Part of this work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (25350520, 22500401, 15K06105) and the US-ARMY ITC-PAC Research and Development Project (FA5209-15-P-0175).

动物保健，准备和实验方案是由东京农工大学的动物研究委员会的批准。对于这种方法，将大鼠圈养在受控环境中（24℃，12小时光照/黑暗周期），与食物和水可随意获得。 1.传统多光谱漫反射成像系统的构建<ol><li>摩9个的窄带光学干涉滤光器具有的中心波长500,520，540，560，570，580，600，730，和760纳米至机动滤光轮的过滤孔。 </li><li>构建使用宽带白色光源，机动滤光轮与上述组窄带干涉滤光片，光导，聚光透镜，视频变焦透镜，和一个单色CCD照相机的多光谱成像系统。光学元件，在图 2中所示的布局，可以被称为用于第是施工程序。 注:照明的角度是大约45°相对于样品表面。 </li><li>转动卤素灯光源经由一个干涉滤光器，光导，和聚光透镜来照射样品的表面。 </li><li>打开CCD相机的操作软件。 </li></ol> 2.动物的制备注:在这个协议中，并没有用于未来的实验大鼠和它多光谱图像的测量后，立即处死。 <ol><li>感应腔室的入口端口连接到麻醉机的与管出口端口。感应腔室的出口端口连接到麻醉机的进气口与第二管。 </li><li>放置老鼠进入吸气室和诱导具有5.0％异氟烷麻醉。在深度，使得老鼠并没有脚趾捏回应维持麻醉。大号奥尔〜2.0％异氟烷使用麻醉机上的旋钮。 </li><li>在立体定位框架固定所述大鼠头部。附上麻醉喉舌的立体框架。 </li><li>吸嘴的吸入口连接到麻醉机用管出口端口。吸嘴的出口端口连接到麻醉机用管入口端口。 </li><li>剃光头区域使用外推剪，直到出现在皮肤表面的预期切口部位。 </li><li>使形成纵向切口长大约20mm沿着所述头部的使用外科手术刀中线（ 图1（a））和暴露的皮下结缔组织（ 图1（b））。 </li><li>使用锋利刮匙或钳子去除皮下结缔组织并将其拉向头部两侧，以暴露颅骨（ 图1（c））。 </li><li>挖颅骨椭圆形沟颅sutur内es（冠状缝合，矢状缝合和羊角缝合）使用高速钻（ 图1（d） ）。 </li><li>使用高速钻机慢慢均匀地挖掘沟内的颅骨。 </li><li>用钳子的尖端轻轻地压在稀疏头骨的表面上，以估计脑血管出现后的骨厚度和强度。如果变薄的颅骨区域容易下压，则用高速钻机终止颅骨骨折。 </li><li>使用钳子或小手术剪刀切割瘦骨头碎片的椭圆形边界线。 </li><li>从脑表面缓慢地轻轻地使用钳子去除稀疏的头骨。 </li><li>用生理盐水轻轻沐浴颅窗，并用约0.1毫米厚的透明玻璃板覆盖。 </li></ol> 3.调节启发氧的分数注意:呼吸系统灰可以通过调节吸入氧的（ 吸入氧浓度）分数来改变。 <ol><li>使用管，一个Y形管连接器（连接器1）的第一端口连接到另一个Y形管连接器（连接器2）的第一端口。 </li><li>吸嘴的吸入口连接到管连接器1的第二端口。 </li><li>使用的管，管连接器1的第三端口连接到一个氧浓度监视器装置。 </li><li>用管，管连接器2的第二端口连接到麻醉机的出口。 </li><li>使用的管，管连接器2的第三端口连接到一个气体混合物装置的出口端口。 </li><li>气体混合物装置的一个进气口连接到一个高压95％O 2 - 5％使用管的CO 2气体圆柱体。 </li><li>气体混合物的设备的另一入口端口连接到一个高压95％N 2 -使用管5％CO 2气瓶。 </li><li>改变气体的流速Ø˚FO 2和N 2使用气体混合物的设备上的旋钮。 </li><li>检查并使用氧浓度监视器装置调节吸入氧浓度。 </li></ol> 4.多光谱漫反射图像采集<ol><li> 参考图像采集 注意:在这个实验中使用的光学部件，例如作为光源，光纤，和检测器有自己的光谱特性。因此，光的通过这些部件通过了强度应记录为参考图像。参考图像是用来自光源的光照亮的标准白色扩散器所拍摄的图像。 <ol><li>放标准白色扩散板在舞台上水平。 </li><li>通过在桶旋转变焦环聚焦白色扩散器的表面上的相机镜头。 </li><li>通过选择适当的值来调整摄像头的积分时间下拉的积分时间列表中相机的操作软件，使最大的光量产生的信号是最大计数的约75％。一边看像素值的直方图，调整积分时间，直到信号强度电平是最大计数的约75％。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。 </li><li>通过旋转滤光轮改变滤波器位置。 </li><li>根据上述方法在其它波长保存的图像。文件名应识别样品和使用的波长（ 例如，W500，W520，W540 W760 ...）。 </li></ol></li><li> 样本图像采集 注意:在9个波长暴露的大鼠脑的漫反射光强度的图像被捕获并保存使用相同的采集条件的个人计算机的硬盘驱动器上。 <ol><li>轻轻地放在将R在舞台上，慢慢调整阶段水平，使相机可以对焦的大鼠脑的表面上。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。 </li><li>通过旋转滤光轮改变滤波器位置。 </li><li>根据上述方法在其它波长保存的图像。文件名应识别样品和波长（ 如，R500，R520，R540 R760 ...）。 </li></ol></li><li> 深色图像采集 注:CCD照相机可以响应于电信号产生的光强度。然而，有一些小的输出由于在电气电路和检测器噪声，即使光不进入到检测器;这就是所谓的暗电流噪声。为了精确测量光的光谱强度时，暗电流成分应记录为暗图像，然后从所测量的信号中减去。黑暗的图像是取n，其中的光路阻断。 <ol><li>关闭卤素灯光源。 </li><li>阻挡光路，以利用屏蔽板的CCD摄像系统。 </li><li>从文件菜单中的"保存"命令，将图像保存到文件中。文件名应确定，样品（ 例如，暗）。 </li></ol></li></ol> 5.可视化血红蛋白含量和光散射参数注:一组多光谱漫反射的图像保存到个人电脑的硬盘驱动器和离线分析。通过在9个波长（500，520，540，560，570，580，600，730，和760纳米）的多光谱漫反射图像的蒙特卡罗模拟19辅助的多回归分析然后进行以可视化的二维氧化血红蛋白浓度，脱氧血红蛋白浓度，总血红蛋白浓度，局部脑氧饱和度，和SCAT的地图的TeringBay动力。具体算法已发表在文献17，18。 <ol><li>来自两个参考图像，并在每个波长处的样本图像中减去暗图像。 </li><li>在每个波长λ归一化由基准图像的样本图像。治疗的归一化的图像作为漫反射率图像R。 </li><li>通过取在每个波长λ的漫反射率图像R的倒数的对数计算的吸光度（或光密度）图像A: <img alt="式（1）" src="/files/ftp_upload/55399/55399eq1.jpg" /> （1） </li><li> y平面示出了用于在脑表面所获得的结构信息，而z -轴示出光谱信息-通过在它们的波长的顺序，其中x堆叠的吸光度图像生成的三维矩阵。 </li><li> PErform在每个xy坐标的吸收光谱A（λ）多元回归分析。 </li><li>使用的吸收光谱A（λ）作为因变量和氧合血红蛋白的摩尔消光系数光谱εHbO2的 （λ）和脱氧血红蛋白εHBR（λ）作为用于步骤5.5的独立变量（公布值εHbO2的 （λ）和εHBR（λ）在表1中提供）。 </li><li>检查三个多重回归的二维地图（图像）系数a HBO， 一个HBr和一个 0。 </li><li>通过堆叠多重回归系数的图像的次序的HBO， 一个HBr和一个 0，生成的三维矩阵，其中该<e米&gt; X - y平面示出了用于在脑表面，而z轴所获得的结构信息示出了多重回归系数。 </li><li>计算氧合血红蛋白浓度C HBO，脱氧血红蛋白浓度C HBr和从该组的多个回归系数a HBO， 一个HBr和 0在每个xy坐标使用以下经验式（他值β的散射电压B HBO，I，βHBR，i和β0，I（I = 0,1,2,3）在表2中提供）: <img alt="公式2" src="/files/ftp_upload/55399/55399eq2.jpg" /> （2） <img alt="公式3" src="/files/ftp_upload/55399/55399eq3.jpg" /> （3） <img alt="公式4" src="/files/ftp_upload/55399/55399eq4.jpg" /> （4） </li><li>检查氧化血红蛋白浓度C HBO，脱氧血红蛋白浓度C HBr和散射功率B的二维地图（图像）。 </li><li>计算由求和ÇHbO2的和C HBR在每个x总血红蛋白浓度C HBT的二维地图- y坐标。 </li><li> y坐标-通过在每个x除以氧化血红蛋白浓度C HbO2的由总血红蛋白浓度C HBT计算局部脑氧饱和度RSO 2的二维地图。 </li></ol>

<ol>
	<li>Bonhoeffer, T., Grinvald, A., Toga, A. W., Mazziotta, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Imaging+based+on+intrinsic+signals:+the+methodology.">Optical Imaging based on intrinsic signals: the methodology.</a> Brain mapping; the methods. , 55-97 (1996).</li><li>Mourant, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+light+scattering+from+biological+cells+relevant+to+noninvasive+optical-tissue+diagnostics.">Mechanisms of light scattering from biological cells relevant to noninvasive optical-tissue diagnostics.</a> Appl. Opt. 37 (16), 3586-3593 (1998).</li><li>Abookasis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cortical+absorption,+scattering,+and+hemodynamic+response+during+ischemic+stroke+using+spatially+modulated+near-infrared+illumination.">Imaging cortical absorption, scattering, and hemodynamic response during ischemic stroke using spatially modulated near-infrared illumination.</a> J. Biomed. Opt. 14 (2), 024033 (2009).</li><li>Lipton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+cell+death+in+brain+neuron.">Ischemic cell death in brain neuron.</a> Physiol. Rev. 79 (4), 1432-1568 (1999).</li><li>Jarvis, C. R., Anderson, T. R., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoxic+depolarization+mediates+acute+damage+independent+of+glutamate+in+neocortical+brain+slices.">Anoxic depolarization mediates acute damage independent of glutamate in neocortical brain slices.</a> Cereb. Cortex. 11 (3), 249-259 (2001).</li><li>Joshi, I., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+anoxic+depolarization+during+ischemia-like+conditions+in+the+mouse+hemi-brain+slice.">Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain slice.</a> J. Neurophysiol. 85 (1), 414-424 (2001).</li><li>Polischuk, T. M., Jarvis, C. R., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+optical+signaling+denoting+neuronal+damage+in+response+to+acute+excitotoxic+insult+in+the+hippocampal+slice.">Intrinsic optical signaling denoting neuronal damage in response to acute excitotoxic insult in the hippocampal slice.</a> Neurobiol. Dis. 4 (6), 423-437 (1998).</li><li>Jarvis, C. R., Lilge, L., Vipond, G. J., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+intrinsic+optical+signals+and+calcein+fluorescence+during+acute+excitotoxic+insult+in+the+hippocampal+slice.">Interpretation of intrinsic optical signals and calcein fluorescence during acute excitotoxic insult in the hippocampal slice.</a> NeuroImage. 10 (4), 357-372 (1999).</li><li>Obeidat, A. S., Jarvis, C. R., Andrew, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+does+not+mediate+acute+neuronal+damage+after+spreading+depression+induced+by+O2/glucose+deprivation+in+the+hippocampal+slice.">Glutamate does not mediate acute neuronal damage after spreading depression induced by O2/glucose deprivation in the hippocampal slice.</a> J. Cereb. Blood Flow Metab. 20 (2), 412-422 (2000).</li><li>Dunn, A. K., Devor, A., Dale, A. M., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+extent+of+oxygen+metabolism+and+hemodynamic+changes+during+functional+activation+of+the+rat+somatosensory+cortex.">Spatial extent of oxygen metabolism and hemodynamic changes during functional activation of the rat somatosensory cortex.</a> Neuroimage. 27 (2), 279-290 (2005).</li><li>Jones, M., Berwick, J., Johnston, D., Mayhew, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+optical+imaging+spectroscopy+and+laser-Doppler+flowmetry:+the+relationship+between+blood+flow,+oxygenation,+and+volume+in+rodent+barrel+cortex.">Concurrent optical imaging spectroscopy and laser-Doppler flowmetry: the relationship between blood flow, oxygenation, and volume in rodent barrel cortex.</a> Neuroimage. 13 (6), 1002-1015 (2001).</li><li>Jones, M., Berwick, J., Mayhew, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+blood+flow,+oxygenation,+and+volume+following+extended+stimulation+of+rodent+barrel+cortex.">Changes in blood flow, oxygenation, and volume following extended stimulation of rodent barrel cortex.</a> Neuroimage. 15 (3), 474-487 (2002).</li><li>Zhou, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffuse+optical+correlation+tomography+of+cerebral+blood+flow+during+cortical+spreading+depression+in+rat+brain.">Diffuse optical correlation tomography of cerebral blood flow during cortical spreading depression in rat brain.</a> Opt. Express. 14 (3), 1125-1144 (2006).</li><li>Bouchard, M. B., Chen, B. R., Burgess, S. A., Hillman, E. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+multispectral+optical+imaging+of+cortical+oxygenation,+blood+flow,+and+intracellular+calcium+dynamics.">Ultra-fast multispectral optical imaging of cortical oxygenation, blood flow, and intracellular calcium dynamics.</a> Opt. Express. 17 (18), 15670-15678 (2009).</li><li>Jones, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+multispectral+reflectance+imaging+and+laser+speckle+flowmetry+of+cerebral+blood+flow+and+oxygen+metabolism+in+focal+cerebral+ischemia.">Simultaneous multispectral reflectance imaging and laser speckle flowmetry of cerebral blood flow and oxygen metabolism in focal cerebral ischemia.</a> J. Biomed. Opt. 13 (4), 044007 (2008).</li><li>Kawauchi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffuse+light+reflectance+signals+as+potential+indicators+of+loss+of+viability+in+brain+tissue+due+to+hypoxia:+charge-coupled-device-based+imaging+and+fiber-based+measurement.">Diffuse light reflectance signals as potential indicators of loss of viability in brain tissue due to hypoxia: charge-coupled-device-based imaging and fiber-based measurement.</a> J. Biomed. Opt. 18 (1), 015003 (2013).</li><li>Yoshida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multispectral+imaging+of+absorption+and+scattering+properties+of+in+vivo+exposed+rat+brain+using+a+digital+red-green-blue+camera.">Multispectral imaging of absorption and scattering properties of in vivo exposed rat brain using a digital red-green-blue camera.</a> J. Biomed. Opt. 20 (5), 051026 (2015).</li><li>Nishidate, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Cerebral+Hemodynamics+and+Tissue+Morphology+of+In+Vivo+Rat+Brain+Using+Spectral+Diffuse+Reflectance+Imaging.">Evaluation of Cerebral Hemodynamics and Tissue Morphology of In Vivo Rat Brain Using Spectral Diffuse Reflectance Imaging.</a> Appl. Spectrosc. , (2016).</li><li>Wang, L. -. H., Jacques, S. L., Zheng, L. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MCML-Monte+Carlo+modeling+of+photon+transport+in+multi-layered+tissues.">MCML-Monte Carlo modeling of photon transport in multi-layered tissues.</a> Comput. Methods Programs Biomed. 47 (2), 131-146 (1995).</li><li>Tsytsarev, V., Premachandra, K., Takeshita, D., Bahar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cortical+electrical+stimulation+in+vivo:+Fast+intrinsic+optical+signal+versus+voltage-sensitive+dyes.">Imaging cortical electrical stimulation in vivo: Fast intrinsic optical signal versus voltage-sensitive dyes.</a> Opt. Lett. 33 (9), 1032-1034 (2008).</li><li>Arnold, T., Biasio, M. D., Leitner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyper-spectral+video+endoscope+for+intra-surgery+tissue+classification+using+auto-fluorescence+and+reflectance+spectroscopy.">Hyper-spectral video endoscope for intra-surgery tissue classification using auto-fluorescence and reflectance spectroscopy.</a> Proc. SPIE. 8087, 808711 (2011).</li><li>Basiri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+multi-spectral+camera+in+the+characterization+of+skin+wounds.">Use of a multi-spectral camera in the characterization of skin wounds.</a> Opt. Express. 18 (4), 3244-3257 (2010).</li><li>Nishidate, I., Maeda, T., Niizeki, K., Aizu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+melanin+and+hemoglobin+using+spectral+reflectance+images+reconstructed+from+a+digital+RGB+image+by+the+Wiener+estimation+method.">Estimation of melanin and hemoglobin using spectral reflectance images reconstructed from a digital RGB image by the Wiener estimation method.</a> Sensors. 13 (6), 7902-7915 (2013).</li></ol>

The authors have nothing to disclose.

在这个协议中最关键的步骤是去除减薄头骨区域，使颅窗口的;这应该小心进行，以避免意外出血。这个步骤是为了获得高品质的多光谱扩散高精度反射图像重要。建议手术过程如果可能的话，使用立体显微镜。明胶海绵小块止血有用。 这篇文章中描述的光学系统穿过位于所述光源的前面的干涉滤光器的单色的光。这可以通过将所述滤光轮在摄像机镜头或CCD摄像机的前面进行?...

多光谱漫反射成像是用于获得在皮质组织固有的光学信号（IOSS）的空间图的最常用的技术。 IOSS在体内脑电波中所见的主要归因于三个现象:在光吸收的变化和由于皮质血流动力学散射特性，这取决于降低或线粒体细胞色素氧化在吸收变化和变型中通过形态学改变诱导的光散射特性1。 在可见光（VIS）到近红外（NIR）的光谱范围内的光被有效地吸收，并通过生物组织散射。 体内脑的扩散反射光谱的特征在于，吸收和散射光谱。减小的散射系数μ在VIS到NIR波长范围内的结果的脑组织的S&#39;以单调散射光谱表现出在较长波长荷兰国际集团较小幅度。减小的散射系数μ频谱S"（λ）可以近似为在幂律函数2，3的形式为μS"（λ）=α×λ-b。散射能力b为在活组织2,3与生物散射体的大小。组织和减少活皮层组织的生存能力的形态改变可影响生物散射体4，5，6，7，8，9的大小。 多光谱漫反射成像光学系统，可以从白炽灯里容易地构建GHT源，简单的光学部件，和一个单色电荷耦合器件（CCD）。因此，各种算法和多光谱漫反射成像光学系统已被用来评估皮质血流动力学和/或组织形态10，11，12，13，14，15，16，17，18。 这篇文章中描述的方法，被用于可视化两者血液动力学和体内使用常规多光谱漫反射成像系统的大鼠脑组织的光散射性质。这种方法比其它技术的优点是，以评估在两个脑血流和皮层组织时空变化的能力形态，以及它适用于各种脑功能障碍的动物模型。因此，该方法将是适当的创伤性脑损伤，癫痫发作，中风和缺血调查。

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			<td height="36" style="height:36px;width:289px;">150-W halogen-lamp light source</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td style="width:119px;">LA-150SAE</td>
			<td style="width:167px;"></td>
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			<td height="36" style="height:36px;">Light guide</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>LGC1-5L1000</td>
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			<td height="36" style="height:36px;">Collecting lens</td>
			<td style="width:281px;">Hayashi Watch Works Co., Ltd, Tokyo, Japan</td>
			<td>SH-F16</td>
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			<td height="21" style="height:21px;">Interference filters l@500nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
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			<td height="21" style="height:21px;">Interference filters l@540nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65096</td>
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			<td height="21" style="height:21px;">Interference filters l@560nm</td>
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			<td height="21" style="height:21px;">Interference filters l@570nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
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			<td height="21" style="height:21px;">Interference filters l@580nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#65646</td>
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			<td height="21" style="height:21px;">Interference filters l@600nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
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			<td height="21" style="height:21px;">Interference filters l@730nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
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			<td height="21" style="height:21px;">Interference filters l@760nm</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>#67777</td>
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			<td height="21" style="height:21px;">Motorized filter wheel&#160;</td>
			<td>Andover Corporation, NH, USA</td>
			<td>FW-MOT-12.5</td>
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			<td height="21" style="height:21px;">16-bit cooled CCD camera</td>
			<td>Bitran, Japan</td>
			<td>BS-40</td>
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			<td height="22" style="height:22px;">Video zoom lens</td>
			<td>Edmund Optics Japan Ltd, Tokyo, Japan</td>
			<td>VZMTM300i</td>
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			<td height="36" style="height:36px;width:289px;">Spectralon white standard with 99% diffuse reflectance</td>
			<td>Labsphere Incorporated, North Sutton, NH, USA</td>
			<td>SRS-99-020</td>
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simultaneous evaluation of cerebral hemodynamics and light scattering properties of the in vivo rat brain using multispectral diffuse reflectance imaging

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

从体内大鼠脑获取的漫反射的代表性光谱图像示于图3中的图像，在500，520，540，560，570和580nm处清楚地看到血管的密集网络在大脑皮层。的血管，在600，730中的图像中观察到的周围组织，和760纳米之间的对比度恶化反射光中的时间更长，NIR波长下吸收血红蛋白。 图4示出了暴露的大鼠脑的用于?...

Watch this Scientific Journal Video about Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging at J

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

脑血流和体内大鼠脑组织的光散射特性的同时评估用常规多光谱漫反射成像系统证明。

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

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

simultaneous-evaluation-cerebral-hemodynamics-light-scattering

Research

JoVE Journal

Neuroscience

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

7.6K 次观看.  Tokyo University of Agriculture & Technology. 脑血流和体内大鼠脑组织的光散射特性的同时评估用常规多光谱漫反射成像系统证明。 

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

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 ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Evaluation of Bioenergetic Function in Cerebral Vascular Endothelial Cells

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types that comprise the BBB; these cells have a very high-energy demand, which requires optimal mitochondrial function. In the case of disease or injury, the mitochondrial function in these cells can be altered, resulting in disease or&#160;the opening of the BBB. In this manuscript, we introduce a method to measure mitochondrial function in CVE cells by using whole, intact cells and a bioanalyzer. A mito-stress assay is used to challenge the cells that have been perturbed, either physically or chemically, and evaluate their bioenergetic function. Additionally, this method also provides a useful way to screen new therapeutics that have direct effects on mitochondrial function. We have optimized the cell density necessary to yield oxygen consumption rates that allow for the calculation of a variety of mitochondrial parameters, including ATP production, maximal respiration, and spare capacity. We also show the sensitivity of the assay by demonstrating that the introduction of the&#160;microRNA, miR-34a, leads to a pronounced and detectable decrease in mitochondrial activity. While the data shown in this paper is optimized for the bEnd.3 cell line, we have also optimized the protocol for primary CVE cells, further suggesting the utility in preclinical and clinical models.

Endothelial cell mitochondria are critical to maintain blood-brain-barrier integrity. We introduce a protocol to measure bioenergetic function in cerebral vascular endothelial cells.

在脑血管内皮细胞生物能量功能的评价

The integrity of the blood-brain-barrier (BBB) is critical to prevent brain injury. Cerebral vascular endothelial (CVE) cells are one of the cell types ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

生活成像的脑皮层细胞使用二维培养系统

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

漫反射光谱分析: 在拇指下进行毛细管填充试验

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

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

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

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

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

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