Name: two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver
Uploaded: 2018-02-06T14:30:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

这项研究得到了韩国政府资助的韩国国家研究基金会 (NRF) 的一笔赠款 (MSIP; 对 Y-m 的赠款2016R1A2B4008199H.) 和大韩民国卫生 & 福利部 (赠款号: HI14C1324)。

所有程序都是按照韩国世大学医学院机构动物保育和使用委员会的指导方针进行的。LysM-绿色荧光蛋白 (gfp; gfp/+) 老鼠8和 CX3Cr1-GFP (gfp/+)9小鼠在8-12 周龄时被用于活体成像。所有的手术工具都是用70% 酒精蒸压和消毒的。
1. 具有辅助工具的定制设计的小鼠肝脏成像室
<ol><li>小鼠肝脏成像室设计<ol>
<li>为小鼠肝脏成像 (图 1) 制作如下尺寸的腔室: 内腔 (120 mm 长度 x 160 mm 宽度 x 30 mm 高度), 外腔 (100 mm 长度 x 100 mm 宽度 x 24 mm 高度), 螺栓 (Ф 3 mm x 30 mm 高度), 螺母 (Ф 4 mm)和翅形螺母 (Ф 4 mm)。</li>
		<li>测量固化剂和弹性体基在1:9 的比例, 使共120克硅。</li>
		<li>将液体硅胶混合物放入250毫升锥形瓶中, 并将其混合均匀。然后把烧瓶放进干燥, 在真空泵的真空下, 将气泡取出约1小时。</li>
		<li>倒入液体硅胶混合成一个定制设计的老鼠室高达10毫米的高度, 让它治愈了几天 (图 1A)。</li>
	</ol>
</li>
	<li>覆盖片和肝固定床的制备<ol>
<li>在金属框架上, 用硅胶胶水把盖子玻璃贴上。让它干1天 (图 1B)。</li>
		<li>为了有效地控制水浸透镜的蒸馏水, 一个永久性的堵水结构是必不可少的。使 1 mm 四舍五入硅胶胶水和硬化过夜。</li>
		<li>混合固化剂和弹性体基础, 然后倒入一个6孔板在一个高度约8-10 毫米. 执行硬化过程, 如所述, 在 1.1. 2-1. 1.4。</li>
	</ol>
</li>
</ol>2. LPS 的制备
<ol><li>制备无菌1x 磷酸盐缓冲盐水 (PBS), 以稀释从沙门氏菌 enterica血清型肠炎的 LPS 粉末。(1x PBS: 137 毫米氯化钠, 2.7 毫米氯化钾, 10 毫米 Na2HPO4, 1.8 mm,2PO4)。</li>
	<li>预热 PBS 在37° c 水浴, 并仔细打开 LPS 粉末瓶 (25 毫克)。</li>
	<li>使用吸管的援助, 倒入无菌 PBS (5 毫升) 到 LPS 粉瓶, 并轻轻搅拌或暂停解决方案, 准备库存解决方案。 注: 在 LPS 粉末液化后, 苛刻的涡流会引起气泡。在1.5 毫升的管子中制造等分时, 避免气泡使损失降到最低。</li>
	<li>储存在20° c 的股票 lps 溶液, 并将其稀释为1毫克/毫升 lps 溶液注射。</li>
	<li>使用1毫升注射器将1毫克/毫升 LPS 溶液 (20 毫克/千克) 注入非麻醉小鼠的腹膜中。对于控制动物, 注入相同体积的 PBS。</li>
	<li>等待2小时足够的炎症反应。</li>
</ol>3. 得克萨斯红葡聚糖用于染色血管的制备
<ol><li>准备两个15毫升锥形管和10毫升无菌 PBS。</li>
	<li>在3毫升的 PBS 中稀释得克萨斯红葡聚糖粉 (25 毫克), 并将其混合均匀。将混合物转移到一个空的15毫升锥形管。</li>
	<li>将剩余的 PBS (7 毫升) 转移到含有葡聚糖混合物 (3 毫升) 的15毫升锥形管中。</li>
	<li>将0.45 µm 注射器过滤器连接到10毫升注射器的尖端。注射器过滤器应该替换针。</li>
	<li>过滤葡聚糖混合物 (10 毫升)。均匀按压注射器以避免溢出。</li>
	<li>500µL 2.5 毫克/毫升右旋糖酐溶液在轻堵塞的1.5 毫升管和存储在20° c。解冻后, 将其储存在4° c。</li>
</ol>4. 手术过程
<ol><li>在手术前用70% 酒精消毒手术台和所有器械。然后, 设置用于肝脏手术的工具, 并将加热板调整到37° c (图 2A, B)。</li>
	<li>对于麻醉, 腹腔注射30毫克/公斤的 tiletamine/唑和10毫克/公斤的嗪。这些解决方案可以混合和稀释在 PBS 的同时注射。通过轻轻按压鼠标的脚趾来确认麻醉。 当没有反射时继续下一步。</li>
	<li>用少量的眼膏来预防小鼠视网膜的干燥。</li>
	<li>使用脱毛膏 (图 2C) 在腹部周围移除体毛。</li>
	<li>注入200µL 的得克萨斯红葡聚糖溶液 (浓度2.5 毫克/毫升) 通过尾静脉或复古轨道注射使用胰岛素注射器 (图 2D)。</li>
	<li>用标记标记右下区域。这条线应该从剑的过程到肋骨的末端。用钳子和剪刀切开表皮 (图 2E, F)。</li>
	<li>用微剪刀和镊子打开腹腔, 仔细地暴露肝脏。使用棉签 (图 2G, H) 将左外侧叶滚出。</li>
	<li>将500µL 的无菌 PBS 放在腹腔内, 防止肝脏干燥。</li>
	<li>将鼠标放在一个定制设计的房间的右卧位位置, 并在硅床上涂抹少量的组织胶。然后, 用棉签仔细地将肝脏附着 (图 2I)。 注: 由于肝组织极其易碎, 不机械拔肝。用棉签轻轻地滚出来。</li>
	<li>几秒钟后, 再用500µL 的无菌 PBS 湿肝, 避免肝脏干燥。将金属框架放置在肝脏上, 并将其固定在螺母上 (图 2J)。</li>
</ol>5. 双光子显微镜对肝脏的活体成像
<ol><li>运行禅宗软件 (卡尔蔡司) 打开激光, 设置波长为 880 nm, 并设置绿色, 和红色通道。根据荧光强度调整激光功率 (图 3A)。发射过滤器信息如下: Green/Alexa488, 500-550 毫微米和红色或 Alexa 555, 575-610 毫微米。</li>
	<li>将腔室置于成像阶段, 并将20X 水浸泡物镜靠近盖板玻璃。</li>
	<li>将硅胶加热器固定在鼠标体内以保持其体温 (图 3B)。</li>
	<li>把500µL 的蒸馏水放在金属架上, 然后将物镜靠近肝脏 (图 3C)。</li>
	<li>调整焦点并确定成像区域的深度和时间。</li>
	<li>进行成像。</li>
	<li>对于长期成像, 在成像过程中每小时注入半剂量的 tiletamine/唑和嗪合剂。在这一阶段, 保持37° c 小鼠的体温是必不可少的。始终保持对鼠标的眼睛, 因为麻醉时间不同于每一个鼠标。 注: 此方法不适用于术后恢复及影像学检查。我们在手术后立即牺牲鼠标。</li>
	<li>检查踏板反射每30分钟的稳定麻醉。</li>
</ol>6. 数据分析
<ol><li>打开数据分析软件 Volocity, 然后将库中的原始数据导入到软件中。</li>
	<li>从图像中删除噪音以适应情况并拍摄快照。 注意: 要获得平滑图像, 请使用 "精细" 或 "中位" 滤镜。此外, 使用 "对比度增强" 功能使深色或模糊图像更清晰。</li>
	<li>将编辑的项目调整为 100%, 并以 JPEG 或 TIFF 文件的格式导出为电影的快照图像和 AVI 文件。</li>
</ol>

<ol>
	<li>Girard, J. R., 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=Fuels,+hormones,+and+liver+metabolism+at+term+and+during+the+early+postnatal+period+in+the+rat.">Fuels, hormones, and liver metabolism at term and during the early postnatal period in the rat.</a> J Clin Invest. 52 (12), 3190-3200 (1973).</li><li>Marques, P. E., 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=Imaging+liver+biology+in+vivo+using+conventional+confocal+microscopy.">Imaging liver biology in vivo using conventional confocal microscopy.</a> Nat Protoc. 10 (2), 258-268 (2015).</li><li>Dasari, S., Weber, P., Makhloufi, C., Lopez, E., Forestier, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Microscopy+Imaging+of+the+Liver+following+Leishmania+Infection:+An+Assessment+of+Hepatic+Hemodynamics.">Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics.</a> J Vis Exp. (101), e52303 (2015).</li><li>Honda, 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=Intravital+imaging+of+neutrophil+recruitment+in+hepatic+ischemia-reperfusion+injury+in+mice.">Intravital imaging of neutrophil recruitment in hepatic ischemia-reperfusion injury in mice.</a> Transplantation. 95 (4), 551-558 (2013).</li><li>Jenne, C. N., Wong, C. H., Petri, B., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+spinning-disk+confocal+microscopy+for+the+intravital+analysis+of+platelet+dynamics+in+response+to+systemic+and+local+inflammation.">The use of spinning-disk confocal microscopy for the intravital analysis of platelet dynamics in response to systemic and local inflammation.</a> PLoS One. 6 (9), e25109 (2011).</li><li>Ritsma, L., 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=Intravital+microscopy+through+an+abdominal+imaging+window+reveals+a+pre-micrometastasis+stage+during+liver+metastasis.">Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis.</a> Sci Transl Med. 4 (158), 158ra145 (2012).</li><li>Sierro, F., 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+Liver+Capsular+Network+of+Monocyte-Derived+Macrophages+Restricts+Hepatic+Dissemination+of+Intraperitoneal+Bacteria+by+Neutrophil+Recruitment.">A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment.</a> Immunity. 47 (2), 374-388 (2017).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 719-726 (2000).</li><li>Jung, 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=Analysis+of+fractalkine+receptor+CX(3)CR1+function+by+targeted+deletion+and+green+fluorescent+protein+reporter+gene+insertion.">Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.</a> Mol Cell Biol. 20 (11), 4106-4114 (2000).</li><li>Heymann, F., 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=Long+term+intravital+multiphoton+microscopy+imaging+of+immune+cells+in+healthy+and+diseased+liver+using+CXCR6.Gfp+reporter+mice.">Long term intravital multiphoton microscopy imaging of immune cells in healthy and diseased liver using CXCR6.Gfp reporter mice.</a> J Vis Exp. (97), (2015).</li></ol>

作者声明没有竞争的金融利益。

由于其功能的重要性, 许多组3,10都积极研究了肝脏。例如, Heymann et al. 提出了一种微妙的方法使用琼脂糖。虽然这种方法被认为是高度精确的, 琼脂糖设置需要时间。然而, 用我们的方法, 所有的程序可以在30分钟内进行, 尽量减少其他混杂因素。第二, 稳定肝脏是非常重要的, 因为心跳会扰乱视频。为了消除这种现象, 我们将小鼠重新定位到合适的卧位位置。
该协议的另一个关键步骤是生产金属盖架及其在金属棒上的位置。我们所面临的最重要的障碍是如何通过适当的压力来确定如何克服肝脏的碎。最初, 当只用蝶形螺母来保存盖子滑动时, 血液循环受阻, 肝脏无法承受盖板滑动的重量。相反, 我们设计了一种方法, 在将盖板沿杆放置之前, 先将螺栓向下滚动。由于盖子滑动由螺栓支持, 它给了一些空间为肝脏厚度和特此, 没有按下来坚硬入肝脏并且保持平衡和固定。使用 LysM-GFP 小鼠6, 我们证实, 这种设计可以使中性粒细胞在窦和中央静脉的可视化。CX3Cr1-GFP 小鼠5也用于该模型, 以观察 LCMs. LCMs 在肝脏的表面经常被发现。
肝基座或硅床的高度, 也是必不可少的。如果太高, 肝脏的血液循环就会被打乱。当肝脏的高度太低时, 很难维持肝脏的稳定固定。因为所有的老鼠都有不同的体型, 所以我们用一个6孔的平板来生产不同高度的硅床, 类似于细胞培养。通过这种方式, 我们可以根据每个鼠标的大小来定制会议厅。总之, 我们设计了一个新的简单的小鼠肝脏活体成像协议, 研究炎症条件下肝脏免疫细胞的形态和运动。
这个新的协议可能是非常有益的研究人员在这个领域谁想要观察急性免疫反应的肝脏。然而, 由于这种方法, 研究人员不能进行长期的成像超过6小时, 由于肝固定的组织胶。此协议是一次性的唯一方法, 因此, 应立即牺牲小鼠成像后。当肝脏与硅床分离时, 肝脏最终会从床上脱落, 导致出血过多。因此, 重新插入和缝合腹部区域是不可能的未来观察。

肝脏是哺乳动物的主要代谢器官;它充当能量、荷尔蒙和解毒的控制塔1。由于其重要性, 研究人员已经进行了许多体外和体内实验来阐明炎症过程中肝脏的变化。活体显微镜已被用来捕捉活体图像从肝脏2 。的确, 各种肝脏成像方法已经被引入2,3,4,5,6。然而, 为了发展一个更简化的手术方法, 并实现目标器官和免疫细胞的稳定, 我们设计了一个简单的协议, 可以指导研究人员减少他们的活体成像的努力。
在本研究中, 我们介绍了一个稳定和新的成像室和外科技术, 可用于减少出血和可能感染。我们证实肝脏保持完好超过2小时。利用这种方法, 我们成功地确定了 LCMs7的形态和脓毒性条件下的中性粒细胞。由于这种方法最大限度地减少物理和生物混杂因素, 如颤抖和意外炎症在外科手术过程中, 我们预计, 这种方法可以用来观察急性反应的免疫细胞在肝脏响应像 LPS 的试剂

<table><tbody><tr><td>Custom designed chamber</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Metal cover slide</td><td>Live Cell Instruments</td><td></td><td>Custom-designed size</td></tr><tr><td>Cover glass</td><td>Electron Microscopy Sciences</td><td>#72204-03</td><td></td></tr><tr><td>Sylgard 184 Silicone elastomer kit</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Erlenmeyer flask</td><td>DURAN</td><td>21 216 36</td><td></td></tr><tr><td>Cell culture plate (Flat type)</td><td>HYUNDAI Micro Co.</td><td>H31006</td><td></td></tr><tr><td>Desiccator</td><td>ADARSH</td><td>55204</td><td></td></tr><tr><td>High Q BOND SE 5 mL</td><td>BJM LAB</td><td></td><td>To make semi-permanent dam</td></tr><tr><td>Flexible Silicone Rubber Heaters</td><td>Omega</td><td>SRFR/SRFG SERIES, M1250/0800</td><td></td></tr><tr><td>DC power supply</td><td>Toyotech</td><td>DP30-03A</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td></td><td></td><td>Home-made</td></tr><tr><td>Lipopolysaccharides (LPS) from Salmonella enteria serotype enteritidis</td><td>Sigma-Aldrich</td><td>L6011</td><td></td></tr><tr><td>Texas Red Dextran</td><td>Thermo Fisher Scientific</td><td>D1830</td><td></td></tr><tr><td>15 mL High-clarity polypropylene&lt;br/&gt; conical tube</td><td>FALCON</td><td>14-959-49B</td><td></td></tr><tr><td>0.45 &amp;mu;m filter (Minisart Syringe Filter)</td><td>Sartorius</td><td>16555k</td><td></td></tr><tr><td>1.5ml Micro Tube, Amber</td><td>Axygen</td><td>MCT-150-X</td><td>For light-blocking</td></tr><tr><td>1 mL syringe</td><td>Korea Vaccine</td><td>KV-S01</td><td></td></tr><tr><td>3 mL syringe</td><td>Korea Vaccine</td><td>KV-S03</td><td></td></tr><tr><td>10 mL syringe</td><td>Korea Vaccine</td><td>KV-S10</td><td></td></tr><tr><td>0.3 mL insulin syringe</td><td>Becton Dickinson</td><td>324900</td><td></td></tr><tr><td>Hair removal cream 100 mL</td><td>Body natur</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>ABDI</td><td></td><td></td></tr><tr><td>Zoletil 50 5 mL</td><td>Virbac</td><td></td><td></td></tr><tr><td>Rompun 10 mL</td><td>Bayer</td><td></td><td></td></tr><tr><td>Heating plate</td><td>Live Cell Instruments</td><td>PH-S-10</td><td>Custom-designed size</td></tr><tr><td>DC controller</td><td>Live Cell Instruments</td><td>CU-301</td><td></td></tr><tr><td>Microdessection Scissors</td><td>Harvard Apparatus</td><td>72-5418</td><td></td></tr><tr><td>Scissors</td><td>Roboz</td><td>RS-5882</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-5137</td><td></td></tr><tr><td>Vet bond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>ZEN software (Black Edition)</td><td>Carl Zeiss</td><td></td><td>Program for LSM 7 MP</td></tr><tr><td>LSM 7 MP</td><td>Carl Zeiss</td><td></td><td></td></tr><tr><td>Volocity</td><td>PerkinElmer</td><td></td><td>Analysis program</td></tr></tbody></table>

two-photon intravital imaging of leukocytes during the immune response in lipopolysaccharide-treated mouse liver

脓毒症是一种严重的感染, 可能导致器官衰竭和组织损伤。虽然与脓毒症有关的死亡率和发病率极高, 但没有对直接治疗或与器官有关的机制进行详细的实时检查。肝脏是管理人体内毒素和感染的关键器官。本文旨在对内毒素血症诱导后的小鼠肝脏进行活体成像, 以跟踪免疫细胞的运动, 如中性粒细胞和肝囊巨噬细胞 (LCMs)。因此, 我们设计了一种新的手术方法, 以接触肝脏微创手术。小鼠腹腔注射脂多糖 (LPS), 一种常见的内毒素。利用我们的新的手术方法, 接触和活体成像的小鼠肝脏, 我们发现, 中性粒细胞招募在 LPS 治疗 LysM 绿色荧光蛋白 (GFP) 小鼠肝脏增加与磷酸盐缓冲生理盐水治疗的肝脏。LPS 治疗后, 中性粒细胞数量随着时间的推移显著增加。此外, 使用 CX3Cr1-GFP 小鼠, 我们成功地可视化肝居民巨噬细胞称为 LCMs。因此, 为了研究新的试剂对控制免疫移动性的有效性在体内, 确定中性粒细胞和 LCMs 在肝脏中的运动和形态学, 可以让我们发现在器官衰竭和组织损伤中所造成的治疗作用白细胞在脓毒症中活化。

一个简单和非常稳定的成像室设计。室的底部布满了硅胶, 防止滑鼠的身体和器官。此外, 由于硅具有适当的弹性, 它可以防止因盖板滑动 (图 1A、 C) 的压力而引起的预期损坏。其次, 采用组织安全胶 (兽医键) 固定在环形硅胶床上提取肝脏。这种方法使我们能够避免因心跳或呼吸运动而引起的颤抖。最后, 根据小鼠的身体尺寸和突起肝脏的位置 (图 1B-D), 将金属盖板放置并固定在螺栓和螺母上。当麻醉被正确地执行, 这些成像设置可能持续长于6小时。
在开始手术之前, 老鼠的体温是通过将动物放在预先设置为37° c (图 2A) 的加热室来控制的。其次, 为了防止二次感染, 所有必要的工具, 为小鼠手术和成像被消毒70% 酒精和/或蒸压, 以防止继发感染 (图 2B)。然后, 鼠标的头发被删除, 特别是从腹部地区, 与脱毛膏 (图 2C)。在做切口之前, 皮肤用聚维酮碘消毒。血液流动被标记与得克萨斯红色葡聚糖通过静脉注射 (图 2D)。一条线开始在剑过程和结束在右肋骨被画了。这条右下切口有助于减少出血。用手术刀或微剪刀, 解剖开始;腹部没有打开太多, 以避免从腹腔排出的肠子 (图 2E, F)。用镊子和微解剖剪刀将脂肪和组织层去除, 切口区域用消毒的 PBS 和棉签清洗 (图 2G)。左外侧叶, 最大的四肝叶, 被取出与棉签, 然后连接到硅床使用组织债券 (兽医债券)。由于肝脏组织非常脆弱, 肝脏被移出使用棉签 (图 2H, I)。在使用金属盖玻璃后, 肝脏受到成像。为了防止干燥, 消毒 PBS 被应用于肝脏和盖子玻璃之间的空间 (图 2J)。当需要时, 盖玻璃的高度由坚果调整。
双光子显微镜配备了 X, Y, Z 轴千分尺控制器和黑色窗帘屏蔽 (图 3A)。激光是稳定的模式锁定在禅宗软件。为了获得稳定的成像, 应防止靶器官的漂移。因此, 除了成像室和外科方法外, 电子加热垫的导线从成像阶段得到了保护, 并由粘胶带 (图 3B) 组织好。最后, 水浸泡透镜的盖子玻璃的顶部添加了足够的蒸馏水 (图 3C)。
血流清楚地显示在屏幕上后, 静脉注射得克萨斯红葡聚糖。为了同时捕获红色和绿色, 我们进行了 880 nm 波长的成像。根据活体成像分析, 适当数量的中性粒细胞总是在 PBS 状态下循环血液。而 LPS 治疗小鼠的中性粒细胞数量显著增加。注意, 许多中性粒细胞在血液中循环, 但它们没有在炎症状态下迁移到血管外。(图 4A,补充电影 1)。在 LPS 治疗的小鼠中, 中性粒细胞更牢固地附着在血管的腔表面, 从而更频繁地检测血管中的中性粒细胞 (图 4A,补充电影 1)。除了中性粒细胞的运动外, CX3Cr1-GFP 小鼠的 LCMs 在肝组织表面观察, 而不是在组织深处 (图 4B)。LCMs 显示, 即使在 LPS 治疗的小鼠肝脏也没有运动或活化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57191/57191fig1.jpg"> 图 1.设计肝脏室和辅助工具.(A) 将硅胶混合物倒入定制设计的鼠室。(B) 金属盖板的尺寸 (1 英寸 = 25.4 mm, Ф = 直径)。(C) 图像显示塑料定制设计的肝腔内填充有机硅弹性体。(D) 其他部分用于组装肝脏室。固定螺栓和螺母由铁制成。箭头表示螺栓和螺母分别在 (C) 和 (D)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57191/57191fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57191/57191fig2.jpg"> 图 2.肝外科手术步骤.(A) 在37° c 和直流温度控制器上进行手术前的金属和橡胶加热板。(B) 肝脏外科的整体手术和辅助工具 (a. 棉签和容器, B. 70% 酒精, c. 纸巾, d. 剃须刀, e. 定制设计的室, g. 手术带, h. 国产肝基座, i. 翅螺母和螺母, j. 金属盖滑动, k. 显微解剖剪刀, 钳子, 剪刀, 3 毫升注射器, o. 标记, p. 0.3 毫升胰岛素注射器, q 1 毫升注射器)。肝脏手术的程序 (C-J)。(C) 在腹部部位涂抹脱毛膏。(D) 用德克萨斯红葡聚糖对小鼠进行逆行眼眶注射。(E) 用记号标记切口部位。(F) 用钳子和剪刀切割表皮。(G) 在腹腔内切开切口。(H) 轻轻拉出左外侧叶。(I) 用兽医键将耳垂连接到肝脏底座。(J) 最后设立的形象。螺母固定在金属盖板上, 然后用蝶形螺母固定盖板。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57191/57191fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57191/57191fig3.jpg"> 图 3.双光子显微镜和成像阶段的设置.(A) 双光子显微镜 (LSM 7 MP) 和成像阶段的看法, 可以在 x 和 y 方向移动。(B) 用胶带覆盖老鼠的肝脏成像室附有橡胶加热垫。(C) 在物镜和盖玻璃之间用吸管将蒸馏水滴掉。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57191/57191fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57191/57191fig4.jpg"> 图 4.活体小鼠肝脏中性粒细胞和肝囊巨噬细胞的可视化.(A) LysM-GFP 小鼠肝脏中中性粒细胞的延长聚焦快照。该控制图像是由 PBS 处理的小鼠。lps 图像是由 lps 治疗的小鼠。图像获得使用 20 x/1.0 NA 水浸泡透镜 (视野 [视] = 424.3 µm x 424.3 µm, 深度 = 40 µm, 切片间隔 = 1 µm, 时间间隔 = 六十年代, 周期 = 31)。缩放条 = 50 µm. (B) CX3Cr1-GFP 小鼠肝脏 LCMs 的内、表面积快照。图像获得使用 20×/1.0 NA 水浸泡透镜 (视 = 424.3 µm x 424.3 µm, 深度 = 40 µm (内)/10 µm (表面), 切片间隔 = 1 µm, 时间间隔 = 六十年代 (内)/二十年代 (表面), 周期 = 31)。缩放栏 = 50 µm.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57191/57191fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/57191/57191mov1.jpg"> 辅助影片1。PBS 治疗肝与 LPS 治疗肝的比较。在两种不同条件下的明显差异是很好的观察。在 LPS 治疗的肝脏中, 中性粒细胞数量明显增加, 运动性降低。观察了这两种情况下电影的稳定性。血流被标记为得克萨斯红葡聚糖。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57191/Movie_1.avi" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver at JoVE.com

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

我们建立了一种新的小鼠肝脏双光子成像的手术方案。利用这一技术, 我们确定了肝内毒素诱导中的内毒素血症的详细结构。我们预期这种方法可以用来确定各种试剂治疗肝白细胞迁移的有效性。

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

two-photon-intravital-imaging-leukocytes-during-immune-response

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  Yonsei University College of Medicine. 我们建立了一种新的小鼠肝脏双光子成像的手术方案。利用这一技术, 我们确定了肝内毒素诱导中的内毒素血症的详细结构。我们预期这种方法可以用来确定各种试剂治疗肝白细胞迁移的有效性。

视频: Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白细胞的归位和迁移的非侵入性的成像在体内</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

科研

免疫与感染

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

小鼠腘窝淋巴结的活体成像

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染:肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

在Mesenterial静脉的活体显微镜白细胞，内皮细胞和血小板白细胞相互作用小鼠

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

生物工程

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

医学

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

<meta charset="utf8"></head><body>小鼠肺移植中细胞动力学的实时监测

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>作者聚焦：通过活体成像推进肺移植免疫学

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

移植小鼠肺的活体双光子显微镜检查

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

脂多糖治疗小鼠肝脏免疫应答中白细胞的双光子活体成像

摘要

探索更多视频

此视频中的章节

白细胞的归位和迁移的非侵入性的成像在体内