenic escherichia coli infection in the neonatal rat

动物宿主和细菌病原体之间的相互作用的研究是唯一有意义的，如果所用的感染模型中复制了自然感染的主要特征。本协议描述由于在新生大鼠neuropathogenic 大肠杆菌 K1全身感染的建立和评价程序。胃肠道的定植导致传播沿感染的肠淋巴血 - 脑当然病原体和模型显示强烈的年龄依赖性。 大肠杆菌的菌株大肠杆菌 O18:K1具有增强毒力的乳鼠产生极高的速度定植，易位血室和脑膜浸润下，通过脉络丛中转。如在人类宿主，中枢神经系统的渗透是伴随着局部炎症和一个总是致命的结果。该模型是经过验证的效用的机制研究发病机制，治疗性干预措施的评价和细菌毒力的评估。

该E.此处描述的大肠杆菌 K1的全 ​​身感染模型复制许多在人体中的自然感染的特征。细菌被摄入，定殖于胃肠道，建立器官特异性疾病与脑24的相关的炎症之前经由肠系膜淋巴结移位到血液舱。重要的是，该模型会显示较强的年龄依赖性;如示于图3中 ，2日龄（P2）的幼鼠极易受到侵入性疾病，但是在一个7天期间，动物变得越来越难熔感染，但不向GI道定植17。从胃肠道定植到血液舱的部位转运后，细菌可以在脉络丛25主要进入中枢神经系统之前被可视化的血液样品中的荧光显微术（ 图4）。在某些动物中，有大量的入侵等主要脏器如肺，脾，肾25。 细菌数目在组织中可以个别幼仔25但之间有很大差异时的生物负载的得分为存在或不存在是有高度再现性的关于机关的入侵。随着12幼崽作为一个单一的测试队列一窝，使用G *电源软件功率计算确定该样本的大小等同于寻找基于生存的使用从队列六只动物和&gt; 99％的概率，如果所有的效果了98.6％的概率12顷考虑。模型，因此适用于专为新生儿细菌性感染的治疗量身新试剂的评价，并已用于在程序中来评价解聚酶EndoE的胶囊，其选择性地移除从细菌表面24-26的K1胶囊的治疗潜力。它也可用于研究宿主细菌相互作用的轻拍该冲击E的hogenesis 大肠杆菌 neuropathogens;在这方面，已经采用了E的研究大肠杆菌 A192PP殖民化和传播。业已证明， 大肠杆菌大肠杆菌 A192PP细胞坚持的P2，P5和P9幼仔大量胃肠道;定植在这三组的时间方面是非常相似的（ 图5），并反映了细菌的复制和维持肠道内人口密度的能力。 将临床分离株A192的毒力是由新生大鼠连续传代，以确保动物使用很少或没有冗余增强。E.大肠杆菌 A192殖民P2新生鼠100％的效率，在动物的35％，引起菌血症和生产的25％27产生致命的影响。在传代衍生A192PP定居于胃肠道，产生菌血症，引起致命性在所有的P2幼崽。因此，该模型可用于研究二的毒力fferent K1菌株相对于它们从定植部位侵入中枢神经系统和其他器官系统的容量。在这种情况下，Pluschke和同事23中使用的乳鼠感染模型来确定的95 大肠杆菌的能力大肠杆菌 K1株人源性引起肠道定植后菌血症;他们观察到的广泛的变化既殖和侵入的能力的效率，支撑大肠杆菌的克隆性质大肠杆菌 K1 neuropathogens。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig1highres.jpg" width="600" /> 图1.材料组织收集:（A）秤，（B）包含必要的媒体预称重管，（C）手术台上，统治者和针头牵制动物，（D）70％（V / V）乙醇和PBS进行消毒的组织采集设备， <s仲&gt;（E）的组织采集用试剂盒，包括一个大剪刀为断头和剪子和镊子和各种尺寸和形状，（F）中的冰刀片，以保持组织中，（G），70％（体积/体积）的乙醇灭菌在手术台上，并包围。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52018/52018fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig2highres.jpg" width="600" /> 图2.分离胃肠道及肠系膜淋巴系统。 （A）的把手的肠系膜淋巴系统以细解剖镊的中央质量。（B）的把手小肠的近端部分，并且拉方向相反。（℃）的胃肠道和肠系膜淋巴系统将完全分隔符即<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52018/52018fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig3highres.jpg" width="500" /> 图3.生存以下E.口服年龄介乎2天（P2）九天（P9）新生幼鼠大肠杆菌 A192PP，说明全身性感染的壮龄相关性，每个组代表24例新生儿。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/52018/52018fig4highres.jpg" width="600" /> 大肠杆菌的图4.荧光图像大肠杆菌 A192PP细胞从P2的小狗血涂片感染细菌之后口服。在细菌表面的脂多糖抗原Ø是STained用兔抗O18多克隆抗体和Alexa546标记的羊抗兔第二抗体。在K1胶囊可视化与EndoE-GFP试剂。事实上，血液样本中检测到的所有细菌所显示的保护K1胶囊。图片由安德烈Zelmer博士抓获。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52018/52018fig4highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/52018/52018fig5highres.jpg" width="500" /> 图5. E.细菌接种。DNA的大肠杆菌 A192PP肠道定植以下管理是从整个肠道大肠杆菌中提取大肠杆菌 K1的菌落形成单位/ g（菌落形成单位/克）通过定量聚合酶链反应（qPCR的）来确定针对polysialyltransferase（NEUS）基因组织如别处所述17 </suP&gt;。 LOD:检测限。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 特征 </td><td> 健康 </td><td> 不良 </td></tr><tr><td>皮肤的颜色</td><td>粉红色</td><td>淡/黄</td></tr><tr><td>敏捷（翻正反射） </td><td>小狗立刻就扭转落后的位置</td><td>难以扭转落后的位置（&gt; 3秒），或者不能达到</td></tr><tr><td>上腹部轻微的压力</td><td>没有声音</td><td>声音激荡</td></tr><tr><td>胃/乳线</td><td>可见，白</td><td>不可见</td></tr><tr><td>温度</td><td>温暖</td><td>比较冷* </td></tr><tr><td>重量</td><td>每天1.5-2克增益</td><td>没有增重或减重</td></tr><tr><td>当放置在笼子里的行为</td><td>对母亲的动作，并开始˚Feeding </td><td>不能走向母亲，并显示喂养困难</td></tr></tbody></table> 表1. 7分的评分系统:列出的第3得分通常观测到的初步迹象。 *新生儿全身性感染的经验体温升高（&gt; 2℃）。然而，由于缺乏对动物的敏捷性，以达到其母亲以维持体温，不健康的动物可能变得垫料分离和感觉冷到裸手。

Watch this Scientific Journal Video about Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat at JoVE.com

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

这里，一个程序被描述为建立系统性感染的乳鼠大肠杆菌 K1的培养物。这种非侵入性的过程允许定植在胃肠道的，病原体向全身循环的移位，以及侵袭中枢神经系统的脉络丛。

Neuropathogenic的非侵入型<em&gt;大肠杆菌</em&gt;感染乳鼠

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

non-invasive-model-neuropathogenic-escherichia-coli-infection

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

10.9K 次观看.  University College London. 这里，一个程序被描述为建立系统性感染的乳鼠大肠杆菌 K1的培养物。这种非侵入性的过程允许定植在胃肠道的，病原体向全身循环的移位，以及侵袭中枢神经系统的脉络丛。 

视频: Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

围产期缺血缺氧缺血性脑病模型

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

科研

生物学

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

“<em&gt;枸橼酸rodentium</em&gt;鼠标产品型号：研究感染性结肠炎的病原体和宿主的贡献

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

免疫与感染

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

医学

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

肾内注射<em&gt;大肠杆菌</em&gt;在肾性肾炎大鼠模型中

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human intestinal epithelium, leading to increased intestinal permeability, impaired restitution, and increased cell death. Although there are numerous animal models of NEC, response to injury and therapeutic interventions may be highly variable between species. Furthermore, it is ethically challenging to study disease pathophysiology or novel therapeutic agents directly in human subjects, especially children. Therefore, it is highly desirable to develop a novel model of NEC using human tissue. Enteroids are 3-dimensional organoids derived from intestinal epithelial cells. They are ideal for the study of complex physiologic interactions, cell signaling, and host-pathogen defense. In this manuscript we describe a protocol that cultures human enteroids after isolating intestinal stem cells from patients undergoing bowel resection. The crypt cells are cultured in media containing growth factors that encourage differentiation into the various cell types native of the human intestinal epithelium. These cells are grown in a synthetic, collagenous mix of proteins that serve as a scaffold, mimicking the extra-cellular basement membrane. As a result, enteroids develop apical-basolateral polarity. Co-administration of lipopolysaccharide (LPS) in media causes an inflammatory response in the enteroids, leading to histologic, genetic, and protein expression alterations similar to those seen in human NEC. An experimental model of NEC using human tissue may provide a more accurate platform for drug and treatment testing prior to human trials, as we strive to identify a cure for this disease.

Enteroids are emerging as a novel model in the study of human disease. The protocol describes how to simulate an enteroid model of human necrotizing enterocolitis using lipopolysaccharide (LPS) treatment of enteroids generated from neonatal tissue. Collected enteroids demonstrate inflammatory changes akin to those seen in human necrotizing enterocolitis.

一种新的坏死性肠结肠炎人上皮肠素模型

Necrotizing enterocolitis (NEC) is a devastating disease of newborn infants. It is characterized by multiple pathophysiologic alterations in the human ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

革兰阴性细菌败血症的新生儿成像模型

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

新生儿 大肠杆菌 分离菌血症肠道转吞作用的评估

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Neuropathogenic的非侵入型

摘要

探索更多视频

此视频中的章节

围产期缺血缺氧缺血性脑病模型

“

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

肾内注射

一种新的坏死性肠结肠炎人上皮肠素模型

革兰阴性细菌败血症的新生儿成像模型

新生儿大肠杆菌分离菌血症肠道转吞作用的评估

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Neuropathogenic的非侵入型

摘要

探索更多视频

此视频中的章节

围产期缺血缺氧缺血性脑病模型

“

早产儿脑病建模使用产前缺氧缺血大鼠羊膜腔内毒素

肾内注射

一种新的坏死性肠结肠炎人上皮肠素模型

革兰阴性细菌败血症的新生儿成像模型

新生儿 大肠杆菌 分离菌血症肠道转吞作用的评估

胎儿接触产妇炎症的穆林模型，研究急性胆管炎对新生儿肠道发育的影响

一种新生儿BALB/c坏死性小肠结肠炎小鼠模型

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

新生儿大肠杆菌分离菌血症肠道转吞作用的评估