Name: in vivo three-dimensional two-photon microscopy to study conducted vascular responses by local atp ejection using a glass micro-pipette
Uploaded: 2019-06-07T13:00:00.000+00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette at JoVE.com

这项研究得到了伦德贝克基金会、诺和诺德基金会、丹麦独立研究理事会的支持|医学和NORDEA基金会向健康老龄化中心提供资助。

丹麦国家伦理委员会根据欧洲理事会《保护用于实验和其他科学目的的脊椎动物公约》中规定的准则,批准了涉及动物的所有程序,符合《到达准则》。这是一个终端程序,小鼠在麻醉恢复前被安乐死。
1. 手术前准备
<ol><li>用70%乙醇清洁手术台和周围所有区域。手术前彻底清洁和干燥手术工具。</li>
	<li>麻醉NG2DsRed小鼠(Tg(Cspg4-DsRed.T1)1Akik/J;两性;4-8个月大)通过排前注射氯胺酮+木黄素(60毫克/千克=10毫克/千克)溶解在消毒水中,pH 7.4。每25分钟服用补充剂量的氯胺酮(30毫克/千克),直到手术完成。定期检查尾巴和脚趾捏反射,以验证麻醉的深度。</li>
	<li>在小鼠背面的四个不同位置注射盐水,总体积为 1 mL,以防止实验期间脱水。为防止眼睛干燥,请涂抹眼部润滑剂。使用直肠温度探头和加热毯将体温保持在37°C。</li>
</ol>2. 外科手术
<ol><li>气管 切开
	<ol>
<li>将鼠标放在鼠标背面。在计划的切口下注射0.04 mL 0.5%的利多卡因,等待2分钟。分离下颌腺和甲状腺肌,然后暴露气管。</li>
		<li>用剪刀在两个气管环之间进行气管切开术。将长度为 16.2 mm、直径为 1 mm 的小金属管插入气管。将管子连接到机械呼吸机和气盖仪,以监控末端呼气CO2(图1A)。</li>
	</ol></li>
	<li>导管插入
	<ol>
<li>在计划切口下下注射0.5%利多卡因(0.04 mL),等待2分钟。</li>
		<li>使用细尖钳轻轻将动脉与静脉分开。用微血管夹停止血液流向上游。用10-0尼龙缝合两条血管来收缩下游的血流。</li>
		<li>使用微解剖剪刀在动脉和静脉中做一个小切口。将塑料导管插入两个血管。通过拧紧缝合线(8-0)固定导管或10-0尼龙缝合线取决于容器大小),而不损害导管流明。拆下微血管夹并关闭皮肤。</li>
		<li>通过动脉导管监测血压。使用血气分析仪测量每个实验前后动脉血样(50 μL)中的血气水平(正常值:pO2,90-120 mmHg;pCO 2,35-40 mmHg;pH,7.25-7.45)。使用静脉导管输液和麻醉。</li>
	</ol></li>
	<li>开颅 术
	<ol>
<li>把毛毛从老鼠头上的耳之间去掉。在计划切口下注射0.04 mL 0.5%利多卡因皮下,等待2分钟。</li>
		<li>用10%的氯化铁溶液擦拭头骨,去除骨膜。用盐水彻底冲洗头骨。用氰丙烯酸酯胶水和活化剂将金属头杆粘到头骨上(图1A)。 注:金属头杆长75毫米,宽13.5毫米,中心有一个直径为5毫米的圆孔。</li>
		<li>钻一个4毫米直径的颅骨切除术,中心在0.5毫米的后面,3毫米在右感觉桶皮层的右脑膜。用细尖容器扩张器拆下杜拉。</li>
		<li>用0.75%的角胶覆盖暴露的皮层,溶解在人工脑脊液中(aCSF;pH = 7.4),冷却至35°C。用10-15°角的玻璃盖玻片覆盖80-90%的颅骨,以10-15°为角度覆盖金属头杆(图1B),允许插入和放置玻璃微移液器。</li>
		<li>为了尽量减少脑脉动,用氰丙烯酸酯胶和活化剂将玻璃盖板的两个角粘附在金属头杆上。小心地应用激活剂,以防止进入暴露的大脑。之后用盐水彻底冲洗粘合的玻璃盖玻片。</li>
	</ol></li>
	<li>执行胡须垫刺激探头的插入。将一组定制的双极电极(8 mm 长度和 0.25 mm 厚度)切入面部左侧,以刺激三叉神经对侧到颅骨的副轨道。将阴极靠近间断位轨道,并将阳极插入粘胶肌肉7。在 20 s 的列车中,在 20 s 的 2 Hz 下,以 1.5 mA 的强度执行 1 ms 的电刺激。</li>
	<li>手术后,将0.05 mL荧光素异异体-dextran(FITC-dextran,4%w/v,分子量[MW] 50,000)放入股骨静脉,为血浆贴上标签。停止氯胺酮,将麻醉切换到连续静脉输注β-氯洛(17%w/v,最终浓度),与FITC-dextran(2%w/v,最终浓度)混合在0.02 mL/10 g/h。等待约半小时,以稳定新的麻醉状态。 注:FITC-dextran和β-氯酮均溶解在0.9%盐碱中。</li>
</ol>3. 第一次双光子成像会话
<ol><li>将鼠标转移到商用双光子显微镜(材料表)的舞台上。在红色荧光蛋白(RFP,图1C)和绿色荧光蛋白(GFP,图1C)模式下,以5倍目标拍摄暴露皮层的照片。使用 RFP 图片作为"地图",用于在下一个会话中插入玻璃微移液器。 注: GFP"地图"用于区分静脉/静脉与动脉/动脉。</li>
	<li>使用双光子显微镜和 25x 1.0 数值孔径 (NA) 水浸物物用压电电机执行双光子成像。将激发波长设置为 900 nm。搜索皮层,跟随每个穿透动脉,并找到它的水平分支(第1阶毛细血管)。</li>
	<li>图像穿透动脉及其1阶毛细血管在休息和胡须垫刺激期间。请参阅第 5 节中的详细成像参数。</li>
	<li>在 RFP"地图"上用 >5% 的血管扩张标记 1阶毛细血管的位置(图1C)。具有 <5% 血管扩张的位置被视为在胡须桶皮层区域之外。 注:如果在实验中使用野生型小鼠而不是 NG2DsRed 小鼠,则 GFP 图像将用作"地图"。</li>
</ol>4. 插入玻璃微移液器
<ol><li>使用移液器拉拔器(材料表)准备玻璃微移液器,用于膨胀。玻璃微移液器的电阻为3-3.5 MΩ,片长为4.5-5 mm。装载混合1mM ATP和10μM Alexa 594的移液器,以便在荧光灯和双光子显微镜下可视化移液器尖端。</li>
	<li>将玻璃微移液器安装到贴片夹支架上,并将其连接到空气泵。将泵保持压力设置为 0 psi。</li>
	<li>使用红色外观荧光照明,用 5x 目标聚焦移液器尖端。将玻璃微移液器放入甘蔗上方的 ACSF 中。将空气泵的保持压力调节到 0.2 psi。可以看到一个小的红色云从ACSF的移液器尖端弹出。这是为了防止在移液器插入过程中堵塞。</li>
	<li>选择 RFP"地图"中标记的位置之一作为目标。将移液器尖端移到覆盖玻璃边缘的水平平面上,其距离为 30 μm,大致指向 x-y 平面中的目的地。</li>
	<li>小心地将移液器尖端以 z 轴在盖玻璃滑片下下。然后开始将玻璃移液器推进到目的地,直到在大脑表面上方 +500 μm。经常在盖玻璃边缘、移液器尖端和大脑表面之间切换对焦,确保有足够的可用空间来移动移液器。</li>
	<li>将目标切换为 25 倍。重新居心移液器尖端,并在 RFP"地图"上进一步向目的地推进移液器尖端。将移液器尖端保持在甘蔗色层中。</li>
	<li>当移液器尖端在大脑表面上方 ±100 μm 时,将成像模式切换到双光子显微镜。专注于要吹嘘的目标位置。记下其 x、y 坐标(x 0, y0) 及其深度低于曲面 (z0)。计算大脑表面插入点的坐标 (xi, yi) 如下: xi = x0 = z0 / 晒黑 | yi = y0 其中*是移液器支架和水平平面之间的角度。</li>
	<li>将移液器尖端置于大脑表面的坐标(xi,yi)。轻轻地慢慢插入移液器,直到它达到 (x0, y0, z0)。如果容器位于插入路径上,则拔下移液器并重新计算其他插入坐标和路径;或稍微转动舞台板(如图 1 A所示)。</li>
	<li>将泵保持压力设置为 0 psi,将喷射压力设置为 10-15 psi。在双光子成像期间,在目标位置将 200-400 ms 的浮松 ATP。如讨论部分所述,调整每个移液器的膨胀压力和持续时间,并随时间延长。</li>
</ol>5. 超堆栈双光子成像
<ol><li>使用双光子显微镜和 25 x 1.0 NA 水浸物使用压电电机执行实验。将激发波长设置为 900 nm。过滤发出的光,以收集DsRed(围生)/四甲基二甲胺异氰酸二聚氰胺(TRITC-dextran)染色血浆的红光和FITC-dextran(血浆)/GCaMP6(GFP,星化钙)的绿光。</li>
	<li>在感兴趣位置产生高分辨率 z 堆栈,其中包含穿透性动脉、一阶毛细管、第二阶毛细管和可能相邻的荧光细胞(例如,圆环状细胞、星形细胞)。通过尽可能包括血管的 3D 结构,确定超堆栈成像的 x_y_z 体积。每个堆栈的总高度可能从 30-50 μm 不等,而 x-y 平面大致覆盖 60 μm x 40 μm 的区域。</li>
	<li>在成像软件中设置图像堆栈,由 8-10 个平面组成,平面间距离为 4-5 μm。压电电机目标在 z 轴上的每一个电平停止,以获取每个平面的图像。采样速率为每堆栈 1 s,x-y 平面中的像素分辨率为 0.2-0.3 μm(图 2A)。一个记录通常包括 10 个预浮图栈和 150 个后吹捧图像堆栈。</li>
</ol>6. 数据处理
<ol><li>使用定制的分析软件处理数据。通过最大强度投影将每个图像堆栈拼合到一个图像中(图 2B)。绘制一个矩形感兴趣区域 (ROI),宽度垂直于 3 μm,垂直穿过感兴趣容器(图 2C)。</li>
	<li>为了尽量减少红血球阴影和皮层轻微振动的干扰,通过将矩形 ROI 投影到一条线中,从而平均矩形 ROI,然后表示血管段的平均轮廓。为每个最大强度图像执行此操作。</li>
	<li>将轮廓线绘制为 2D 图像,x 轴按时间顺序表示最大强度图像(图2D,上面板)。使用有源轮廓算法(Chan-Vese分割)查找容器8和9的边缘(以红色曲线表示;图 2D,上面板)。</li>
	<li>计算直径的时间过程随着血管边缘之间的差值而变化(图 2D中上下红色曲线之间的垂直距离,下面板)。将直径标准化更改为预膨胀直径基线,直径曲线的图曲线随时间而变化。为每个 ROI 执行此操作 (图 2E)</li>
	<li>容器响应振幅定义为膨胀后的最高/最低峰值振幅。响应延迟定义为半最大振幅的延迟(图2G)。通过沿血管放置节点(图2F)并测量每个 ROI 和穿透动脉之间的地理距离,手动跟踪骨架血管结构。通过将距离除以延迟差异来计算 CVR 速度。</li>
</ol>7. 病毒载体注射
<ol><li>为了同时研究星形钙反应,按照标准的立体定向病毒载体注射程序,将0.6μL病毒载体(pZac2.1 gfaABC1D-lck-gCaMP6f,Addgene)注入NG2DsRed小鼠的体感皮层10.</li>
	<li>三周后,小鼠躯体感觉皮层中的星形细胞表达基因编码的钙指标GCaMP6f。遵循上述外科手术和小鼠双光子成像。为了区分星形细胞和血管亮度,TRITC-dextran代替FITC-dextran用于染色血管氧化铝红色。</li>
</ol>

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作者没有什么可透露的。

血管研究的一个挑战是精确测量血管直径。我们在这里描述的方法使用电动压电物镜,通过双光子显微镜进行快速和重复的超堆栈成像。首先,这种方法允许反复检查穿透性动脉、1阶和第二阶毛细血管,而不会失去焦点,导致在体内毛细血管中发现缓慢进行的血管反应。其次,结合病毒载体注射技术,使我们能够研究星形钙在三维的反应,这是研究血流调节所必需的。
此方法并非没有限制。首先,在3D成像之前定义一个窄矩形视场,以实现高时间分辨率。这通常将成像限制为毛细血管的前三个顺序。其次,双光子显微镜的激光必须完全对齐。激光错位会错误地使每个帧去中心,并增加容器直径测量。第三,3D成像的采样率能够捕获缓慢的CVR和减缓星形细胞的钙变化。每堆1-2赫兹仍然不够快,无法研究星形细胞12、13中的快速CVRs11或快速钙信号。
此外,为了影响局部感兴趣的血管,我们优化了将玻璃微移液器精确插入小鼠大脑皮层特定位置的程序。成功应用此方法有几个关键步骤。首先,必须仔细调整玻璃微移液器支架的角度。它应该允许微移液器在目标下方自由操纵,玻璃盖滑片位于暴露的皮层上方。其次,在将玻璃微移液器插入角胶层和皮层时,需要一个很小的保持压力,以保持移液器尖端的未堵塞。但是,必须小心,不要施加太大的保持压力,以免化合物在膨胀前泄漏。第三,移液器的膨胀应在大脑上方的甘蔗层中进行测试,以找到最佳压力和持续时间,在一次喷发时释放足够的膨化化合物,同时不引入由于机械引起的细胞和容器的明显位移压力。
为了更精确地测量CVR速度,应提高体积扫描速度。还有其他新开发的体积扫描显微镜,例如,带有声学光学导流板 (AOD) 的双光子显微镜扫描速度高达每秒 5 卷,具有相同的空间分辨率和体积大小(电子邮件通信)。光片显微镜扫描甚至更大的体积大小(350 μm x 800 μm x 100 μm),每秒10卷14。
我们的鼠标准备也有局限性。急性小鼠手术可能有潜在的并发症。止痛药和麻醉可能会影响神经血管反应。急性神经炎症也可以触发,导致皮质组织和血管中的微胶质活化和白细胞化。虽然玻璃微移液器的尖端尺寸通常非常小(<1 μm),但插入过程和ATP膨胀也可能在插入后0.5-1小时内触发微胶质迁移和插入部位的拥抱。
总之,将三维成像与双光子显微镜和局部膨胀玻璃微移液相结合是研究神经血管活动及其机制的先进工具。

大脑有高能量消耗率。人体消耗的氧气和葡萄糖的20%专门用于大脑功能,而大脑只占身体总量的2%。维持正常的大脑功能需要在复杂的血管网络中通过血液流动提供充足和高效的氧气和营养。局部大脑活动和脑血流(CBF)是强强耦合的,取决于神经元、星形细胞、坐体、平滑肌细胞(SMC)和内皮细胞(ECs)1的功能特性。最近,从穿透动脉中分枝的毛细血管的前几条订单已经出现为"热点"2,显示出毛细血管血流的积极调节。在用玻璃微移剂3的ATP的胡须刺激和局部弹出(喷出)期间,在小鼠躯体感觉皮层的这个"热点"上发现了缓慢的血管反应(CVR)。
虽然通过双光子激光扫描荧光显微镜在体内成像已被广泛用于研究大脑皮层的神经血管反应,但大多数研究测量了血管直径,并研究了其在二维 (2D) x-y 平面。挑战是:首先,脑血管及其拥抱的星形细胞、围细胞和SMC在三维(3D)中构建分支。因此,在 3D 中研究它们的交互作用至关重要。其次,即使是少量的聚焦漂移也会影响容器直径和细胞荧光信号的精确测量。最后,CVR 在三个方面是快速而深远的。3D 体积扫描是检测 CVR 和揭示其机制的最佳选择。我们在双光子显微镜中实现了压电运动目标,以研究小鼠体内的躯体感觉皮层,通过对获得的图像进行最大强度投影,从而进行精确的直径测量。
玻璃微移液器经常用于体内脑研究,例如,散装有机染料4,记录EEGs5和贴片夹紧6。然而,限制仍然存在。通常,玻璃微移液器的尖端放置不精确,或者微移液器不用于局部干预。在这里,我们优化了微移液器插入和局部弹出的过程。
此外,3D 双光子显微镜和基因编码荧光指标的结合为在 3D 范围内研究神经血管耦合提供了前所未有的机会。在这项研究中,我们利用了这一点,将携带星形细胞特定基因编码钙指标的病毒载体注射到小鼠躯体感觉皮层中。通过组合不同的荧光标记,同时成像星形细胞和容器直径。
总体而言,我们提出了通过玻璃微移液器和快速双光子超堆栈成像进行局部喷射(浮肿)的优化方法,从而能够精确测量毛细管直径变化。此外,我们的方法提供了一个新的工具,可以同时研究星形细胞和血管直径反应中的Ca2+反应的三维轮廓。

<table><tbody><tr><td>Agarose</td><td>Sigma&amp;ndash;Aldrich</td><td>A6138</td><td>Apply upon exposed cortex for protection</td></tr><tr><td>Alexa 594</td><td>Life Technologies</td><td>A-10438</td><td>Stain puffing compound to red fluorescent color</td></tr><tr><td>ATP</td><td>Sigma-Aldrich</td><td>A9187</td><td>Vasodilator and vasoconstrictor, puffing compound</td></tr><tr><td>Cyanoacrylate glue and activator</td><td>Loctite</td><td>Adhesives and SF7452</td><td>Glue for metal piece and coverglass</td></tr><tr><td>Eye lubricant</td><td>Neutral Ophtha, Ophtha A/S, Denmark</td><td></td><td>Keep the mouse eyes moisterized</td></tr><tr><td>FITC-dextran</td><td>Sigma-Aldrich</td><td>FD500S</td><td>Blood serum dye, green fluorescent color</td></tr><tr><td>NG2DsRed mice</td><td>Jackson Laboratory</td><td>8241</td><td>These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter</td></tr><tr><td>pZac2.1 gfaABC1D-lck-GCaMP6f</td><td>Addgene</td><td>52924-AAV5</td><td>Astrocyte specific viral vectors carrying genetically encoded calcium indicators</td></tr><tr><td>TRITC-dextran</td><td>Sigma-Aldrich</td><td>52194</td><td>Blood serum dye, red fluorescent color</td></tr><tr><td>&lt;strong&gt;List of Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Air pump</td><td>WPI</td><td>PV830</td><td>Give air pressure to pipette puffing</td></tr><tr><td>Blood gas analyzer</td><td>Radiometer</td><td>ABL 700</td><td>Measure levels of blood gases&amp;nbsp;</td></tr><tr><td>Blood pressure monitor</td><td>World Precision Instruments</td><td>BP-1</td><td>Monitor aterial blood pressure</td></tr><tr><td>Body temperature controller</td><td>CWE</td><td>Model TC-1000</td><td>Keep the mouse body temperature in physiological range</td></tr><tr><td>Capnograph</td><td>Harvard Apparatus</td><td>Type 340</td><td>Monitor the end-expiratory CO&lt;sub&gt;2&lt;/sub&gt; from the mouse</td></tr><tr><td>Electrical stimulator</td><td>A.M.P.I.</td><td>ISO-flex</td><td>Apply whisker pad stimulation</td></tr><tr><td>Mechanical ventilator</td><td>Harvard Apparatus</td><td>D-79232</td><td>Mechanically ventilate the mouse in physiological range</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-97</td><td></td></tr><tr><td>Two-photon microscope</td><td>Femtonics Ltd</td><td>Femto3D-RC</td><td></td></tr><tr><td>&lt;strong&gt;List of Surgical Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anatomical tweezer&amp;nbsp;</td><td>Lawton</td><td>09-0007</td><td></td></tr><tr><td>Angled and balanced tweezer</td><td>S&amp;amp;T AG</td><td>00595 FRAS-18 RM-8</td><td></td></tr><tr><td>Iris scissor</td><td>Lawton</td><td>05-1450</td><td></td></tr><tr><td>Micro vascular clamp</td><td>S&amp;amp;T AG</td><td>462</td><td></td></tr><tr><td>Mouse vascular catheters</td><td>Verutech</td><td>100828</td><td></td></tr></tbody></table>

in vivo three-dimensional two-photon microscopy to study conducted vascular responses by local atp ejection using a glass micro-pipette

维持正常的大脑功能需要由复杂的血管网络充分而有效地供应氧气和营养。然而,大脑血流(CBF)的调节不完全理解,特别是在毛细管水平。双光子显微镜是一种强大的工具,广泛用于研究CBF及其调控。目前,由于缺乏体内双光子显微镜研究(1)三维CBF反应,(2)血管反应,以及(3)血管网络内的局部干预,这一领域受到限制。在这里,我们描述了一种使用双光子显微镜的3D体内方法,用于研究通过玻璃微移液器局部弹出ATP引起的血管反应。我们的方法使用快速和重复的超堆栈双光子成像,通过获得的图像的最大强度投影提供精确的直径测量。此外,我们还表明,该方法也可用于研究3D星形钙反应。我们还讨论了玻璃微移液器插入和双光子超堆栈成像的优点和局限性。

手术后,小鼠被运送到双光子显微镜(图1A)。在目标位置的目标血管附近插入了一个含有1 mM ATP的玻璃微移液器(图1B)。
我们执行超堆栈成像,同时给出 1 mM ATP (图 2A,补充视频 1) 的浮肿。每个图像堆栈通过最大强度投影拼合到一个图像(图 2B)。矩形 ROI 垂直放置在容器上,以测量 ATP 膨胀时容器直径的变化(图2C)。使用陈-Vese分割测量容器直径(图2D)。在单次吹吸记录中,覆盖每个 ROI 的标准化直径变化,以比较不同船段的响应(图 2E)。通过手工绘制血管骨架(图2F)计算了从每个ROI到穿透动脉的距离。在单次吹吸记录中,每个 ROI 的振幅和膨胀和延迟是在从 ROI 到穿透动脉的计算距离上绘制的(图 2H-K)。膨胀时的血管扩张以14.69 μm/s(上游)和2.8 μm/s(下游)的速度线性传播,从第1和第2阶毛细血管的交界处开始(图2H)。血管收缩也以3.92 μm/s线性传播,从穿透性动脉开始(图2I)。血管扩张和血管收缩的最大振幅发生在1阶毛细血管中(图2J-K)。
此外,结合星形细胞特异性病毒载体携带荧光钙指标和双光子超堆栈成像,研究了星形钙对ATP膨胀的反应(图3A,补充视频2).矩形ROIs被放置在星形索塔和工艺。ATP诱导细胞内钙在星形过程中上升,但不是在星形细胞索马塔 (图3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59286/59286fig1.jpg"> 图 1:体内双光子三维成像图和玻璃微移液器膨胀图。(A) 麻醉小鼠的头部固定在金属棒上,并机械通风。端潮CO2由显像仪监控。胡须垫刺激和微移液膨胀都用于诱导血管直径变化。玻璃微移液器支架安装在舞台板上。股动脉和静脉被导管监测血压和血液气体,并分别注入麻醉和荧光。(B) 玻璃盖玻片以一定角度覆盖颅骨,允许移液器自由移动。膨胀微移液器放置在穿透性动脉及其毛细血管附近,并含有 10 μM Alexa 594(玻璃微移液器中的红色)和 1 mM ATP 的混合物。双光子成像是一种快速和重复的3D体积扫描,包括穿透性动脉和前几阶毛细血管,以及相邻的星形细胞和圆环。(C) 显示了使用表荧光照明的红色荧光蛋白 (RFP) 和绿色荧光蛋白 (GFP) 的代表性图像。在移液器插入过程中,它们用作"地图"。红色圆圈在胡须垫刺激期间用 >5% 血管化标记第 1阶毛细血管的位置。比例尺:50 μm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59286/59286fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59286/59286fig2.jpg"> 图 2:通过微移液器吸气诱导血管扩张,然后收缩。(A) 在一个代表性的图像堆栈中的平面级联,包括穿透性动脉和 1st和 2阶毛细血管。围冰体标有红色荧光素(NG2-DsRed),容器流明标有FITC-dextran(绿色)。(B) 图像堆栈的最大强度投影。(C) 多个独特的感兴趣区域 (ROIs) 垂直放置在血管上,以测量容器的直径。(D) 面板 C 在深蓝色 ROI 处随时间而具有代表性的荧光强度。两条红色曲线定义血管壁的边缘。两条红色曲线之间的垂直距离是容器直径,显示为时间函数(底部)。(E) 标准化直径在每个 ROI 随时间的变化,以响应 1 mM ATP 膨胀。测量基于单个实验。(F) 血管骨架是通过将节点沿血管放置来手动追踪的。(G) 扩张或收缩的振幅分别定义为记录过程中最大的正或负血管反应。膨胀和收缩的延迟报告为膨胀后达到一半正或负最大值的时间。(H-K)图表显示膨胀延迟 ( H)、收缩延迟 (I)、 扩张振幅 (J) 和面板 C 中显示的所有 ROI 的振幅 (K ) 相对于每个 ROI 与穿透性动脉。虚线表示上行和下游导电响应的线性拟合。p.a.:穿透性动脉。比例尺:10 μm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59286/59286fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59286/59286fig3.jpg"> 图 3:通过微移液器吸收ATP膨胀诱导星形钙活性。(A) 携带荧光钙指标的天体细胞病毒载体在实验前三周注射在小鼠躯体感觉皮层中。图为绿色标有TRITC-dextran(红色)和星形钙的容器氧化铝。(B) 多个 ROIs 被放置在星形寄索和工艺中.在 1 mM ATP 膨胀时,细胞内钙在星形过程中增加,但在 somata(虚线盒)中增加,其中钙水平大于平均值加 1.5 x 标准偏差被定义为显著。比例尺:10 μm。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59286/59286fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>
<img alt="Supplementary Video 1" src="/files/ftp_upload/59286/59286movie1.jpg"> 补充视频1:时间序列电影从容器的超堆栈成像中扁平化,以响应1 mM ATP的膨胀。绿色:FITC-dextran,染色容器流明。红色:NG2DsRed,染色过周冰。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59286/Supplementary_video_1_slow.avi" target="_blank">请点击此处观看此视频。(右键单击下载。</a>
<img alt="Supplementary Video 2" src="/files/ftp_upload/59286/59286movie2.jpg"> 补充视频2:时间序列电影从星形钙的超堆栈成像扁平化,以响应1 mM ATP的膨胀。绿色:天体化钙。红色:TRITC-dextran,染色容器流明。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59286/Supplementary_video_2_slow.avi" target="_blank">请点击此处观看此视频。(右键单击下载。</a>

Watch this Scientific Journal Video about In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette at JoVE.com

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

我们使用玻璃微移液器和快速的双光子超堆成像方法提供优化的局部弹出程序,从而能够精确测量毛细管直径变化,并在三维中调查其调节。

在 Vivo 三维双光子显微镜中,利用玻璃微移器研究本地 ATP 喷射的血管反应

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

该协议旨在研究由局部弹出ATP引起的脑毛细血管反应缓慢。该技术的优点是通过局部移液器弹出来模拟局部突触活动，通过 3D 双光子显微镜保持聚焦的微量容器。该方法还可用于研究脑神经瘤和胶质活动在休息和刺激三个维度。演示程序将是迈克尔 L

in-vivo-three-dimensional-two-photon-microscopy-to-study-conducted

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.4K 次观看.  University of Copenhagen. 该协议旨在研究由局部弹出ATP引起的脑毛细血管反应缓慢。该技术的优点是通过局部移液器弹出来模拟局部突触活动，通过 3D 双光子显微镜保持聚焦的微量容器。该方法还可用于研究脑神经瘤和胶质活动在休息和刺激三个维度。演示程序将是迈克尔 L

视频: In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

在肿瘤血管生成中使用荧光共聚焦电视显微镜活体成像

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

科研

医学

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

一种新型的高分辨率<em&gt;在体内</em&gt;成像技术研究肿瘤生长及治疗颅内结构的动态响应

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

神经科学

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

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

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

生物学

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

在 Vivo 三维双光子显微镜中,利用玻璃微移器研究本地 ATP 喷射的血管反应

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