Name: diffuse optical spectroscopy for the quantitative assessment of acute ionizing radiation induced skin toxicity using a mouse model
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Description: Watch this Scientific Journal Video about Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model at JoVE.com

This work was supported by research grants awarded to SKL from Abbott CARO (Canadian Association of Radiation Oncologists) Uro-Oncologic Radiation Awards and the Alan E. Tiffin Foundation. EK was supported by the Frederick Banting and Charles Best Canada Graduate Scholarship, the Scace Graduate Fellowship in Prostate Cancer Research and Paul Starita Graduate Student Fellowship.

下面的方法是按照新宁研究所动物护理伦理委员会的指导方针。 1.漫反射光谱系统<ol><li>使用先前已描述的手持式，光纤探针和便携式光谱采集系统（Kim等人 2010）收集漫反射光谱和在图1（以及相关字幕）为完整性1,2-简要回顾。 </li></ol> 2.急性放射性皮肤损伤的小鼠模型的制备<ol><li>顺序6周龄小鼠（优选无毛，如无胸腺或SKH-1），并允许他们在动物设施一​​个星期开始实验前适应环境。储备至少3的非照射对照组小鼠和5只小鼠为一照射组。 </li><li>基线DOS测量和照射前，用耳钻或永久的M标记标记老鼠尾部arkings。如果老鼠是不是裸照，胁肋皮肤2厘米补丁删除一个2厘米的头发，但是这可能会导致皮肤过敏。 </li></ol> 3.弥漫性光学光谱数据采集<ol><li>接通电源到电子设备。 </li><li>对于老鼠的皮肤，在25毫秒采集时间，25信号平均值和1棚车滤波器带宽输入设置了采集软件的信号参数。这些参数报价噪声采集时间和信号之间的合理平衡。 </li><li>使用自定义编程的采集软件，在两个源-检测器间距自动获得一个背景阅读，R BG（LED关闭）和漫反射，R MEAS（260微米，520微米），点击"获取"按钮。收购总时间大约为2秒。 </li><li>按执行测量前的房间电灯开关关掉所有荧光房间的灯。 注:Fluoresc耳鼻喉科室内光线与检测到的信号干扰（这些灯产生一个随时间变化的光强度，因而它难于减去作为背景信号）。虽然白炽灯泡，可以采用保持灯光从DOS探头的距离，以避免高本底水平（和信号差的噪声）。 </li></ol> 4.动物麻醉和基线测量DOS <ol><li>通过确保所有连接都完好，液体异氟烷水平是充足的准备麻醉机。使用麻醉诱导室带有附加管和鼻锥，可轻松抵达的DOS探头被录音下来消毒，轻声软垫的表面。 </li><li>通过用4％的异氟醚诱导持续30秒麻醉小鼠之一笼在感应腔室的时候。降低的异氟烷量至2％为下2分钟。验证鼠标是通过观察从没有捏后肢脚趾响应麻醉。 </li><li>很快移动一个鼠标到无菌的DOS探测区，将其放置在其一侧，系其吻到鼻锥和打开鼻锥管麻醉（2％异氟烷）的流动。 注:如果过程需要超过1长 - 2分钟，对眼睛适用兽医药膏，以防止干燥。 </li><li>获取小鼠皮肤测量之前，通过用70％乙醇擦拭消毒探头。不要消毒皮肤。 </li><li>轻轻把探头侧面的皮肤并确保避免局部血管分散开。握住手工探头的测量的持续时间。 </li><li>通过以下在模具上的5点形成了2厘米（被照射的区域）探测的约2厘米的侧面皮肤区域获得的反射率的数据。保持这种探测图案，面积，探针为所有后续测量一致的压力和身体侧（左或右）。 注:完整的扫描大约需要60秒。探头压力应该足以获得扫描不分散升OCAL血管。 </li><li>将鼠标移动到恢复笼，并在移动鼠标旁边的DOS探测区域。直到所有的老鼠都被测量4.6 - 重复步骤4.2。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。 </li></ol> 5.动物辐照注:此协议要求使用照射的，与动物制备可能需要调整，以满足照射设备的需要。期间照射，仅侧面皮肤的小面积应暴露于辐射束。所述照射应该位于无菌设施并返回小鼠其无菌容纳区域时，应观察到适当笼灭菌。 <ol><li>准备麻醉机（如步骤4.1 - 4.2），以等待辐照前麻醉在感应室一次一个鼠标。 </li><li>从感应室，根取下鼠标TLY捏皮肤的侧面，地点在磁带和拉伸皮肤下，形成皮瓣。 </li><li>鼠标放置到一个有机玻璃阶段并覆盖与自定义引线夹具主体（一个工作的设计是与底部的矩形框和至少一个端部开放的，具有侧窗口，以允许侧翼皮肤穿过被拉到一起）。转危为安夹具窗口皮瓣和轻轻拍打大盘搬上了舞台。 注:定制引线夹具是小到足以固定所述鼠标。如果自定义夹具并没有完全固定的小鼠，然后用额外的阻尼和/或施用氯胺酮（80 - 100毫克/千克）和赛拉嗪 - 通过腹膜内注射（10 12.5毫克/公斤），以保持固定在整个照射的小鼠程序。 </li><li>将带有夹具和鼠标进入辐照有机玻璃阶段。确定的设定（从X射线源，电压，持续时间和电流皮肤的距离），并提供所需的剂量（ 例如，从160 kVp的X-11厘米射线源2.5分钟6.3 MA）。 注意:请谨慎使用通过以下机床使用准则，以避免烧伤和DNA损伤的X射线源。 注:无胸腺裸鼠发展湿性脱皮14天左右为响应35戈瑞照射后，但与17 Gy的只有轻微的修修补补脱屑。 </li><li>就拿设备和鼠标了辐照，删除屏蔽，取出磁带，并放入一个单独的恢复笼。返回鼠标到其正常共享笼它已经从麻醉中恢复后。重复步骤5.2 - 5.4所有小鼠，并在对照组小鼠进行假手术。 </li><li>照射后，房子在他们的常规条件下的动物。如果异常行为的发展（ 例如 ，弯腰驼背的姿势，这可能意味着疼痛），请教兽医来诊断问题。缓解疼痛可包括施用为0.1mg /由兽医公斤丁丙诺啡皮下或遵医嘱。如果减肥超出正常B的20％ODY质量，房子就分别在自己的笼子里，并提供高营养的食物。 </li></ol> 6.后续DOS测量<ol><li>监控并采用定量DOS的技术，测量皮肤的反应强度。皮肤的变化和以前的工作目视检查表明，在DOS参数大的变化能约6预期（相对于基线） -以下辐照3,4 12天。然而，由于明显的变化可能发生，甚至早于或晚视型号而定，其他测量时间点可能是有用的调查。 </li><li>设置DOS设备和部分所述3.Prepare麻醉机，并获得如在第4节DOS测量校准。 </li></ol> 7.收购后处理注:使用在高性能的软件环境中创建一个自定义程序执行以下部分的所有步骤。标准化的命名修道院离子对每个光谱采集文件被用来允许批处理。所有步骤在图2中示出。 <ol><li>减去所有的测量光谱包括背景阅读基线（本底噪声）。 </li><li>减去背景阅读，R BG（LED熄灭），在步骤3.3从测量频谱，R MEAS获得。 注:本文的其余所有光谱被认为是本底噪声和本底减去称为R 科尔 。 </li><li>转换- [R 科尔绝对反射率，R ABS，如第1参考文献1,2描述。 <ol><li>获得相对反射率测量，Rrel，在脂肪乳-20％幻影（费森尤斯卡比，瑞典）随着3％等分试样馏分高达48％体模（ 即，3％，6％，9％，...，48％）和创建Rrel的情节与脂肪乳的浓度。 </li><li>产生R个绝对的对和绝对阴谋＃956; s&#39;的使用反射扩散方程14。 </li><li>同时匹配曲线的峰和调整Rrel x轴，以匹配后的R 绝对 x轴。 </li><li>在给定的波长和源 - 检测器分离，使用比例y轴: <img alt="图1" src="/files/ftp_upload/53573/53573eq2.jpg" /> 注:在下面的部分，所有测量接头将引用与R 腹肌 。 </li></ol></li></ol> 8.光谱数据拟合注:以下部分概述了用于提取小鼠皮肤的功能参数利用的理论和拟合算法。对于使用的所有理论，请参考以下文章14-18和参考文献。假定所有方程在高端科学的软件环境（含有预编程模块）在物理或工程实验室常用的编程。 <ol><li>编程描述一函数bsorption光谱，μ皮肤中的使用等式感兴趣的光谱范围有关个人的发色团的总和的一个 （λ）: <img alt="图1" src="/files/ftp_upload/53573/53573eq4.jpg" /> 这里， 高度 b是总血红蛋白浓度（g / L），而氧含量2是无单位的氧饱 ​​和度范围从0到1。 </li><li>获取氧， <img alt="图1" src="/files/ftp_upload/53573/53573eq5.jpg" />和脱氧， <img alt="图1" src="/files/ftp_upload/53573/53573eq6.jpg" />从上线收集Prahl 19的血红蛋白谱（存储为文本文件）。 </li><li>编程描述皮肤的散射光谱的函数， <img alt="图1" src="/files/ftp_upload/53573/53573eq7.jpg" /> ，使用幂律依赖，其中，A（-1）为μs&#39;的在λ0 = 1毫微米和k的值是一个介质相关的功率因数16。 </li><li>方案的基础上从基准14，纳入从步骤8.2的光谱方程方程漫反射的正向模型的数学函数- 8.3到正向模型函数（ 即 ，R（R，μ 一个 （λ），μs&#39;的 （λ ））= R（R，H b，申通快递2，A，K）。 注:虽然有各种型号，稳态扩散理论方程提供了组织上的光分布的一个简单而准确的描述。 </li><li>程序的功能是正方形的正向模拟反射光谱的从第8.4节和测量反射光谱的差异。 </li><li>迭代地改变H B，氧含量2，A，和k，直到在第最小二乘差函数8.5最小。 Matlab的lsqcurvefit可用于自动执行此步骤。 </li><li> Repea经T 8.5 - 8.6，以获得DOS参数（H B，申通快递2，A和K）用于所有测量反射率数据集。 </li><li>情节DOS参数使用每只小鼠的组3的平均对相应的唯一计量基准的相对变化 - 5规范化探针点测量。这些图是利用MATLAB的绘图命令创建的。 </li></ol> 9.视觉放射性皮炎记分周期<ol><li>监控并使用定性分级量表得分皮肤反应强度（见道格拉斯和福勒分级标准20）照射后，每48小时（一个也可以观察到的变化3 - 24小时以下的照射）。双盲研究人员的理想选择。收购了手持相机和参考尺度（即统治者）的照片可以帮助评估。 </li><li>注:照射后每两天打进皮肤可以帮助确定最佳DOS测量时间FOr是模型。更频繁的得分可能因型号和研究问题产生的重要数据。 </li><li>绘制每组的中位数在每个时间点。在特定时间点或每一个曲线下位总面积比较组。 </li><li>老鼠一直沿袭到所期望的皮肤愈合的点（ 例如 ，4周）后，安乐死通过适当的（批准）方法的老鼠。 </li></ol>

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The authors have nothing to disclose.

定量评估使用光学生物标志物辐射皮肤毒性一个DOS方式已经呈现。视觉皮肤毒性评分系统需要专业的培训，即使这样很容易发生间观察员变性和主观性。 DOS系统和分析软件使用简单，只需要很少的培训和解释在皮肤的生理变化返回目标函数的参数。此外，代替描述皮肤损伤的外观作为一个单一的参数，在DOS提供了丰富的信息在频谱形状，光学性质和功能性/微结构参数，提供灵敏度和特异性在当前定性评分方法没有可用的附加的程度。节1和7突出用于获得可用于光生物标志物的定量嵌合绝对频谱数据的主要处理步骤。背景和基线减法是至关重要的，以允许用户执行在DOS测量正常光照条件下。部分8提供了必要的模型和前和X射线照射后描述无胸​​腺小鼠所需方程。这里，合适的吸收剂的选择是对所测量的光谱的精确描述是至关重要的。这是建议用户在文献彻查主宰的波长范围，并在一个特定的研究构建一个光学生物标志物的拟合模型之前使用的兴趣组织的主要吸收。最后，第3-5节描述DOS采集过程中裸鼠的处理。为了避免破坏当地的血管，用温柔的力量来放置DOS探头小鼠皮肤表面。 虽然相比于高光谱摄像系统3,4相对便宜，所描述的DOS方式的明确限制是用来测量漫反射使用点探针。这种反射几何生活必需品与皮肤接触的温柔和有通过，如果不采用一致的探针肌肤压力脉管分散引入测量不确定度的潜力。在DOS探头的未来设计可结合有压力传感器，以保持一致的结果。此外，当使用靠近源 - 检测器间隔（&lt;2-3毫米）的允许特定于皮肤表面的光学探测深度，改进的特异性正值相比二维超光谱成像的空间分辨率的损失。为了减少这种限制，有5点扫描象限捕捉整体照射体积使用。尽管缺乏空间分辨率，在小鼠中5以前的工作已经表明光生物标志物的能力平均的稀疏区域区分不仅照射和未照射的皮肤上，同时保留皮肤介入药物如Vasculotide 6的影响。 应当指出的是，虽然整个系统的设计可以被修改为不同的皮肤<html>模型，根据光谱底层和散射形状可能需要进行优化。具体地说，在氧和脱氧血红蛋白以及描述一个无胸腺小鼠模型中，相同的模型向深色皮肤的应用可能需要增加黑色素的最佳拟合。此外，在DOS的扩展带宽更高波长&gt; 950nm的将需要添加水，其中占主导地位在更高的波长。另外，动物模型具有不同的皮肤厚度可能需要不同的源 - 检测器分离，以优化深度灵敏度。最后，毛功能使得算法简单。虽然非无毛模型可能是最佳的某些研究问题，他们将需要的DOS测量之前脱毛，以及由该方法对皮肤的刺激会影响结果。为研究，其中总的免疫功能是至关重要的，免疫活性无毛小鼠（ 例如，SKH-1）可作为一个更好的模型由于其euthymic性质。 耳鼻喉科"&gt;用于DOS探针测量的重要考虑因素是一致的RT和照射区域的估计。温度的波动会影响组织的Hb和STO 2级。每一个数据采集时间测量的一组3个非辐照的动物可以用作基线这在参数值意外环境波动可以被归一化，另外，被照射的区域可以是难以估计（如果皮瓣制剂并不一致）损伤开始表现视觉约5天（40戈瑞）之前，如果使用黑色永久标记点的辐射暴露的皮肤的界限，避免过多的油墨使用以防止墨水弄脏，这可能会危及读数。 该系统的另外一个特点是，以从散射性质分离的吸收的能力。而替代高光谱成像系统还提供监测oxyHb和Hb浓度，超光谱成像i的自由空间几何的能力<html> Ş无法解决散射的变化。此限制可能会导致在返回oxyHb，血红蛋白不准确和STO 2参数如果散射显著变化的发生是由于红斑（发红）。另外，使用DOS的散射变化的监测可以提供用于红斑评估附加的光学生物标志物。 如图6中所示，来自勒芒等人 （2014）的初步结果表明，和k证明以下不与从其他替代方法观察到的趋势，如视觉评分系统关联电离辐射的时间的趋势。这表明散射变化不会在视觉上描述的方式体现和实际上可以描述一个单独的生物过程。因此，相对于其他方法，在DOS为浅散射变化的分辨率高，对调查新颖的皮肤损伤的生物标记物可能是从通常的基于血红蛋白测量单独的途径。 jove_content"&gt;虽然我们的模型使用（即在临床上使用，而不是多个较小分馏剂量）大单辐射剂量，这模仿急性人类皮肤放射毒性21的病理生理学。可以设想，与进一步的优化，DOS可能提供对辐射引起的皮肤反应的自动化，标准化得分定量方法。掌握这一技术后，未来的应用可能包括监控保留皮肤的治疗方法之间的差异（ 例如，皮肤辐射防护控制和实验性治疗之间的比较oxyHb水平，或伤口愈合推广），而理想的动物模型中的高通量药物筛选，DOS系统可能是适应于临床环境由于容易使用性和在正常光照条件下，测量的能力，在这种情况下，探头设计可能需要小的修改用稍大光极分离占人的皮肤的厚度增加。临床DOS系统将允许介入疗法，能最大限度地减少痛苦的皮肤反应，提高患者的舒适度和遵守的在线评估。在将来，有可能是有趣的，扩大的基于DOS的量化到的慢性辐射诱导的皮肤损伤（ 例如，纤维化）的功能。

在放射治疗（RT）的规划和交付技术改进现在允许高度保形治疗剂量被递送至肿瘤区域，同时不放过正常周围的结构。然而，当高剂量目标是在靠近皮肤的急性和有时严重毒性是不可避免的。如果足够严重，由此产生的正常组织的损伤可在RT治疗效果和生命1,2病人的生存质量产生负面影响。 尽管有害后果，放射性皮肤红斑目前管理仍然是特异性的，用人忽视导致损坏底层的生物机制药膏或软膏。这些方法是基于减少的症状，而不是原因。此外，介入治疗的时间和施用通过辐射皮肤损伤评估的定性和主观性质复杂。虽然一些认可组织（RTOG，EORTC）提供可视化分级的建议，机构在选择首选的得分有所不同，从而掩盖了荟萃分析的目的正常组织的毒性比较。此外，这种分级系统是原油和容易出现跨观察员变异，使得辐射损伤的严重程度差异可能是在研究评价毒性削减战略分辨。 而不是视觉上描述照射皮肤红斑的程度，另一种方法是测量定量描述发生在器官底层生理变化的参数。血液中的血红蛋白（Hb）的，组织氧饱和度（STO 2）或氧化血红蛋白（oxyHb）水平已被用作代理对小鼠3-6射线诱导的红斑。以下的照射下，总Hb水平经受波动，但oxyHb或氧含量2经历一个特性早急剧上升，接着下跌，另一种更持续上升3,6。当刺激物是用来诱发皮肤红斑，血管oxyHb水平直接与局部红斑和炎症7的严重程度相关。 漫射光光谱（DOS）的使用近红外光，以提供对重要组织成分的生化和微观结构元件的功能的信息。这个定量的，非侵入性的光学技术提供以测量经由Hb浓度和STO 2的功能替代物红斑期间发生血管细胞因子诱导的血管扩张的方法。与对照的临床评分方法8-11比较DOS测量参数最近的研究表明该技术的潜力用于克服固有的限制，以当前分级 系统。 在这里，我们描述了一个内部​​的，可移植的，DOS系统，该系统采用功能性替代物定量DETE在辐射诱发的皮肤毒性电视机的差异在临床前小鼠模型5。所述平台可以提供与早期检测和介入药物反应的细微差异，灵敏度高标准化红斑计分的手段。此外，只有轻微的修改，该仪器可能最终在临床上用于实时床边监控使用。

<table><tbody><tr><td>Nude mice</td><td>&lt;em&gt;e.g.,&lt;/em&gt; Charles River</td><td>Athymic nude Crl:NU(NCr)-Foxn1nu, or immunocompetent nude Crl:SKH1-Hrhr</td><td></td></tr><tr><td>Small animal irradiator&amp;nbsp;</td><td>&lt;em&gt;e.g.,&lt;/em&gt; Faxitron X-Ray Corp.</td><td>Faxitron CP160</td><td></td></tr><tr><td>Animal anaesthesia&amp;nbsp;</td><td></td><td></td><td>If using isoflurane vaporizer machine with induction chamber, need tube and nose cone.</td></tr><tr><td>Lead jig and plexiglass stage</td><td>Custom made</td><td></td><td>If irradiator device exposes whole animal body to radiation, lead shielding must be used to expose only the skin flap.</td></tr><tr><td>Medical tape&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Permanent marker/ear puncher</td><td></td><td></td><td></td></tr><tr><td>Matlab</td><td>Mathworks Inc., Natick, MA</td><td></td><td>With StatisticsToolbox&amp;nbsp;</td></tr><tr><td>Labview</td><td>National Instruments, Vaudreuil-Dorian, QB</td><td></td><td></td></tr><tr><td>DOS system</td><td></td><td></td><td></td></tr><tr><td>Optical multiplexer</td><td>Ocean Optics, Dunedin, FL</td><td>Model MPM-2000</td><td></td></tr><tr><td>Spectrometer</td><td>Ocean Optics, Dunedin, FL</td><td>Model S200</td><td></td></tr><tr><td>White light source</td><td>Ocean Optics, Dunedin, FL</td><td>Model LS-1</td><td></td></tr><tr><td>Intralipid-20%</td><td>Kabi Pharmacia, New York, NY</td><td></td><td></td></tr><tr><td>Reflectance standard</td><td>INO, Quebec City, QB</td><td></td><td></td></tr></tbody></table>

diffuse optical spectroscopy for the quantitative assessment of acute ionizing radiation induced skin toxicity using a mouse model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

在DOS反射技术提供了一个客观的替代评估辐射引起的皮肤毒性的传统的定性方法。以下毒性剂量形式存在于所测量的反射光谱的大小和形状都变化辐射的视觉变化在皮肤外观。两者都与在潜在的细胞组织和生理组织状态的功能变化。在本节中，由勒芒等人2014 5从以前发表的著作代表性的结果进行了综述。 图3（左）显示代表光谱（细蓝线）在6天后40戈瑞照射皮肤红斑的裸鼠模型中的260微米源分离测量。相比预辐照（ 图3，右面板），在约550-650纳米的频谱形状差异观察到的，LIK伊利由于增加在氧合血红蛋白。在绝对反射一小的上升还可以看出，是相关的增加组织散射功率。第6天以下的辐射观测频谱相关的视觉皮肤得分0.75。 在选择波长后的照射的反射率变化的评价不使用完整的反射光谱，并且还带有噪声敏感度的潜在问题。然而，装配完整的频谱允许整个数据集被转换成直观的光学生物标志物（H B，氧含量2）。 图3示出了使用等式测得的数据的所得到的拟合（绿色实线）（薄嘈杂线）呈现在第4节优秀协议观察，证实此基础上的发色团的选择和散射形状充分说明小鼠皮肤模型。 图4示出了在皮肤的相对变化氧含量2在经照射的小鼠队列不同的时间点（6,9，12天）（N = 8），而图5示出了相应的质皮肤反应分数。在氧含量2所述的逐步增加观察相比在所有3天（P &lt;0.05）的照射前的值是统计学上不同。这些趋势反映了目视观察增加皮肤损伤程度在12天那峰（平均得分〜3）证实氧含量2的电势作为视觉得分替代（ 图5）。 应当注意的是，没有统计学显著变化被视为为任何用于返回的光生物标记在测量12天以非照射对照组（n = 3）（数据未显示）。 所述的和k的变化，也可以在一段时间（ 图6）监视，并且这些都表明皮肤的散射特性响应于辐射正在发生变化。 <img alt="图1" src="/files/ftp_upload/53573/53573fig1.jpg" /> 图1. DOS仪器。 （A）的漫反射率的测量几何的图（B）的光纤探头:该光学探针包括被捆绑到一个18G的金属针和间隔260微米相距200微米的芯光纤的线性阵列的。两个源纤维耦合到两个宽带发光二极管，而检测光纤连接到光分光计。由每个源顺序地转动，光谱仪可以在260微米的距离收集漫反射和520微米来自每个源的纤维（C）的完整DOS系统包括笔记本，附着光纤探针和视框:一个自动化数据采集程序被用来驱动光谱的顺序集合。电子被安置在连接到通过SMA连接器的光纤探针的采集盒。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53573/53573fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53573/53573fig2.jpg" /> 。 图2 光谱的处理所有的X轴尺度是在纳米（A）原料的相对光谱，基线读数大约900之间-纳米和大约等于背景信号1000（B）的相对背景读数（℃。 ）背景和基线扣除相对小号<html> pectra（D）绝对校准按照（c）所示的处理光谱缩放频谱。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53573/53573fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53573/53573fig3.jpg" /> 非辐照（左）和辐照（右）小鼠皮肤6天 图3. 典型 的 白光反射光谱照射后，测量（嘈杂蓝色）和配合（纯绿色）是典型的观察之间具有很好的一致性。两个关键的差异，看到两组:1）绝对反射整体有所上升，2）550之间的光谱形状发生明显变化 - 600纳米。与来自勒芒等人 2014年5权限。&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53573/53573fig4.jpg" /> 图4. 以下40戈瑞辐射变化小鼠皮肤的氧化部分，两组之间的基线标准化平均差（每鼠）是显著为6天（专栏1），9（专栏2）和12（专栏3 ）。从勒芒等人2014 5权限。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53573/53573fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53573/53573fig5.jpg" /> 图5.平均定性皮肤反应得分（N = 8）如下40戈瑞天的函数照射小鼠皮肤 。从勒芒等人。2014年5改编。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53573/53573fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53573/53573fig6.jpg" /> 在接下来的6天40戈瑞辐射（专栏1），9（专栏2）和12（专栏3）。在A（左侧）的变化和K（右侧） 图6.相对变化和小鼠皮肤的k个 6日（专栏1，左，右两侧）被认为是显著（p &lt;0.026）。从勒芒等人2014 5权限。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53573/53573fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model at JoVE.com

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

diffuse-optical-spectroscopy-for-quantitative-assessment-acute

Universidad San Sebastian

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JoVE Journal

Medicine

8.0K 次观看.  University of Toronto. We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

视频: Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

科研

癌症研究

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

免疫与感染

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

医学

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery.
The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

发育生物学

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

生物学

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

测量DNA损伤与修复小鼠脾细胞慢性后<em&gt;在体内</em&gt;暴露于β和γ辐射剂量很低

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Antitumor Efficacy Analysis of PDT: A Technique to Determine the Phototoxic Potential of a Photosensitizer in Tumor Bearing Mice

Source: Chang, J. E., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a> J. Vis. Exp. (2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. This video describes the in vivo anti-tumor efficacy analysis using photosensitizer containing nanoparticles in a tumor-bearing mouse, to determine the phototoxic potential of the photosensitizer.

PDT 的体内抗肿瘤疗效分析:一种确定光敏剂在荷瘤小鼠中的光毒性潜力的技术

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles. J. Vis. Exp. (2016).
Photodynamic ...

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

摘要

探索更多视频

此视频中的章节

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

4D多模态成像

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法

测量DNA损伤与修复小鼠脾细胞慢性后

PDT 的体内抗肿瘤疗效分析:一种确定光敏剂在荷瘤小鼠中的光毒性潜力的技术

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

摘要

探索更多视频

此视频中的章节

激光微辐照在哺乳动物细胞单、双链断裂修复中的应用

4D多模态成像

表皮黑色素和防止晒斑的人性化小鼠模型的诱导药物

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

在鼠耳皮肤的免疫反应的活体多光子成像诱导缺血再灌注损伤

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法

测量DNA损伤与修复小鼠脾细胞慢性后

PDT 的体内抗肿瘤疗效分析:一种确定光敏剂在荷瘤小鼠中的光毒性潜力的技术

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C）损伤的反应