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
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?...

<ol>
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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"...

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 border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1162">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Agarose</td>
			<td style="width:263px;">Sigma&#8211;Aldrich</td>
			<td style="width:152px;">A6138</td>
			<td style="width:524px;">Apply upon exposed cortex for protection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 594</td>
			<td>Life Technologies</td>
			<td>A-10438</td>
			<td>Stain puffing compound to red fluorescent color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Vasodilator and vasoconstrictor, puffing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyanoacrylate glue and activator</td>
			<td>Loctite</td>
			<td>Adhesives and SF7452</td>
			<td>Glue for metal piece and coverglass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye lubricant</td>
			<td style="width:263px;">Neutral Ophtha, Ophtha A/S, Denmark</td>
			<td style="width:152px;"></td>
			<td>Keep the mouse eyes moisterized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-dextran</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">FD500S</td>
			<td>Blood serum dye, green fluorescent color</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NG2DsRed mice</td>
			<td style="width:263px;">Jackson Laboratory</td>
			<td style="width:152px;">8241</td>
			<td style="width:524px;">These transgenic mice express an red fluorescent protein variant (DsRed) under the control of the mouse NG2 (Cspg4) promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pZac2.1 gfaABC1D-lck-GCaMP6f</td>
			<td style="width:263px;">Addgene</td>
			<td style="width:152px;">52924-AAV5</td>
			<td>Astrocyte specific viral vectors carrying genetically encoded calcium indicators</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC-dextran</td>
			<td>Sigma-Aldrich</td>
			<td style="width:152px;">52194</td>
			<td>Blood serum dye, red fluorescent color</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">List of Equipments</td>
			<td style="width:263px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Air pump</td>
			<td>WPI</td>
			<td>PV830</td>
			<td>Give air pressure to pipette puffing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Blood gas analyzer</td>
			<td style="width:263px;">Radiometer</td>
			<td>ABL 700</td>
			<td>Measure levels of blood gases&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Blood pressure monitor</td>
			<td>World Precision Instruments</td>
			<td>BP-1</td>
			<td>Monitor aterial blood pressure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Body temperature controller</td>
			<td>CWE</td>
			<td>Model TC-1000</td>
			<td>Keep the mouse body temperature in physiological range</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">Capnograph</td>
			<td>Harvard Apparatus</td>
			<td>Type 340</td>
			<td>Monitor the end-expiratory CO2 from the mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Electrical stimulator</td>
			<td style="width:263px;">A.M.P.I.</td>
			<td>ISO-flex</td>
			<td>Apply whisker pad stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td>D-79232</td>
			<td>Mechanically ventilate the mouse in physiological range</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Micropipette puller</td>
			<td style="width:263px;">Sutter Instrument</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Two-photon microscope</td>
			<td>Femtonics Ltd</td>
			<td>Femto3D-RC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">List of Surgical Instruments</td>
			<td style="width:263px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anatomical tweezer&#160;</td>
			<td>Lawton</td>
			<td>09-0007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Angled and balanced tweezer</td>
			<td>S&#38;T AG</td>
			<td>00595 FRAS-18 RM-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iris scissor</td>
			<td>Lawton</td>
			<td>05-1450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro vascular clamp</td>
			<td>S&#38;T AG</td>
			<td>462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 2<s...

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

Research

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In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

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Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

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In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

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

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 ...

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy

可视化蛋白质激酶 在头部固定行为小鼠中使用 Vivo 双光子荧光寿命成像显微镜的活性

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. ...