我们谨向瑞典国防部 (信息) 和灾难医学和创伤治疗中心 (KMC) 的林雪平工作人员表示谢意, 感谢他们的亲切支持。

这里描述的研究遵循了当地的道德准则, 并得到了林雪平地区伦理审查委员会的批准 (许可证编号 2015/99-31)。
1. 知情同意和甄别
<ol><li>获得有关问题的知情同意。</li>
	<li>根据包含/排除标准进行筛选。 注: 纳入标准为: (一) 健康成人 > 18 岁, (二) 能够理解书面和口头信息, (三) 提供口头和书面同意。排除标准是: (一) 无法或不愿意提供知情同意, (二) 持续的皮肤状况, (iii) 心血管疾病, (iv) 可干扰血管功能的药物。口服避孕药是允许的。如果主题使用口服避孕药, 应在主题的协议文件中注明, (五) 前额无疤痕和无擦伤皮肤, (六) 在试验开始前2小时不摄入咖啡因或烟草, (vii) 没有剧烈的体力活动至少 2 h pr开始测试。</li>
</ol>2. 驯化和设备设置
<ol><li>在测试开始前, 让测试对象适应至少20分钟的仰卧位。 注: 主题和设备的正确定位显示在补充图1中。</li>
	<li>
使用指定的软件和数码相机设置毛细管填充测试的相机记录 (见材料表)。
	<ol>
<li>将 USB 电缆连接到照相机和计算机, 并将摄像机设置调整为视频模式。</li>
		<li>打开摄像机, 确保它连接到计算机。</li>
		<li>启动远程控制的照相机软件 (请参见材料表)。</li>
		<li>关闭环境光源, 将计算机屏幕从测量区域中关闭。</li>
	</ol>
</li>
</ol>3. 数据采集
<ol><li>单击 "生命视图" (Sic) 按钮, 然后按 "开始"。</li>
	<li>在 "视频" 模式和 "TiVi" 模式之间切换, 方法是单击单选按钮 (这不会影响录制, 只有屏幕显示)。确保在摄像机和计算机屏幕上显示主体前额的实时视频流。</li>
	<li>调整相机的高度约15厘米直接以上的主体的额头。</li>
	<li>确保照相机的焦点设置为 "AF" (自动对焦)。</li>
	<li>要求受试者保持他/她的头仍然在测试期间和克制谈话。在测试过程中, 请告知这个问题, 让他/她的眼睛闭上是 OK 的, 说: "拜托, 把你的头放在这个枕头上, 保持你的头部在测试期间。请不要在20秒内说, 测试将持续。如果你觉得光线太亮, 你可能会一直闭着眼睛。</li>
	<li>录制时请勿移动相机。</li>
	<li>在 "生命视图" 窗口中找到并单击 "HD 记录" 以开始二十年代的连续录制. 注意记录的持续时间 (秒), 可以在摄像头显示的时候看到;记录的前5秒是获得正常的前额毛细血管血浓度。</li>
	<li>在基线记录的5秒结束时, 对 5 s 的测量区域应用一个牢固的烫漂压力. 使用食指和前额之间的塑料茶匙来进行压力平衡和温度隔离。</li>
	<li>释放压力, 并迅速从测量区域撤回考官的手指和塑料茶匙。</li>
	<li>继续录制另一个十年代, 以捕获随后的充血响应。</li>
	<li>通过单击 "生命视图" 窗口中的 "停止" 停止录制。</li>
	<li>录制停止后, 将打开一个窗口, 用于保存和命名文件 (以。选择视频的文件夹和名称。在将视频下载到计算机上的指定文件夹时, 请勿将照相机与计算机断开。</li>
</ol>4. 数据分析
<ol><li>使用专用的分析软件继续执行图像分析和曲线构造 (请参见材料表)。</li>
	<li>通过双击桌面图标启动分析软件。</li>
	<li>软件启动后, 在主屏幕上选择 "影片页面"。</li>
	<li>在 "影片页面" 窗口中, 单击 "加载影片"。等待软件上传和分析电影。这可能需要几分钟时间, 这取决于视频文件的大小。</li>
	<li>一旦电影的上传和分析完成, 点击 "另存为图像", 等待软件分析视频。 注意: 这将自动在包含视频序列的计算机上创建一个新文件夹, 并将其分为单独的 jpeg 图像。</li>
	<li>将视频分割成单独的 jpeg 图像后, 在主屏幕窗口中查找并选择 "裁剪图像页面"。</li>
	<li>在 "裁剪图像页面" 中, 单击 "第一张照片", 然后导航到包含视频中的 jpeg 照片的文件夹, 然后选择要分析的第一张照片;该软件将自动选择该批的以下照片。</li>
	<li>单击 "编辑" 菜单, 并为感兴趣的区域选择圆形或矩形标记。</li>
	<li>在 "选择照片" 窗口中, 找到 "实际照片" 框, 并键入在批中的第一张照片的名称, 调查员的手指完全从测量区域收回。要查找此照片, 请查看包含来自视频的单个照片的文件夹。</li>
	<li>通过单击并按住鼠标左键并在位于脱皮区域边框内的照片窗口中绘制圆形或矩形, 选择感兴趣的区域。所选的感兴趣区域将自动应用于批次的所有照片。</li>
	<li>单击 "文件" 菜单, 选择 "保存 ROI" 以保存所选区域的位置, 以备将来参考。</li>
	<li>单击 "曲线跟踪器" 按钮, 等待软件分析感兴趣的区域。</li>
	<li>一旦 "曲线跟踪器" 窗口打开, 在测试期间所选的感兴趣区域的红血球浓度变化将显示为曲线。通过单击不同的单选按钮, 可以更改屏幕上的演示文稿。这不会影响原始数据。</li>
	<li>单击 "导出数据" 并保存原始数据记录以进行进一步分析。</li>
	<li>在原始数据记录中, 导航到名为 "平均强度 TiVi 值" 的列。该列旁边的 "图像编号"、"总时间" 和 "毛细管重填时间" 在 1 s、0 s 和 0 s 之间生成三新列, 分别为 1 s、0.02 s 和 0.02 s 的增量。 注: "毛细管重填时间" 与随后对热烫压力释放的反应时间相关, 并以平均值表示第一张照片, 调查人员的手指完全从测量区域收回。</li>
	<li>删除基线和毛细管重填时间之间的值, 因为这些数值只包含由研究者的食指和塑料茶匙在烫漂过程中产生的噪音。</li>
	<li>计算基准测量的平均值 (记录的前5秒)。</li>
	<li>查找等于或超过计算平均值基线值的第一个值, 并记下 "毛细管填充时间" 列中的时间。此时间点表示 "返回到基线1的时间"。</li>
	<li>找到压力释放后产生的值的最高平均强度值, 并记下 "毛细管重填时间" 列中的时间点。这个时间点代表了 "高峰时间"。</li>
	<li>查找小于或等于基线测量的计算平均值的第二个值, 并记下 "毛细管重填时间" 列中的时间点。此时间点表示 "返回到基线2的时间"。</li>
</ol>

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WheelsBridge AB 的财务支持没有参与研究的进行。作者约翰是受雇于约特兰郡议会, 但有一个版税协议与 WheelsBridge AB。资深作家综合教育有一个全职的学术职位, 但也有限的参与 WheelsBridge AB。

为了使系统得到最佳的结果, 必须控制环境因素造成的变化。所有的环境光必须关闭。照相机必须与测量区域垂直对齐。为了保证一个恒定的测量区域, 对象不应该移动或谈话在测量期间。出于同样的原因, 相机最好安装在一个立场, 以避免移动和保持一个恒定的距离测量区域。测试对象应避免咖啡因的17, 烟草18, 和硬练习19至少两个 h 之前的测?...

危重病人的评估和分诊集中在经典生命体征血压 (BP)、心率 (HR)、呼吸率 (RR)、氧饱和度和体温1。这些参数的变化在循环恶化过程中显得相对较晚。例如, 在出血, 血压下降将不会发生, 直到血液损失变得中度到严重的2, HR 增加也可以是一个不敏感的和不标记3。
毛细管重填试验 (CR 测试) 可以提供早期的循环崩溃的征兆, 因为重填时间在重要征兆之前被相信改变, 并且临床出现冷, 阴湿黏的和斑驳的皮肤1,4,5. 毛细管填充试验通常是由应用, 然后释放一个坚定的漂白压力的皮肤与时间 (以秒计) 的血液返回到脱皮区域。根据指导原则, 重填应该在2秒内发生, 在5秒的牢固的压力 (例如, 由医生的手指) 在正常健康仰卧患者6。测试的基本原理是, 较慢的填充时间将表明皮肤灌注不良, 可能是由许多关键事件之一, 如脓毒症, 失血, 急性心力衰竭, 或体温过低造成的。
目前, 在执行 CR 测试的艺术方法的状态上没有达成一致意见6,7,8,9,10。有争议的问题包括缺乏标准化的实际漂烫机动和依赖主观 (即, 裸眼) 评估的填充端点7,9,11。此外, 有迹象表明, 性别影响 CR 时间12,13。温度, 环境和皮肤, 是已知的影响毛细管填充时间, 但在何种程度上是不清楚的。最后, 使用不同的测量点, 外围或中央, 可能是一个进一步的原因变化的结果, 在这方面的研究很少14,15。
在目前的工作中, 我们使用了一个光学生物工程系统, 以记录血液的返回过程和随后的充血反应, 在 CR 试验中看到。该系统利用漫反射极化光谱学, 更详细地量化和描述, 比可能的肉眼, 毛细管填充的时间和过程。该系统包括一个标准的数码相机, 装有一个外部的光环与92白色 led, 和专门开发的软件。镜头和两个偏振过滤器, 连接正交在前面的 led, 阻挡光, 已直接反映在皮肤表面, 只允许光已成为偏的组织, 以达到相机。这就产生了一个 "亚表皮" 的组织图像, 深度约0.5 毫米。图像分为其颜色平面, 并计算每个像素的红色和绿色内容, 生成一个对应于红细胞组织浓度的值16。在视频模式下, 系统的时间分辨率为0.02 秒。

<table>
	<tbody>
		<tr>
			<td>TiVi701 Camera</td>
			<td>WheelsBridge AB</td>
			<td>TiVi701 Camera, version 1.5.1</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>TiVi700</td>
			<td>WheelsBridge AB</td>
			<td>TiVi700 Analysis, version 1.2.9</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Canon EOS 700D</td>
			<td>Canon U.S.A., inc.</td>
			<td>Canon EOS 700D</td>
			<td>Digital SLR Camera</td>
		</tr>
		<tr>
			<td>Camera stand</td>
			<td>Manfrotto</td>
			<td>681B</td>
			<td>Modified camera stand to hold the digital camera in position</td>
		</tr>
		<tr>
			<td>Camera stand</td>
			<td>Disa Denmark</td>
			<td>9020B</td>
			<td>Modified camera stand to hold the digital camera in position</td>
		</tr>
	</tbody>
</table>

diffuse reflectance spectroscopy: getting the capillary refill test under one's thumb

毛细管重填试验在1947年引入, 以帮助估计危重病患者的循环系统状况。通常情况下, 在正常健康的仰卧位患者释放5的牢固压力 (如, 由医生的手指) 后, 应在2秒内发生重填。较慢的填充时间表示皮肤灌注不良, 这可能是由于败血症、失血、灌注和体温过低造成的。自其介绍以来, 该试验的临床效用已被争论。倡导者指出它的可行性和简单性, 并声称它可以显示血管状态的变化早于生命体征的变化, 如心率。另一方面, 批评家强调, 在测试中缺乏标准化, 肉眼评估的高度主观性, 以及测试对周围环境因素的敏感性, 明显降低了临床价值。本工作的目的是详细描述重新灌装事件的过程, 并提出了潜在的更客观和准确的终点值的毛细管填充试验使用漫极化光谱学。

拍摄的毛细管重填过程产生了大量的数据不可能获得肉眼评估。我们建议在这里新的端点, 以进一步提高 CR 测试的可用性, 作为早期的指标恶化的循环状态。我们称这些端点为: "基线"、"血零" (或 ")"、"返回基线1的时间" (或 "tRtB1")、"时间到峰值" 或 "Tpk"。"基线" 值是通过计算在 5 s 基线测量期间获得的所有值的平均值来获得的。"血零" 是指在压力释放后立即从脱皮区域的第?...

Watch this Scientific Journal Video about Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb at JoVE.com

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

该协议描述了如何使用漫极化光谱学可以提高临床效用的毛细管填充试验。我们建议使用漫反射光谱视频和新的信息端点对健康志愿者毛细血管充盈的过程进行更详细的分析。

漫反射光谱分析: 在拇指下进行毛细管填充试验

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

diffuse-reflectance-spectroscopy-getting-capillary-refill-test-under

Research

Education

JoVE Core

JoVE Journal

Medical-Surgical Nursing

Neuroscience

Unit 1

An Overview of the Cardiovascular System: Anatomy, Physiology, Assessment, and Abnormalities

SCIENTISTS IN ACTION

9.0K 次观看.  Region Östergötland. 该协议描述了如何使用漫极化光谱学可以提高临床效用的毛细管填充试验。我们建议使用漫反射光谱视频和新的信息端点对健康志愿者毛细血管充盈的过程进行更详细的分析。

视频: Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

在小鼠嗅球成像气味诱发的活动，利用光学反射和自体荧光信号

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

科研

神经科学

The Mouse Forced Swim Test

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental manipulations that are aimed at rendering or preventing depressive-like states. Mice are placed in an inescapable transparent tank that is filled with water and their escape related mobility behavior is measured. The forced swim test is straightforward to conduct reliably and it requires minimal specialized equipment. Successful implementation of the forced swim test requires adherence to certain procedural details and minimization of unwarranted stress to the mice. In the protocol description and the accompanying video, we explain how to conduct the mouse version of this test with emphasis on potential pitfalls that may be detrimental to interpretation of results and how to avoid them. Additionally, we explain how the behaviors manifested in the test are assessed.

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

小鼠强迫游泳实验

The forced swim test is a rodent behavioral test used for evaluation of antidepressant drugs, antidepressant efficacy of new compounds, and experimental ...

The Tail Suspension Test

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test.

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

悬尾实验

The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

大鼠桶内皮层 7 t 的功能磁共振波谱在晶氏体活化过程中的应用

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte function is not fully known. Pericytes have been suggested to be involved in capillary development, regulation of endothelial barrier tightness and trancytosis activity, regulation of capillary tone and to play crucial roles in certain brain pathologies.
Pericytes are challenging to investigate in the intact brain due to the difficulties in visualizing processes in the brain parenchyma, as well as the close proximity to the other cells of the NVU. The present protocol describes a method for isolation and culture of primary bovine brain capillary pericytes and their following usage in calcium imaging studies, where effects of agonists involved in brain signaling and pathologies can be investigated. Cortical capillary fragments are allowed to attach to the bottom of culture flasks and, after 6 days, endothelial cells and pericytes have grown out from the capillary fragments. The endothelial cells are removed by gentle trypsinization and pericytes are cultured for 5 additional days before passaging.
Isolated pericytes are seeded in 96-well culture plates and loaded with the calcium indicator dye (Fura-2 acetoxymethyl (AM)) to allow for measurements of intracellular calcium levels in a plate reader setup. Alternatively, pericytes are seeded on coverslips and mounted in cell chambers. Following loading with the calcium indicator (Cal-520 AM), calcium live-imaging can be performed using confocal microscopy at an excitation wavelength of 488 nm and emission wavelength of 510-520 nm.
The method described here has been used to obtain the first intracellular calcium measurements from primary brain capillary pericytes, demonstrating that pericytes are stimulated via ATP and are able to contract in vitro.

Brain capillary pericytes are essential players in the regulation of blood-brain barrier properties and blood flow. This protocol describes how brain capillary pericytes can be isolated, cultured, characterized with respect to cell type and applied for investigations of intracellular calcium signaling with fluorescent probes.

脑毛细管中培养细胞细胞钙测量和钙成像研究

Pericytes are associated with endothelial cells and astrocytic endfeet in a structure known as the neurovascular unit (NVU). Brain capillary pericyte ...