The authors have no acknowledgements.

必要的实验开发协议是按照国家规定执行，由当地的伦理科委批准（Comitato迪Bioetica迪雅典耀），都灵，意大利都灵大学的机构内工作。 1.准备<ol><li>麻醉<ol><li>将适当的异氟醚室内的动物麻醉吧。 </li><li>填充混合的 O 2和异氟醚气为兽医用途的室中的2.5％在一个2升的气体室中的浓度，并等待约3分钟的大鼠入睡。检查麻醉通过脚趾捏的效果。 </li><li>一旦麻醉生效后，取出大鼠和称重。 </li><li>散布在该动物的眼睛的薄层水溶性眼用凝胶，以保护它们，以保持眼生理水化。 </li><li>奠定了老鼠倒在超声和光声成像站台面。我n阶维持麻醉效果，迅速鼻适当掩模提供恒定麻醉流量（异氟烷2％氧1L /分钟-2.5％）的内部定位。 </li></ol></li><li>剃须动物<ol><li>传播的毛发去除奶油一致层上的头的表面上，注意覆盖周围的耳朵和颈部。允许奶油行事几分钟，轻轻取出用抹刀。轻轻地取出所有残余膏用湿海绵准确地清洁皮肤。 注：动物皮毛俘获空气产生负面影响超声基于成像的采集，从而它必须被除去一定尽可能。 </li></ol></li><li>定位动物<ol><li>安排在传播鹰立场动物。监测生命体征，通过对操作面适当生命参数的传感器装置（如果它们存在）。运用电极膏滴一些用于专业用途后依靠传感器的爪子。 注：在麻醉下，确保重要参数具有值如下：大鼠体温≈37.5℃，每分钟（BPM）的心脏搏动250和350与呼吸频率之间变化被包括在每分钟40-80次呼吸的范围内。 </li><li>最后拧紧防过敏人造丝补丁四肢。如果有必要，再传播一层薄薄的水溶性眼用凝胶，保护动物的眼睛。 </li></ol></li></ol> 2.图像采集从查看的时空点<ol><li>定位动物<ol><li>保持动物处于俯卧位置，稍转动它的身体的一侧，具有大约45°的相对于该矢状体轴线的倾斜角。用小棉花纱布卷为代表正确安排处置（ 图2a）。 </li><li>提高动物的头，稍微旋转一侧（ 图2a）。用棉卷的立场保持鼻子以及inser泰德进入麻醉面罩。 </li><li>倾斜的台面以约30°的相对于水平面的角度。 </li><li>转动成像换能器在约30°与相对于垂直平面的夹角。 </li></ol></li><li>超声波和光声解剖学和血管图像采集<ol><li>打开成像扫描，输入B模式图像采集和正确设置所有的图像采集参数，尊重实验（ 图3a）可能给出的要求。 注：设置发射中心频率尽可能低（16兆赫， 图3b），为了具有最大的穿透深度可以为换能器。 </li><li>处置一致层（约1厘米厚）低变应原性水溶性超声传输凝胶对动物的头（ 图2b）。覆盖换能器头有一薄层同一凝胶的，并把它接触到对RA的层吨。用温暖的凝胶，以减少局部低温。 </li><li>开始于B模式图像采集和实时调整换能器的定位，通过识别解剖学参考和定心的感兴趣的区域，以在监视器中点。确保消除气泡在包封于凝胶层的任何级别，是因为他们的负面影响收购。 </li><li>放置换能器，将其调整到虚拟轴线耳连接到眼（ 图4a），以获得最佳的光束聚焦。获得不同的视图内部脑容量，通过顺时针或逆时针旋转（ 图4b和c）。 </li><li>最终固定在机械支架传感器确保稳定的好方法的位置和调整方向。 </li><li>确保感兴趣的脑区域局部化以10mm深度相对于美激光传感器源以接收一个最佳pH耳声的响应信号（ 图5）。然后将美国波聚焦的指示器恰好在所分析的区域的中心。 注：在对感兴趣的领域的研究，避免激活所述呼吸门的选项，以加速的定位过程。 </li><li>进入彩色多普勒模式，以可视化的内部脑血管在高敏感的方式。 </li><li>一旦定位，以适当的方式来可视化通缉区域被设定时，激活所述呼吸门的选项，以避免相关的运动（ 图6a）不希望的效应。 </li><li>选择在彩色多普勒模式（ 图6b）的有用采样参数集，在这种方式来区分血流的速度和方向获取图像，直到穿透深度为几毫米。 </li><li>输入脉冲多普勒模式，并获取图像来检测动脉搏动和动脉一个区分第二脉。 </li><li>输入功率多普勒模式并设置采集参数（ 图7），以散射所造成的磁通运动事件的数量的基础上执行的信号量化，并因此在流速评价分歧。 </li><li>进入光声模式，妥善细化采集参数（ 图8a），以收集有关血液中的总血红蛋白含量，或氧化程度的一个特定区域的数据。通过将整个波长光谱上产生激光激发（从680纳米至970纳米， 图8b）中，存在于组织内不同的化学状态总的血红蛋白的吸收可进行定量。通过在单一的特定波长（ 图8c）进行信号采集，可以分离由于氧和脱氧的纯物质的吸收的明显信号的贡献。 </li></ol></li></ol>从查看的枕骨点3.成像<ol><li>定位动物<ol><li>保持动物俯卧位，降低了动物的头，用小棉花纱布卷作为横向站在正确安排处置。 </li><li>转并行成像换能器到动物头部的横向平面（ 图9）。 注：通过这种方式，收购将通过枕骨孔的头骨的基础上居中。通过改变探针的方向（ 图9）的倾斜角度，将有可能获得根据设置倾角在不同视图中的内部容器的图像。 </li></ol></li><li>超声波和光声解剖学和血管图像采集<ol><li>输入B模式图像采集，设置所有的图像采集参数如先前报道（ 图3）和传播所需的超声凝胶层上的探针和对动物的颈背。 </li><li>安排传感器保持几乎水平，在ORD呃被沿着主体的解剖后部到前部轴定向。它指向的口鼻部的前侧和倾斜稍微向前。 </li><li>开始图像获取中乙模式和彩色多普勒模式（ 图3和6）。精确地调整换能器的位置并如前所述从凝胶涂层除去气泡。如果可能的话，固定传感器上的坚定立场，以控制罚款方式的方向和选择最佳的倾斜角度，以获得所需的解剖区域的图像。 </li><li>可视化内部脑血管能量多普勒模式中，通过适当地设定采集参数（ 图7）。 </li><li>本地化强烈脉动动脉通过脉冲多普勒模式。从脉，这反过来的特点是血流量脉动水平低区分它们。 </li><li>收集血流速度数据和方向彩色多普勒模式，通过充分适应ACQuisition参数（ 图6）。 </li><li>完整深脑血液动力学特征数据集，通过添加（ 图8），通过光声采集获得的血液化学信息。通过在特定的评估血参数的量，如O 2饱和度百分比和总血红蛋白含量（HBT），是通常通过将激光的激发波长在750和850纳米（ 图8c）测定执行此。 </li></ol></li></ol> 4.结束采集和动物去除注：正确考虑专用于图像采集过程（从步骤1到步骤3），其进行与施加到动物的麻醉剂量主要限制的全部时间。 <ol><li>保存所有采集到的数据，把激光脉冲关闭退出光声采集模式和距离传感器。 </li><li>同时保持ANI在麻醉作用发作，开始轻轻去除眼睛的保护胶用湿棉签进行清洁。用刮刀和几个纸巾从头部和鼻子完全除去超声凝胶，然后清洗它们用湿海绵。要小心，不要损坏娇嫩的皮肤剃光。 </li><li>取出用于固定四肢，并从该监测生理参数的传感器断开它们的粘合贴剂。迅速从收购台面转移动物到一个不同的笼子里。 </li><li>举办​​动物在一个小笼子从麻醉中恢复。确保动物不应以防止侵略在这一阶段共享笼</li><li>将在红外光恢复笼，以保持动物的温暖。等到它已经恢复了足够的意识，保持胸骨斜卧。检查动物的一般健康状况，将其移动到动物饲养室前。 </li></ol>

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所提出的协议进行了优化，以提供在小动物的高度有效的脑的成像性能。图像可以在不同的方式通过精确以下关于采集参数和头骨椎间孔传感器定位的迹象被收购。特别是，在颞侧的定位是最关键的，因为美国和激光必须居中尽可能精确，以正确地穿透孔，这是比枕骨1小。然而，由于本实验设置，关于生理或病理甚至竞赛血流动力学特征都可以访问，甚至在深脑区域，这通常是困难的表征可以被评估。 由于成功的图像采集取决于换能器的定位的精确度，这种依赖必须仔细考虑，因为它可能会影响成像性能。例如，一些感兴趣的解剖结构，可以没有完全包含在收购成像平面，并从提供的只是一个片面的眼光可能会导致次优的图像他们的身份。此外，在三维模式（3D模式）进行的美国和PA成像采集将是与先前描述的实验设置不兼容，因为它需要的换能器，以沿着一个预先定义的自动化的路径移动。最后，由于自然解剖变异，颅骨开口的尺寸可以显著动物中发生变化，从而对采集过程不可预知的影响。这一事实使图像质量取决于每个个体的特性。因此，不可能以应用此策略，一些动物具有设计实验方案时，需要​​考虑。 具体地讲，一个显着的利益被寻址到血流动力学，由于在确定其基本作用全身用药后28-29生物分布的药物或其他外源性分子。在分子成像领域中的应用性的影响是多方面的，包括从血池造影剂的验证，以图像监视的药物递送研究需要超声诱导的血脑屏障开口30。所有这些研究目的必定从协议，考虑到的最小侵袭中获益，而没有任何附加的外科手术，死亡或不希望的副作用的风险大大降低，在相同的动物模型中的长期监测是可行的。 总之，所提出的协议可以使从业者能够高效地图像和正确解释解剖地形和正常或病理脑组织的研究使用的动物模型中的血管图案。虽然目前的方法主要限于断层成像皮质25-27，这个设定会给机会吨Ø表明，影响深部脑生理学，通​​过合并由美国和PA成像提供了优势的几个过程。

需要策略以微小描述在小动物的中枢神经系统的脑血液动力学功能，以促进神经1-3的领域。所提出的技术演示了如何以考察血管生物学，安排和功能上的小动物的大脑进行无创声学和光声成像。 光学成像技术使相关的神经活动2,4-5事件的本地化，同时获得无论是在含氧和非含氧州6血红蛋白生成的信号。然而，由于光子的吸收和散射，纯光学成像患有空间分辨率差的和有限的组织穿透深度7-8。相反地，声学提供了机会，以更高的空间的空间分辨率进行更深的成像，但它是由散斑和有限的对比度9-11阻碍。通过结合光子无线功能日超声波，光声技术，改善了成像和单一方法12-16诊断潜力。 大脑的光声成像具有澄清多个问题在神经生物学的潜力，然而，自然保护脑，大大限制了二者的光子和超声波组织穿透17-19的黄芩。此外，骨头促进双方光线和声音造成的灵敏度和图像畸变17-18的损失散射。作为结果，脑超声波和光声成像能够容易地对新生儿动物之前骨化20执行的，但成人脑的深解剖学和生理学仅经过开颅21,22清楚地访问。遗憾的是，所需的颅骨切除术在技术上是坚硬和它的作用可能是有害的一些实验目的从而难以以监测神经疾病进展同样的动物随着时间的推移。因此，非侵入性的方法，将图像的深脑生物学中的小动物模型是非常需要的。在文献光子再循环器17的方法被报导，以此来减少电话损耗，并通过完整颅骨增加透射率，提高光声信号噪声比（SNR）和目标的对比度。 所提出的协议的目的是，没有任何侵入性手术提供皮层下脑声学和光声成像研究使用的啮齿动物（特别是对大鼠）的可靠方法。该过程是基于使用便携式传感装置用于高频超声波和光声成像。而相比之下，断层成像技术23，便携式和定向传感器24能够选择特定颅区与自然减小的厚度，称为裂隙或scissures。主要裂（椎间孔）出现在脊椎动物nimal颅骨是必要的，以找到神经束，血管或其它结构连接内部脑回路到身体的其他部位。主要裂被发现在不同尺寸的骨的孔，可利用作为超声波和激光的具体段落。此类靶向成像减少由骨界面波的反射效应和通过增强成像的穿透深度增大的灵敏度。在这个角度来看，成像换能器可以被布置成垂直于位于颞和头骨上（ 图1）的后头部侧的裂隙中，为了最大程度地会聚于这些区域的超声和光子束。这种取向既增强了信号质量，并强制进行，通过更薄的骨层相对于其它颅取向的信号。因此，发射和反射波发生散射的程度较低，使采集的强烈信号，从更深的始发组织层。与早期的程序，这个实验设置只需要动物剃光头，是没有必要的其他手术时。 与所提出的协议，成像在相对较高的空间分辨率进行，揭示两者，具体参考解剖结构和血管比现有技术方法的当前状态更深，同时还能在动物的皮肤和颅骨保持不变。独特的冠状和轴向图像可以通过利用各种超声成像采集方式（B，能量多普勒，彩色多普勒，脉冲波模式）并行光声成像被收购。参数扩展的剧目可以从这些图像中提取，从而实质和血管解剖一起描绘影响血液循环动力学特征的整个集合。该协议可以用于图像基本皮层薄壁特征在高频超声波乙模式形态，基底和颈内动脉（BA和ICA分别）组成Willis环，大脑中动脉（MCA）和循环设备的其他细节。另外，血流量化，是指流速度，定向运动的描述和氧饱和度数据可以从皮层到脑深部区域收集。 这个新的战略具有巨大潜力的各种应用和满足迫切需要可靠的程序来描绘脑深部的功能，在各种病症的关键。由于其最小的侵袭。此外，所提出的协议可以使中枢神经系统的无数可能的成像研究，特别是那些需要长期监测或涉及娇嫩病理动物模型。

<table border="0" cellpadding="0" cellspacing="0" width="979">
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			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:464px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">High frequency ultrasound and photoacoustic imaging station (VEVO LAZR 2100 system)&#160;</td>
			<td>FUJIFILM VisualSonics Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vevo Compact Dual Anesthesia System (Tabletop Version)&#160;&#160;</td>
			<td>FUJIFILM VisualSonics Inc.</td>
			<td style="width:119px;"></td>
			<td>http://www.visualsonics.com/anesthesiasystem#sthash.opODt 
			Sht.dpuf</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Ultrasound Transmission Gel (Aquasonic 100)</td>
			<td style="width:180px;">PARKER LABORATORIES INC.</td>
			<td style="width:119px;">01-08</td>
			<td>http://www.parkerlabs.com/aquasonic-100.asp</td>
		</tr>
		<tr height="231">
			<td height="231" style="height:231px;width:216px;">Sprague-Dawley rats</td>
			<td style="width:180px;">&#160;Charles River Laboratories</td>
			<td style="width:119px;"></td>
			<td style="width:464px;">Three helathy 6-weeks old Sprague-Dawley rats were purchased by Charles River Laboratories and kept in standard housing (12 h light-dark cycles) with a standard rodent&#160; chow and water available ad libitum. Provided by: http://www.criver.com/</td>
		</tr>
	</tbody>
</table>

non-invasive parenchymal, vascular and metabolic high-frequency ultrasound and photoacoustic rat deep brain imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

此方法允许将图像的两个特定的解剖参考结构和血管以相对高的空间分辨率，比当前技术与动物的皮肤和颅骨完好更深。在我们的实验条件下的PA信号的深度为4.5毫米，轴向分辨率为75微米的具有视场23×15.5厘米。实验光声断层扫描模式19显示的分辨率&lt;1毫米值。 SNR值的范围是从21.6分贝23.8分贝（通过对脑组织和背景随机选择5个不同点获得）。并列在颅骨颞侧的换能器，脑图像可被获取作为横向或甚至冠状切片的换能器的具有的观点得到的横向成像点（ 图4）所选择的定位角度的基础上。表皮，颅骨和脑实质材料是很好的代表在超声波B模式，因为他们有很大的不同在交流方面oustic阻抗（ 图10）。即使它们的配置取决于所选择的角度来看，对实质部分解剖参考位点是可识别的，如裂隙分离皮质脑内部部分和特征形视束（ 图10）。此外，大量的船只都在超声波和光声成像方式可见。颈内动脉（ICA）以沿动物的大脑的外部侧向表面上运行的其他主要大血管的特性的交点可以容易地识别。大血管的路线，诸如ICA的，提供了一个巨大的血液供应，以满足能量和氧气的一贯神经的需要。在ICA，源自颈总动脉（CCA），运行在头部的深度为几毫米的侧面，超出其所有分叉点和最后到达正面头部。 intermedi这中间主要的血液流传播吃了大小船只，被引导的总是小动脉，最终滋养神经元之前。从视图的时间点，所以能够追踪内部脑动脉图案，即分叉成朝向前方和横向侧脑血管。冠状和横向的图像可以具有不同的倾角传感器的相对于该虚拟轴接合的眼睛和动物的耳廓（ 图4）的方向上取得。通过根据图4中所描述的突起倾斜传感器，它有可能获得的大脑中动脉（MCA），该源自ICA和进一步划分成两个或更多分支的分辨图像，即最后环绕皮质瓣（ 图11和12）。对于MCA获得具有如在图4c和ICA的表现为显示在图4b探针倾斜的最佳可视化效果。 多普勒为基础的声学成像显示小的分支，而血电流的方向信息是彩色多普勒采集（ 图13）提供的感谢。 MCA动脉功能是由脉冲波超声技术（ 图14和图15）证实。可以被检测和分析以收集数据有关其分子的氧化状态，并计算血氧饱和度包含血红蛋白的光声信号转换成循环的红血细胞（ 图16和17）。血的氧含量可以关联到的声波数据，以确认的动脉血从静脉血判别。 通过指向枕骨孔的传感器，视觉投影在头部轴向平面（ 图9）和该成像平面上可以可变倾角进行结算。在这种情况下，视脑成像后点可以由被吴，<html>高穿透深度，因为较大的枕骨条目的。 Willis环，在脑深部特征容器的配置，可以本地化，并通过应用所有上述技术研究。基底动脉（BA），对小脑的腹侧运行，最终导致脑和对称地分叉成两个分支。在大脑腹侧这两个分支摊开，然后联合起来再次，因此建立一个环结构（威利斯圈）。此基底深圆是血管基底膜从所有中型血管发生，如后路，中部和大脑前动脉（PCA，MCA和ACA分别），即是一个庞大的血液供应的主要效应器到大脑。在彩色多普勒模式下，中型分支的标识是可行的，能够弯曲的血管段（如PCA）的清晰可视化进入威利斯（ 图18）的圈。 NT“&gt;在脑实质组织中也记录与PA的方式在枕骨凸起（ 图19），以显示在频谱图（ 图20）的血管特性。采用这种光谱可以区分动脉和静脉血管导出的信号。 <img alt="图1" src="/files/ftp_upload/52162/52162fig1highres.jpg" /> 图1：头骨椎间孔和视图像采集各点的位置的大鼠头部轮廓（a）和其中的成像换能器装置可以被放置在颞孔（紫色箭头）并置的位点，并在枕骨孔。 （黄色箭头）的轮廓（b）中 。 <img alt="图2" src="/files/ftp_upload/52162/52162fig2highres.jpg" /> 图2：动物的处置时间图像习得ñ。 （a）在该台面用于图像采集动物的布置：头剃须后，动物被放置在具有稍微倾斜的一侧上的主体上的俯卧位，以便于头部的颞侧露出。台面可可能赋予了加热装置，以保持动物的身体温暖采集过程中。一些棉辊可用于获得该位置，而粘性贴片紧固爪上的传感器，用于生命体征的监测。（二）超声凝胶的一致层覆盖在其上的换能器将在成像期间被定位在头部的面积。 <img alt="图3" src="/files/ftp_upload/52162/52162fig3highres.jpg" width="550" /> 图3：采集参数B模式成像。 （一）说明截图显示面板报告中B用于脑成像的重要采集参数-mode。（二）重要的是，发射频率被设置在低的值（16兆赫），以改善美国组织穿透。 <img alt="图4" src="/files/ftp_upload/52162/52162fig4highres.jpg" /> 图4：从颞孔横向图像采集（一）轴接合外耳到眼睛和倾斜运动（红色箭头），以改变该换能器倾斜和图像采集平面中的虚拟参考;（二）逆时针运动相通过。参考耳朵到眼睛轴与振子的位置的可变倾斜; 三）顺时针运动相对于参考耳朵到眼睛轴与振子的位置的可变倾斜。 <img alt="图5" src="/files/ftp_upload/52162/52162fig5highres.jpg" /> 图5：最优聚焦深度为美国和PA图像采集。虽然在寻找感兴趣的区域，成像焦点深度（由黄色三角形表示）必须设置在从美国/激光源深度约10毫米，以获得最佳的成像性能。 <img alt="图6" src="/files/ftp_upload/52162/52162fig6highres.jpg" width="300" /> 图6：采集参数，彩色多普勒成像模式。 （一）启动图像采集的彩色多普勒模式前，呼吸门选项可以打开，以避免生理呼吸运动产生的神器。（b）一个例证截图显示的彩色多普勒用于脑成像的重要采集参数模式。 <img alt="图7" src="/files/ftp_upload/52162/52162fig7highres.jpg" width="300" /> 图7：收购<html>参数能量多普勒模式成像。一个说明性的屏幕截图显示用于在能量多普勒模式的脑成像重要的采集参数。 <img alt="图8" src="/files/ftp_upload/52162/52162fig8highres.jpg" /> 图8：采集参数，光声成像模式。 （一）面板报告用于在光声模式脑成像重要采集参数。（二）采集一个光声光谱，基于激光的激发范围从680纳米至970纳米，以5nm的波长间隔（称为步骤大小）。（分别为750 nm和850 nm的用于单波光声模式，为去氧化和氧合血红蛋白信号判别三）采集参数。 <html> fig9highres.jpg“/&gt; 图9：从枕骨孔横向图像采集（一）对动物的脖子（黄色箭头）传感器的定位和由此产生的横向成像平面，实际上部分的脑袋上caudo，喙方向;（二）后的传感器定位的观点及图像采集平面。 <img alt="图10" src="/files/ftp_upload/52162/52162fig10highres.jpg" /> 图10：从颞孔为解剖参考文献的个性化的B-模式采集表皮（a）中 ，头骨（b）和实质（C）可以很容易分辨，但也可以检测其他解剖述，例如裂缝（四）周围的腹侧脑深部和视束（E）的特征形状。 ve_content“FO：保持together.within页=”总是“&gt; <img alt="图11" src="/files/ftp_upload/52162/52162fig11highres.jpg" /> 图11：能量多普勒模式，通过收购颞孔血管引用的个性化与ICA的大脑颞侧MCA提高。为了获得这种观点，横向图像被收购的指点传感器上时间和孔转动它反时针方向。 <img alt="图12" src="/files/ftp_upload/52162/52162fig12highres.jpg" /> 图12：能量多普勒模式，通过收购颞孔血管引用的个性化与ICA的大脑颞侧MCA提高。为了获得这种观点，横向图像被收购的指点传感器上时间和孔旋转它顺时针方向。 图13：彩色多普勒模式通过收购颞孔血管引用的个性化与ICA的大脑颞侧MCA提高。血液流的方向信息是由彩色标尺条的方式表达，朝向换能器设备，并远离其磁通动作之间进行区分。 <img alt="图14" src="/files/ftp_upload/52162/52162fig14highres.jpg" /> 图14：脉冲波模式采集通过颞孔血管引用的个性化的血液循环内该被假设识别为动脉血管的动脉性质确认：脉冲波模式提供关于流的速度的变化，它可被相关联的信息心脏搏动的影响（更多INTENSE动脉比静脉）。 <img alt="图15" src="/files/ftp_upload/52162/52162fig15highres.jpg" /> 图15：脉冲波模式，通过收购颞孔用于识别血管参考血管为静脉，其中投产速度的心脏搏动的影响可以忽略不计的个性化由脉冲波模式。 <img alt="图16" src="/files/ftp_upload/52162/52162fig16highres.jpg" /> 图16：光声模式，通过收购颞孔血管引用的个性 。实质内血管中颞脑侧由B型（左）和单波的光声模式（右）可视化。比例条的颜色反映的光声信号的不同强度值，诱发在一个所选波长进行激光激发。邻的rDer到个体化的静脉和动脉，激发波长可设定在750和850纳米，表示该值分别以获得用于脱氧和氧化血红蛋白的光声发射峰。 <img alt="图17" src="/files/ftp_upload/52162/52162fig17highres.jpg" /> 图17：光声模式，通过收购颞孔的氧化和去氧合血红蛋白的歧视 。内部容器在颞侧大脑通过B模式（左）和富氧出血天赋光声模式（右）可视化。比例尺的颜色反映血液中血红蛋白的氧饱和度不同的百分比值。 <img alt="图18" src="/files/ftp_upload/52162/52162fig18highres.jpg" /> 图18：彩色多普勒模式通过收购孔枕血管引用的个性 。弯血管段创建Willis环，位于大脑腹侧方的地下室结构。 <img alt="图19" src="/files/ftp_upload/52162/52162fig19highres.jpg" /> 图19：光声和B模式，通过收购孔枕血管引用的个性 。 Nell&#39;immagine在B型SI possono evidenziare乐strutture anatomiche individuabili骗子拉proiezione occipitaleË内拉corrispondente acquisizione骗子modalitàfotoacustica骗子rilevamento spettrale TRA 670纳米的波长980（CON步迪5纳米）。 <img alt="图20" src="/files/ftp_upload/52162/52162fig20highres.jpg" /> 图20：光声和B模式，通过收购孔枕血管引用的个性 。在奎斯塔想象viene rappresentato罗spettro corrispondeNTE全部TRE的ROI tracciate一个livello德尔parenchima cerebellare;在particolare索诺tracciate一个livello迪特雷strutture vascolari，拉翠tipologia SI differenzia一个livello dell&#39;andamento spettrale（投资回报福厦Ë蔚corrispondono一个strutture vascolari venose; ROI gialla corrisponde广告UNA STRUTTURA vascolare arteriosa）。

Watch this Scientific Journal Video about Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging at JoVE.com

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

non-invasive-parenchymal-vascular-metabolic-high-frequency-ultrasound

Research

JoVE Journal

Neuroscience

12.0K 次观看.  University of Turin. The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

视频: Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

科研

神经科学

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

非侵入性评估人类皮质神经元传播的变化

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

Isolation and Cannulation of Cerebral Parenchymal Arterioles

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves &#8212; innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

分离及脑实质小动脉插管

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

发育中大脑运动皮质的非侵入性调制和机器人映射

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

脑回路非侵入性断开的靶向神经元损伤

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

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 ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

使用经颅功能超声成像的小鼠全脑3D激活和功能连接映射

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages over conventional multi-photon calcium imaging systems: (1) compact; (2) light-weighted; (3) affordable; and (4) allows recording from freely behaving animals. This protocol describes brain surgeries for deep brain in vivo calcium imaging using a custom-developed miniscope recording system. The preparation procedure consists of three steps, including (1) stereotaxically injecting the virus at the desired brain region of a mouse brain to label a specific subgroup of neurons with genetically encoded calcium sensor; (2) implantation of gradient-index (GRIN) lens that can relay calcium image from deep brain region to the miniscope system; and (3) affixing the miniscope holder over the mouse skull where miniscope can be attached later. To perform in vivo calcium imaging, the miniscope is fastened onto the holder, and neuronal calcium images are collected along with simultaneous behavior recordings. The present surgery protocol is compatible with any commercial or custom-built single-photon and two-photon imaging systems for deep brain in vivo calcium imaging.

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Miniscope in vivo calcium imaging is a powerful technique to study neuronal dynamics and microcircuits in freely behaving mice. This protocol describes performing brain surgeries to achieve good in vivo calcium imaging using a miniscope.

立体定向病毒注射和梯度指数晶状体植入术用于深部脑 体内 钙成像

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...