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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。
<p class="jove_content"…Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Agarose | Sigma–Aldrich | A6138 | Apply upon exposed cortex for protection |
| Alexa 594 | Life Technologies | A-10438 | Stain puffing compound to red fluorescent color |
| ATP | Sigma-Aldrich | A9187 | Vasodilator and vasoconstrictor, puffing compound |
| Cyanoacrylate glue and activator | Loctite | Adhesives and SF7452 | Glue for metal piece and coverglass |
| Eye lubricant | Neutral Ophtha, Ophtha A/S, Denmark | Keep the mouse eyes moisterized | |
| FITC-dextran | Sigma-Aldrich | FD500S | Blood serum dye, green fluorescent color |
| NG2DsRed mice | Jackson Laboratory | 8241 | These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter |
| pZac2.1 gfaABC1D-lck-GCaMP6f | Addgene | 52924-AAV5 | Astrocyte specific viral vectors carrying genetically encoded calcium indicators |
| TRITC-dextran | Sigma-Aldrich | 52194 | Blood serum dye, red fluorescent color |
| List of Equipments | |||
| Air pump | WPI | PV830 | Give air pressure to pipette puffing |
| Blood gas analyzer | Radiometer | ABL 700 | Measure levels of blood gases |
| Blood pressure monitor | World Precision Instruments | BP-1 | Monitor aterial blood pressure |
| Body temperature controller | CWE | Model TC-1000 | Keep the mouse body temperature in physiological range |
| Capnograph | Harvard Apparatus | Type 340 | Monitor the end-expiratory CO2 from the mouse |
| Electrical stimulator | A.M.P.I. | ISO-flex | Apply whisker pad stimulation |
| Mechanical ventilator | Harvard Apparatus | D-79232 | Mechanically ventilate the mouse in physiological range |
| Micropipette puller | Sutter Instrument | P-97 | |
| Two-photon microscope | Femtonics Ltd | Femto3D-RC | |
| List of Surgical Instruments | |||
| Anatomical tweezer | Lawton | 09-0007 | |
| Angled and balanced tweezer | S&T AG | 00595 FRAS-18 RM-8 | |
| Iris scissor | Lawton | 05-1450 | |
| Micro vascular clamp | S&T AG | 462 | |
| Mouse vascular catheters | Verutech | 100828 |
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