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。
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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

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

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 the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

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

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

在皮肤中的单神经末梢体内双光子显微镜

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

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

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

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

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 has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

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

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

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

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 ...

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

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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

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.

Two-photon Imaging of Microglial Processes&#039; Attraction Toward ATP or Serotonin in Acute Brain Slices

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 ...

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

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKAR&#945;, is used, which is based on F&#246;rster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKAR&#945; is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

A procedure is presented to visualize protein kinase A activities in head-fixed, behaving mice. An improved A-kinase activity reporter, tAKAR&#945;, is expressed in cortical neurons and made accessible for imaging through a cranial window. Two-photon fluorescence lifetime imaging microscopy is used to visualize PKA activities in vivo during enforced locomotion.

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. ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...