本研究的部分内容是 Pantel 博士论文的一部分, 提交给古腾堡美因茨大学医学院。这项研究得到了 Friedhelm 的支持, 释放了基金会和基金会 Neurochirurgische 研究 (赠款阿尔弗雷德·诺思·怀特黑德)。

动物实验由负责任的动物关心委员会 (Landesuntersuchungsamt 莱茵-Pfalz) 批准并且按照德国动物福利法案 (TierSchG) 进行。所有适用的关于照料和使用动物的国际、国家和机构准则都得到遵守。
在本研究中, 使用了雄性 C57BL6 小鼠 (年龄10-12 周)。简单地, 用异氟醚麻醉下腔内纤维穿孔引起蛛网膜下腔出血。左颈外动脉经手术制备。然后, 将一根灯丝插入颈外动脉, 并通过颈内动脉穿孔的高级 intracranially, 导致蛛网膜下腔出血。颅内压增高是血管 内穿孔成功的标志。另有8、14发表了一份详细的协议, 说明小鼠蛛网膜下腔纤维穿孔模型。
1. 灌注和腔内铸造
<ol><li>本研究在蛛网膜下腔灌注72小时后进行。腹腔注射5µg/g 体重 (生物武器) 咪达唑仑, 30 ng/克芬太尼, 0.5 µg/克 medetomidin 的麻醉。只有在达到足够的麻醉水平后才能继续, 这是由于没有对疼痛刺激的反应而证实的。</li>
	<li>打开胸腔, 用21G 套管穿刺左心室, 打开右心房, 并钳住下降的主动脉, 如其他地方15所述。</li>
	<li>使用以下解决方案执行 transcardiac 灌注: (i) Dulbecco 的磷酸盐缓冲盐水含有氯化镁2和 CaCl2 , pH 7.4 为1克/升葡萄糖, (ii) 4% 多聚甲醛溶液。<ol>
<li>用溶液 (i) 开始灌注2分钟, 并继续 (ii) 溶液4分钟。</li>
		<li>在温度为37摄氏度的情况下注入溶液, 并使用可变灌注率的压力控制泵, 以 70 mmHg 的恒定压力灌注, 这被认为是分析16小鼠血管痉挛的最佳灌注压力。从解决方案 (i) 切换到解决方案 (ii) 时避免压力损失。</li>
	</ol>
</li>
	<li>在溶液灌注 (i.) 和 (ii) 后, 在室温下继续灌注20分钟, 显影浇铸剂 (见材料表), 以恒定速率为0.2 毫升/分。</li>
	<li>允许固化的显影铸件材料在4°c 过夜。然后从头骨中取出大脑, 如前所述17, 将样品转移到4% 粉煤灰溶液中, 并将样品保存在4摄氏度直到微 CT 扫描。</li>
</ol>2. 微计算机断层扫描
<ol><li>把大脑放在塑料管的中心, 用钝的解剖钳。选择一个直径比样品稍大的管子, 以确保物体在图像采集过程中不会移动。用纱布把管子关上。</li>
	<li>将塑料管连接到在 X 射线机舱内的计算机导航控制 (CNC) 定位系统中的微步进电机, 在该机上, 物体绕其水平轴旋转。</li>
	<li>在 X 射线照相术的视野下对齐样品。为了达到最大的放大倍数, 尽量将物体尽可能靠近 x 射线源, 尽可能地最大限度地扩大探测器的距离。</li>
	<li>使用带有以下扫描参数的步进图像采集协议: 将曝光时间设置为每一个投影以优化信噪比 (信噪比)、管电压80伏 (当前38µA)、360°旋转导致1000个投影。</li>
	<li>对于原始数据的重建, 使用一种过滤后投影算法, 应用 Shepp-洛根过滤器与矩阵的 1024年 x 1024 x 1024 素使用重建软件 (见材料表)。为了进一步分析, 将由此产生的 DICOM 数据导入3D 可视化软件 (参见材料表)。</li>
</ol>3. 3 维颅内血管树重建及血管容积的测定
注:使用函数帮助可以找到可视化软件功能的背景信息。
<ol><li>使用函数导入将 Dicom 数据导入可视化软件中。</li>
	<li>用函数Volren可视化容器树。选择可视化阈值, 使大的脑动脉被描绘成尖锐的轮廓。对于属于实验序列的所有数据集, 使用相同的可视化阈值是很重要的。</li>
	<li>实际上解剖基底脑动脉 (威利斯的圈子) 与功能VolumeEdit的周围的血管与游标。然后对所分析的容器段进行实际解剖。因此, 旋转的3维血管树模型, 以便精确地分离所有小分支从主动脉。对于进一步分析, 除要分析的容器段外, 还必须删除所有船只。</li>
	<li>将具有阈值设置的函数Autoskeleton应用于可视化阈值, 从而生成基于中心线的SpatialGraph。</li>
	<li>然后, 应用函数SpatialGraphToLineSet创建行集.通过使用游标手动选择单个子细分市场并单击 "拆分", 将该行设置为单个子细分市场。为了计算单个子细分市场的体积, 这一步是至关重要的。</li>
	<li>使用函数LineSetToSpatialGraph再次创建空间图。</li>
	<li>使用函数SpatialGraphStatistics确定每个子细分市场的长度、体积和直径。对于表示容器直径过程的颜色编码可视化, 请使用函数SpatialGraphView.将段着色设置为 "厚度", 与容器直径相关。对于属于实验序列的所有数据集, 选择相同的颜色映射是很重要的。</li>
	<li>添加子细分市场的长度, 以确定将在进一步分析中包含哪些子细分市场。在本研究中, 我们评估了一个血管段, 包括1毫米的颈内动脉近颈 t 和2.5 毫米的大脑中动脉远端的颈 t。然后添加卷以确定所定义的容器段的容器体积。</li>
</ol>

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作者没有什么可透露的。

小鼠蛛网膜下腔模型是研究蛛网膜下腔的重要工具。脑血管痉挛常被用作实验研究的终点, 用于调查9、11的蛛网膜下腔导致的机制。然而, 定量的脑血管痉挛的小鼠或其他小动物模型的蛛网膜下腔是挑战。通常情况下, 血管痉挛是通过体外测定血管内灌注和铸件7、9、12或通过周长测定确定解剖点的容器直径来量化的。被定义的血管在组织学部分10,11。然而, 这些方法有一些缺点: 血管痉挛仅评估在定义的解剖点;邻近血管的血管痉挛可能会逃避评估。组织学文物呈现另一个错误来源。此外, 评估可以是相当主观的, 因为准确的位置, 测量船只直径是由调查员决定的。
因此, 我们的目标是建立一种方法, 通过计算横断面成像数据7的整个脑血管段的血管体积来量化脑血管痉挛。这里提出的容积法最重要的优点是可以检查整个容器段。这就避免了在测量容器直径时, 需要定义一个点。评估整个血管段的另一个好处是, 它大概提出了一个更客观的参数, 以量化血管痉挛, 而不是确定的位置, 更近或远端血管的血管痉挛可能逃脱的确定点的血管直径评价。使用颜色代码对容器直径进行数字表示, 可以直观地估计血管痉挛的程度。此外, 容量评估导致 vasospastic 血管之间的较大差异相比, 船舶直径的评估, 如代表的结果所示。该方法实现的虚拟重建准确反映了血管解剖。这体现在对代表性系列的评价中, 在显微镜下测量的容器直径和数字重建相似, 再现了以前研究的7的观测结果。然而, 尽管有其优点, 还需要进一步的研究来评估这里提出的方法是否优于传统的血管痉挛分析方法。
这里提出的方法的局限性是, 它提供了更多的时间相比, 对铸件的脑标本或组织学分析 (显微 CT 扫描时间90分钟每脑样本, 数据处理45分钟每脑样本)。此外, 微型 CT 扫描仪的可用性可能会限制其应用。这里检查的动物数量足以证明本手稿中所述议定书的可行性。然而, 如果该议定书应用于治疗研究, 动物数量将必须根据预期对船只体积和直径的影响计算。这项研究的另一个局限, 使用小鼠蛛网膜下腔模型是血管痉挛是确定的体内。这使得纵向研究不可能在不同时间点的 SAH 诱导和血管痉挛之前调查基线值。虽然研究表明, 可以用磁共振断层扫描18、计算机断层成像19或数字减法来描述大鼠颅内大型血管的解剖情况。血管造影20, 这些方法, 我们的知识, 尚未被用来分析脑血管痉挛的小鼠蛛网膜下腔模型的体内。值得注意的是, 脑血管痉挛的数字重建, 随后的容量评估, 这里提出的是不限于使用的活体微 CT 数据。如果未来小鼠的高分辨率血管横断面脑成像应可供使用, 则可用于对体内血管痉挛进行容积分析。

Aneurysmatic 蛛网膜下腔出血 (SAH) 是出血性中风的亚型, 是 neurointensive 护理单位常见的疾病。除了早期脑损伤 (EBI), 其中包括由出血事件本身造成的脑损伤, 另一个重要因素确定患者结局是延迟脑缺血 (DCI), 定义的临床恶化, 通过大脑损伤灌注或脑梗塞与介入或手术程序不相关1,2,3。对 vasospasms 的重要机制一方面是大型脑血管;另一方面, 微循环障碍的微血管和血栓血管痉挛, 与皮质扩张性抑郁症相关的缺血起着作用 (回顾了 Madonald 20141)。因此, 对大型脑血管痉挛的诊断在临床实践中至关重要, 在许多临床和实验研究中显示了一个重要的端点。
尽管小鼠蛛网膜血管痉挛的特点不能直接转移到病人身上, 但在过去几年中, 蛛网膜下腔血管痉挛的鼠模型有越来越重要的意义。在这些模型中, 蛛网膜下腔纤维穿孔4,5,6,7,8, 横断池血管9, 或注射血液进入脑脊液10。 ,11,12。与传统上设计用于研究血管痉挛13的大动物模型相比, 小鼠模型具有许多转基因老鼠菌株的巨大优势。这使得它们成为研究导致血管痉挛和 DCI 的分子机制的绝佳工具。然而, 对小鼠脑血管痉挛的测定具有挑战性。这是因为与大型动物模型相比, 血管痉挛可以用临床成像技术来检查,在活体成像分析小鼠脑血管痉挛尚不可用。因此, 血管痉挛通常是通过10、11或显微镜下的脑血管铸件7,9,12的病理切片来确定的。然而, 这些技术有缺点, 船直径在被定义的点仅被审查。
本文在前人研究7的基础上, 提出了一种对小鼠蛛网膜下腔痉挛模型进行客观、重现性分析的方法。该方法以脑血管灌注、体外 ct 扫描、血管树的数字化重建及对整个脑血管容积的评估为基础。

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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:503px;">Medetomidin&#160;</td>
			<td style="width:363px;">Pfizer, Karlsruhe, Germany</td>
			<td style="width:344px;">n.a.</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Midazolam</td>
			<td>Ratiopharm, Ulm, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fentanyl&#160;</td>
			<td>Curamed, Karlsruhe, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Venofix 21G</td>
			<td>B Braun Melsungen AG, Melsungen, Germany</td>
			<td>n.a.</td>
			<td>21G cannula&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s Phosphate Buffered Saline containing MgCl2 and CaCl2, pH 7.4&#160;</td>
			<td>Sigma-Aldrich, Hamburg, Germany</td>
			<td>D8662&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4% paraformaldehyde solution&#160;</td>
			<td>Sigma-Aldrich, Hamburg, Germany</td>
			<td>100496</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microfil MV-122&#160;</td>
			<td>Flowtech Inc., Carver, MA, USA</td>
			<td>n.a.</td>
			<td>Radiopaque</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Micro-CT system Y.Fox</td>
			<td>Yxlon, Garbsen, Germany</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Reconstruction Studio software version 1.2.8.1</td>
			<td style="width:363px;">TeraRecon, Frankfurt am Main, Germany</td>
			<td style="width:344px;">n.a.</td>
			<td style="width:176px;">Reconstruction software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amira software version 5.4.2&#160;</td>
			<td>FEI Visualization Sciences Group, Hillsboro, OR, USA</td>
			<td style="width:344px;">n.a.</td>
			<td>Visualization software</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:503px;">PHD ultra syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-3</td>
			<td>Pressure controlled pump</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anatomical forceps (blunt)</td>
			<td>B Braun Melsungen AG, Melsungen, Germany</td>
			<td>160323_v&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Infinity X-21</td>
			<td>Deltapix, Maalov, Denmark</td>
			<td>n.a.</td>
			<td>high resolution camera</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DeltaPix Insight software version 2.0.1</td>
			<td>Deltapix, Maalov, Denmark</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">C57BL6 mice</td>
			<td>Charles River, Cologne, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a volumetric method for quantification of cerebral vasospasm in a murine model of subarachnoid hemorrhage

蛛网膜下腔出血 (SAH) 是出血性中风的亚型。出血后发生的脑血管痉挛是决定患者预后的重要因素, 因此经常被视为研究终点。然而, 在小动物对蛛网膜下腔的研究中, 脑血管痉挛的量化是一个重大挑战。在这里, 提出了一种前体方法, 允许量化的整个血管段, 这可以作为一个客观的措施, 以量化脑血管痉挛。第一步, 用显影浇铸剂进行脑血管腔内铸型。然后, 通过微计算机断层扫描获得了横断面成像数据。最后一步涉及虚拟血管树的3维重建, 其次是计算选定容器段的中心线和体积的算法。该方法通过解剖样本的直径比较和虚拟重建, 对脑血管树进行了高度准确的虚拟重构。与血管直径相比, 血管体积突出显示了一系列蛛网膜下腔和假鼠 vasospastic 和非 vasospastic 血管之间的差异。

3维颅内血管树的虚拟重建
3维重建的颅内血管树提供了高度精确的血管解剖 (图 1)。为了评估其准确性, 我们在显微镜下和3维虚拟重建2个解剖上定义的点 (1: 左大脑中动脉 (MCA) 1 毫米远端进行了基于直径的比较测定。颈动脉 T;2: 右 MCA 1 毫米颈动脉 T 的远端)。对于容器直径的显微测定, 使用了具有 Deltapix 洞察力软件版本2.0.1 校准的高分辨率相机 (无穷 X-21、Deltapix)。对这一评估, 分析了10例脑标本 (5 个蛛网膜下腔出血, 5 例假)。这些来自12只小鼠, 其中7个是蛛网膜下腔诱发的, 5 例进行了假手术 (蛛网膜下腔组2例死亡, 分别为术后天数1和 2)。在显微镜下和实际的直径之间没有显著的差异, 表明颅内血管解剖的准确虚拟重建 (显微测定与虚拟重建,平均标准错误: 左 mca 150 9 µm vs左 mca 150 @ 8 µm;右 MCA 153 8 µm vs154 @ 9 µm, 见图 2)。
蛛网膜下腔出血小鼠脑血管痉挛的定量研究
为量化脑血管痉挛, (i) 预定义代表3.5 毫米血管段的体积, 由1毫米颈内动脉 (ICA) 和左侧2.5 毫米 MCA 组成, (ii) 在2个解剖上定义的点 (左和右 MCA) 的血管直径确定在脑标本从蛛网膜下腔和假操作的动物 (n = 5)。与假相比, 在蛛网膜下腔的血管容积明显降低 (36 4 nl vs 71 @ 9 nl, p < 0.05)。与假相比, 蛛网膜下腔血管直径较低 (左 MCA: 140 @ 11 µm vs 160 @ 10 µm, p = 0.11; 右 MCA: 130 @ 16 µm vs 158 @ 13 µm, p < 0.05; 见图 3), 而意义水平只达到分析是正确的 MCA。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57997/57997fig1.jpg"> 图 1。颅内血管树的虚拟重建。(a) 显示有代表性的脑样本;(B)显示相应的几乎重建的血管树。(C)左 MCA 直径的彩色编码可视化。<a href="https://www.jove.com/files/ftp_upload/57997/57997fig1large.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/57997/57997fig2.jpg"> 图 2.血管数字化重建的准确性.与显微镜下测定的3D 重建脑样的平均直径。数据显示为平均值, 即平均值的标准误差。<a href="https://www.jove.com/files/ftp_upload/57997/57997fig2large.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/57997/57997fig3.jpg"> 图 3.蛛网膜下腔血管容积和血管直径.(A)对蛛网膜下腔和假鼠3D 重建血管的 MCA 直径进行比较。(B)蛛网膜下腔和假鼠的血管容积。数据显示为平均值, 即平均值的标准误差。*p < 0.05。<a href="https://www.jove.com/files/ftp_upload/57997/57997fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage at JoVE.com

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

本文的目的是提出一种方法, 允许3维脑血管树重建后, 小鼠的微计算机断层扫描和确定的整个血管段, 可用于量化脑血管痉挛的数量小鼠蛛网膜下腔出血模型。

小鼠蛛网膜下腔出血模型脑血管痉挛量化的容积法

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

a-volumetric-method-for-quantification-cerebral-vasospasm-murine

Research

JoVE Journal

Neuroscience

8.0K 次观看.  Medical Center of the Johannes Gutenberg - University. 本文的目的是提出一种方法, 允许3维脑血管树重建后, 小鼠的微计算机断层扫描和确定的整个血管段, 可用于量化脑血管痉挛的数量小鼠蛛网膜下腔出血模型。

视频: A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

自体血液的注入模型小鼠自发性脑出血

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

科研

神经科学

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

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 ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

轻度宫内灌注圈狭窄的大鼠模型

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

高频颅内双体超声波的苏巴拉赫诺德出血的穆林模型脑血管痉挛分析

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

视交叉前，单次注射自体血以诱导大鼠模型中实验性蛛网膜下腔出血

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

数字液滴PCR法定量分析小鼠视网膜AAV转导效率

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of ...

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

蛛网膜下腔出血结合磁共振成像（MRI）的血管内穿孔模型

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high ...