Name: prevention of heat stress adverse effects in rats by bacillus subtilis strain
Uploaded: 2016-07-11T14:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

This work was supported by the Auburn University fund FOP 101002-139294-2050.

所有的实验程序，由奥本大学IACUC，阿拉巴马州奥本市批准。 1，文化传媒，餐具和细菌培养的制备<ol><li>接种10毫升营养肉汤的与枯草芽孢杆菌 BSB3培养物的单个菌落，在营养琼脂平板上生长过夜的烧瓶中。 </li><li>使500毫升孢子形成介质19（每升:细菌用营养肉汤，8克10％（重量/体积）的KCl，10毫升，1.2％（重量/体积）用MgSO 4×7H 2 O，10毫升，琼脂，15克），在121℃下20分钟的高压釜。允许冷却至50℃，并添加以下无菌溶液:1米的Ca（NO 3）2，1毫升， 0.01M的氯化锰2,1毫升， 1 mM的硫酸亚铁1毫升。 </li><li>至40℃，在室温下20分钟凉爽准备介质和将25 ml接种入各在无菌柜20的无菌培养皿中。让中等固化过夜。 </li><li>传播0.5毫升的B过夜培养芽孢杆菌BSB3到在培养皿中的介质的表面上。文化孵育在37℃5天，直到90％孢子。显示高分辨率显微镜相亮孢子。 </li><li>收获细菌加入1毫升无菌磷酸盐缓冲盐水（PBS）的到板，并使用无菌细胞吊具从表面刮细菌。直到需要存储该悬浮液在冰箱中。 </li><li>在37℃。24小时-通过电镀0.1毫升上孢子形成培养基平板细菌悬浮液的合适的稀释液（10 -5〜10 -6），随后温育18的确认菌的生存能力</li><li>指望平板菌落使用菌落计数器并计算测试悬挂细菌的滴度。制备细菌悬浮液与最终浓度为1×10 8 CFU / ml，在PBS中。 </li></ol> 2.动物<ol><li>使用32只雄性SD大鼠（250 - 300克）。保持与免费获取标准颗粒饲料和水无特定病原体条件下的动物。建立一个平均温度（21±1℃），湿度（50±5％）和12小时光照-黑暗周期。 </li></ol> 3.实验设计<ol><li>经口灌胃11开始治疗的动物，驯化后的2天。使用注射器用口管饲针头。治疗16大鼠B.枯草 BSB3悬浮（1毫升，每鼠）两次两天每天（ 枯草芽孢杆菌组）。治疗16只大鼠用PBS（1毫升，每鼠）两天（PBS组）（图1），每天两次。 </li><li>治疗后，各细分组（每组8只）:1组对照组（PBS /无应力），2-组B.枯草芽孢杆菌 （ 枯草芽孢杆菌 /无应力），3组应力（PBS /热应激）和组4- B.枯草芽孢杆菌 +应力（ 枯草芽孢杆菌 /热应激）。 </li><li>使用电子角测量每只大鼠的直肠温度TRONIC数字温度计。保持组1和2的大鼠在室温下25分钟。在45℃下在气候室组3和4以及相对湿度为25分钟的55％的地方动物。 </li><li>然后，测量使用电子数字温度计各组每只大鼠的直肠温度。放置在室温下的所有动物4小时。 </li><li>在密封的罐子麻醉 - （4％2）观察，直到大鼠深度麻醉（无响应脚趾捏的）麻醉异氟醚老鼠。通过用断头台斩首迅速安乐死只。 </li><li>从每只大鼠20收集主干血（15 - 15毫升）与无热原进入移液器无热原管，以获得血清，并立即采取与镜检吸管的血液样本（每只大鼠7微升）。 </li><li>放管与血液在冰箱中至少30分钟，以允许凝固。离心管中，在20℃7000 XG 10分钟，迅速除去血清吸管。在-20℃下，直到检测每只大鼠成50微升体积和存储获得的分装血清。 </li></ol> 4.手术过程<ol><li>剪切/修剪从电动剪板机尸体的下面的腹部和胸部都皮毛。放置胎体在无菌柜和治疗用70％酒精胎体的剃表面。 </li><li>用无菌解剖刀创建腹部中线切开。使用无菌镊子各组大鼠肝脏取出，肠系膜淋巴结，脾，小肠。 </li></ol> 5.细菌移位分析<ol><li>肝，肠系膜淋巴结和脾的每个样品放入预先称重的无菌管和权衡。计算出样品的重量与样品的管的重量和空管的重量之间的差异。 </li><li>添加无菌PBS到管以得到1:10稀释（重量/体积）的样品和homogenize每个样品用无菌玻璃组织匀浆器以获得均匀的悬浮液21。 </li><li>使在无菌PBS各样品的连续稀释液（10 -1〜10 -4）。板0.1各组织的所有稀释到麦康凯的表面和5％血琼脂和布鲁氏菌血琼脂血红素（0.005克/ L）和维生素K1（0.01克/升）板毫升。 </li><li>孵育麦康凯的5％血琼脂平板和好氧布鲁氏菌血琼脂平板厌氧条件下在37℃22。 </li><li>算需氧细菌的菌落后24小时，并使用菌落计数器48小时温育后厌氧菌菌落。表达结果作为在一克组织的菌落形成单位（CFU）的数目。 </li></ol> 6.组织学分析<ol><li>通过外科手术从每只大鼠中取出小肠样品（步骤4.1 - 4.2）。修复布安固定液的样品，石蜡嵌入，制备6微米的部分和染色部分，使用标准程序23苏木精和曙红。 </li><li>用高分辨率显微镜系统，用于测量每个样品（倍率100倍）24在肠绒毛和总粘膜厚度的高度。分析来自各大鼠4个样品，并在每个样品中至少二十测量。表达微米的结果。 </li></ol> 7.细胞因子检测<ol><li>使用IL-1β商业大鼠ELISA试剂盒; IL-6; TNF-α; INF-γ，IL-10，以根据制造商的协议测量血清中的细胞因子的水平。 </li><li>生成每个组使用标准的相关试剂盒测定样品的标准曲线。通过与合适的标准曲线的实验数据进行比较确定每个样品中的细胞因子的水平。 </li></ol> 8.脂多糖含量<ol><li>使用根据大​​鼠脂多糖ELISA试剂盒分析血清中的LPS水平制造商的协议。通过与标准曲线的值所获得的值的比较，判断样品中的LPS浓度。 </li></ol> 9.血液的高分辨率光学显微镜<ol><li>使用先前描述25,26显微镜系统。使用一个隔振平台作为用于显微镜系统中的基站和摄像机和计算机25来记录实时图像。校准使用理查森滑动如前所述27测试图像。 </li><li>放置7微升新鲜抽取的血液从载玻片上，盖玻片，照片各大鼠和每个样品（倍率1,700X）中记录的72×53.3微米2将 10图像帧。 </li><li>测量使用软件，它提供实时高分辨率直视光学图象囊泡的浓度。检查每个实验条件28最小20的图像帧。 </li></ol> 10. STATIS抽动<ol><li>表达的所有值以平均值和标准偏差。进行统计分析和图形绘制。分析与两样本t检验的结果，在0.05设置显着性水平确定统计学意义。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

暴露在高温下会导致严重的健康状况29。预防和早期发现与热应激的并发症是至关重要的30。 所提出的协议可以用来评估用于预防热应力的不良影响的各种方法的功效。 这个协议中的关键步骤包括:在热处理过程（45℃，25分钟）;无热原的材料的收集和血液以防止肠毒素污染处理期间的使用;保持血清等分在-20℃至避免反复冻融;和细菌易位的分析过程如下无菌操作。该协议建立了与细菌进行预防热应激的副作用口服治疗大鼠。施用和其它化合物的其它途径，该协议可以被修改。例如，TR的时间热应激前eatment可以延长。口服给药可改为直肠给药。被用于通过暴露于升高的温度之前口服治疗动物的预防热应力并发症枯草杆菌细菌。为了固化热应力的并发症，该协议可以通过使用细菌暴露于热治疗后的动物进行修改。 结果表明，体温度的升高而引起的红细胞囊泡的增加，并且这完全被预处理与B防止枯草芽孢杆菌的细菌。因此，在血液中的小泡浓度的暴露于热时的增加可以指示热应力的水平和提供诊断证据红细胞的稳定性以及它们对耐热性。它也是用于评价对热应力的并发症的预防性治疗的功效一个非常有用的，快速的检测。感应Ø˚F热休克蛋白为热应激的标记物被前面讨论的30，但这种方法是费时且不能用于响应的实时诊断热应激。 为防止热应力的不良影响所提出的方法是简单，成本有效的，并且不需要额外的设备或特别的设施。这项工作未来的应用将有助于了解对热应激并发症的保护机制和描述这种保护的新程序。此方法是修路工人，军人，运动员的意义-人，在激烈的户外体育锻炼29将中暑高危人群。所提出的方法允许一个审查热应力的程度，并评价各种方法来保护的人在极端条件下工作的健康。

Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, modulation of the intestinal microbiota can provide a novel approach for prevention of stress-related adverse effects. 
Beneficial bacteria have been used as a promising strategy to modify the gut microbiota for successful management of various clinical conditions, such as diarrhea, inflammatory bowel disease, atopic dermatitis, metabolic disorders7-10. Probiotic effects of beneficial bacteria include production of essential metabolites to support intestinal health, stimulation of the immune system, promotion of lactose tolerance, restoration of epithelial dysfunction. Probiotics were effective in prevention of the stress-related complications in vitro and in animal models11,12. 
Researchers have given more attention to Bacillus bacteria as probiotics because these bacteria support intestinal homeostasis13 and have beneficial effects on the host14. Our previous data demonstrated high efficacy of Bacillus probiotics against pathogens in vitro15,16 and in clinical trials17,18. Here, we aim to evaluate the preventive effect of B. subtilis BSB3 strain against complications after heat stress.

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich,&amp;nbsp;St. Louis, MO</td><td>P4417</td><td></td></tr><tr><td>Ethyl Alcohol&amp;nbsp;</td><td>Pharmco products Inc. Brookfield, CT, USA&amp;nbsp;</td><td>64-17-5</td><td></td></tr><tr><td>Agar</td><td>VWR</td><td>97064-334</td><td></td></tr><tr><td>MacConkey agar plates</td><td>VWR</td><td>470180-742</td><td></td></tr><tr><td>5% blood agar plates</td><td>VWR</td><td>89405-024</td><td></td></tr><tr><td>Brucella blood agar plate with Hemin, and Vitamin K</td><td>VWR</td><td>89405-032</td><td></td></tr><tr><td>Bouin&amp;rsquo;s Fixative</td><td>Electron Microscopy Sciences, Hatfield, PA, USA</td><td>15990</td><td></td></tr><tr><td>IL-10 Rat ELISA kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0101</td><td></td></tr><tr><td>IL-1beta Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0011</td><td></td></tr><tr><td>IL-6 Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC0061</td><td></td></tr><tr><td>INF-gamma Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC4021</td><td></td></tr><tr><td>TNF-alpha Rat ELISA Kit</td><td>Invitrogen, Camarillo, CA, USA</td><td>KRC3011</td><td></td></tr><tr><td>Rat LPS ELISA Kit</td><td>NeoBioLab, MA, USA</td><td>RL0275</td><td></td></tr><tr><td>Environmental Chamber 6020-1</td><td>Caron, Marietta, OH, USA</td><td>6020-1</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Beckman Coulter, Indianapolis, IN, USA</td><td>Optima L-90K</td><td></td></tr><tr><td>Ultra Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Light microscope optical system</td><td>CitoViva Technology Inc., Auburn, AL</td><td></td><td></td></tr><tr><td>Colony counter</td><td>Fisher Scientific , Pittsburgh, PA, USA</td><td>RE-3325</td><td></td></tr><tr><td>Freezer</td><td>Haier, Brooklyn, NY, USA</td><td>HCMO50LA</td><td></td></tr><tr><td>Ultra microplate reader</td><td>Bio-Tek Instrument, Winooski, VT, USA</td><td>ELx 808</td><td></td></tr><tr><td>Auto Strip Washer</td><td>Bio-Tek Instrument, Winooski, VT, USA</td><td>ELx50</td><td></td></tr><tr><td>Pipettes</td><td>Gilson, Pipetman, France</td><td>P100, P200, P1000</td><td></td></tr><tr><td>C24 Incubator Shaker</td><td>New Brunswick Scientific, Enfield, CT, USA</td><td>Classic C24</td><td></td></tr><tr><td>Rocking Shaker&amp;nbsp;</td><td>Reliable Scientific, Inc., Nesbit, MS, USA</td><td>55</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific, Pittsburgh, PA, USA</td><td>875713</td><td>100 mm x 15 mm</td></tr><tr><td>SterilGard III Advance</td><td>The Baker Company, Sanford, ME, USA</td><td>SG403</td><td></td></tr><tr><td>Culture Growing Flasks</td><td>Corning Incorporated, Corning, NY, USA</td><td>4995</td><td>PYREX 250 ml Erlenmeyer flasks</td></tr><tr><td>Optical Spectrometer Genesys 20</td><td>Thermo Scientific, Waltham, MA, USA.</td><td>4001</td><td></td></tr><tr><td>Sony DXC-33 Video camera</td><td>Sony</td><td></td><td></td></tr><tr><td>Richardson test slide</td><td>Electron Microscopy Science, Hatfield, PA, USA</td><td>80303</td><td></td></tr><tr><td>Millipore water purification system</td><td>Millipore</td><td>Direct-Q</td><td></td></tr><tr><td>Image-Pro Plus software</td><td>Media Cybernetics, MD, USA</td><td></td><td></td></tr><tr><td>Triple Beam Balance</td><td>OHAUS Corporation, Parsippany, NJ, USA</td><td></td><td></td></tr><tr><td>Tissue Homogenizer, Dounce</td><td>VWR</td><td>71000-518</td><td></td></tr></tbody></table>

prevention of heat stress adverse effects in rats by bacillus subtilis strain

此研究的目的是评估对热应力有关的并发症的枯草芽孢杆菌菌株的保护作用。三十二个的Sprague-Dawley大鼠用于该研究。动物口服治疗，一天两次两天与B枯草芽孢杆菌菌株BSB3或PBS。第二天在最后一次治疗后，各组被划分和两个实验组（一个用PBS处理和一个与枯草芽孢杆菌处理的）被放置在45℃下25分钟。两个对照组留在室温下25分钟。所有大鼠安乐死，并在各组不同的参数进行了分析。热应力的不良影响是由绒毛高度，并在肠上皮细胞总粘膜厚度的减小登记;从管腔细菌易位;增加红细胞的囊泡和仰角的脂多糖（LPS）在血液中的水平。治疗的保护效力是通过预评估这些副作用公约。该协议被设定为大鼠细菌预防热应力并发症的口服治疗，但该协议可以被修改，并用于其它给药途径和不同的化合物的分析。

热应激后的动物之前，立即的平均体温为36.7±0.07℃，40.3±0.17℃，分别为（P &lt;0.05）。大鼠暴露于热（第3组）导致绒毛高度的显著抑制和总粘膜厚度（图2,3）。 BSB3应变口服从热量的有害影响的应力保护肠道之前。 从肠道细菌易位通过肠系膜淋巴结的细菌分析（MLN），肝脏和各大鼠的脾脏来确定。在1.7×10 3±4.6×10 2 CFU / g组织（图4）的浓度的细菌培养物从MLN只分离和大鼠肝脏用PBS预处理并暴露于热（组3）。从对照大鼠所有测试样品（组1，2）和从热应激动物接到BSB3株（4组）共育。没有细菌从脾样品中分离。 IL-1β无明显变化; IL-6，TNF-α，INF-Γ细胞因子水平在热应激大鼠登记。在IL-10及LPS水平的热暴露显著抬高之前用PBS处理的动物的血清中发现（第3组） - （ 图5，图6）。与B预处理枯草 BSB3（组4）可防止IL-10及LPS的在热应激动物的血清中升高。 从（1.4±0.2）自由囊泡浓度的显著增加×10 6（3.8±0.3）×10 6囊泡微升-1（P &lt;0.001）在热应激前接受PBS的大鼠的血液中发现（图7 ，8）。与BSB3预处理一组动物中的游离囊泡的数量没有变化，发现船尾尔热曝光（图7）。 <img alt="图1" src="/files/ftp_upload/54122/54122fig1.jpg" /> 图1.研究设计。两个组大鼠（16只每组）通过与B口腔管饲法处理枯草 BSB3（ 枯草杆菌组）或用PBS（PBS组），每天两次。在第3天各组由8只大鼠每组细分。从组3和4只大鼠被放置在45℃下25分钟。对照组动物（组1和2）保持在室温。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54122/54122fig2.jpg" /> 用PBS或图2.肠绒毛高度（A）和粘膜厚度（B）在大鼠的测量。大鼠预处理枯草杆菌 BSB3暴露于45℃（压力），或至室温（对照）。每组8只。每个样品中的每个参数的二十测量采取。值被表示为平均值和标准偏差。与显着性水平0.05两样本t检验确定统计学意义的结果进行了分析。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54122/54122fig3.jpg" /> 肠黏膜无黄染的行HE。大鼠图3. 组织学图像 ，用PBS或B. ​​预处理枯草 BSB3并暴露于45℃（压力）或室温（对照）A. PBS /无应力; B。PBS /热应激;Ç。B.枯草 /无应力; D. B.枯草 /热应激。绒毛高度由箭头指示。酒吧200微米。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54122/54122fig4.jpg" /> 图 4. 大鼠组织细菌总数。大鼠PBS或B. ​​预处理枯草 BSB3暴露于45℃（压力），或至室温（对照）。每组8只。肠系膜淋巴结，肝和脾无菌从各大鼠，匀浆去除并接种到琼脂培养基以回收需氧和厌氧细菌。易位菌（需氧和厌氧）的总数被表示为每克组织的菌落形成单位（CFU）。值被表示为<html>均值和标准差。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54122/54122fig5.jpg" /> 图5. 在大鼠血清IL-10的水平。大鼠在用PBS或B预处理枯草 BSB3暴露于45℃（压力），或至室温（对照）。每组8只。从每只鼠获得血清和用市售的大鼠ELISA试剂盒进行分析。值被表示为平均值和标准偏差。与显着性水平0.05两样本t检验确定统计学意义的结果进行了分析。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 帐篷"FO: - together.within页保留="1"&gt; <img alt="图6" src="/files/ftp_upload/54122/54122fig6.jpg" /> 图6. 在大鼠血清浓度LPS。大鼠用PBS或B. ​​预处理枯草 BSB3暴露于45℃（压力），或至室温（对照）。每组8只。从每只鼠获得血清和用市售的大鼠ELISA试剂盒进行分析。值被表示为平均值和标准偏差。与显着性水平0.05两样本t检验确定统计学意义的结果进行了分析。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/54122/54122fig7.jpg" /> 在大鼠血液囊泡的 图7. 数目。大鼠预处理用PBS或B枯草 BSB3暴露于45℃（压力），或至室温（对照）。新鲜抽取的血液一小滴（7微升）置于载玻片上，盖上盖玻片，拍摄和记录的10帧的图像的每个样品（倍率1,700X）英寸囊泡的浓度用软件，它提供了在实时高分辨率直视光图像测量。检查每个实验条件二十图像帧。值被表示为平均值和标准偏差。与显着性水平0.05两样本t检验确定统计学意义的结果进行了分析。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/54122/54122fig8.jpg" /> 图8. 增加的Ve的sicles血中浓度热应激后，暴露于室温（A）或45℃（B）前用PBS组大鼠血液视频图像。新鲜抽取的血液一小滴（7微升）置于载玻片上，盖上盖玻片，拍摄和记录的10帧的图像的每个样品（倍率1,700X）英寸小泡表示为五杆6微米。 <a href="https://www.jove.com/files/ftp_upload/54122/54122fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

热火预防应激在大鼠不利影响<em&gt;枯草芽孢杆菌</em&gt;应变

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

prevention-heat-stress-adverse-effects-rats-bacillus-subtilis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

7.4K 次观看.  Auburn University. We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

视频: Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

大鼠甲基 Seq 平台识别与应力接触相关的表观变化

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

科研

生物化学

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

卡诺哈布迪蒂斯精英热休克反应的测量诱导标准化方法

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

环境科学

Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.

<meta charset="utf8"></head><body>从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

This protocol provides a complete and comprehensive description of the detailed process for the isolation, purification, and identification of bacitracin-producing Bacillus licheniformis from healthy pig feces.

从健康猪的新鲜粪便中分离、纯化和鉴定产酸杆菌肽的地衣芽孢杆菌

Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and ...

生物工程

Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of neurological disorders. Despite strong pre-clinical evidence, TH has not shown efficacy in clinical trials of most neurological disorders. The only successful trials employing therapeutic hypothermia were related to cardiac arrest in adults and hypoxic ischemic injury in neonates. Further investigations into the parameters of its use, and study design comparisons between pre-clinical and clinical studies, are warranted. This article demonstrates two methods of short-duration hypothermia induction. The first method allows for rapid hypothermia induction in rats using ethanol spray and fans. This method works by cooling the skin, which has been less commonly used in clinical trials and may have different physiological effects. Cooling is much more rapid with this technique than is achievable in human patients due to differences in surface area to volume ratio. Along with this, a second method is also presented, which allows for a clinically achievable cooling rate for short-duration hypothermia. This method is easy to implement, reproducible and does not require active skin cooling.

This article describes two methods of whole-body short-duration hypothermia induction in rats. The first, rapid induction method, employs active cooling using fans and ethanol spray for a rapid decrease in temperature. The second method is a gradual cooling method. This is achieved using the combination of isoflurane anesthesia and the reduction of temperature settings on the homeothermic heat mat. This results in a gradual decrease in core body temperature without the use of any external cooling devices.&#160;

使用研究临床相关性和机制的模型在大鼠中短时间体温过低诱导

Therapeutic hypothermia (TH) is a powerful neuroprotective strategy that has provided robust evidence for neuroprotection in pre-clinical studies of ...

医学

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

小鼠劳力性中暑的临床前模型

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

生物学

建立和验证慢性不可预测的轻度应激大鼠模型

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

作者聚焦:建立啮齿动物模型以研究传统蒙古医学中的抑郁因素

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

热火预防应激在大鼠不利影响

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使用研究临床相关性和机制的模型在大鼠中短时间体温过低诱导

小鼠劳力性中暑的临床前模型

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

基于蒙古药的大鼠慢性不可预测的轻度应激

基于蒙古药的大鼠慢性不可预测的轻度应激

基于蒙古药的大鼠慢性不可预测的轻度应激