German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

使用人体组织，必须审查和机构审查委员会或等效的批准。这里所描述的工作被批准由当地伦理委员会批准了科学检查。这项工作是根据赫尔辛基宣言进行。人类巩膜标本采自世界各地的捐助者的眼中（最大验尸时间24小时），在科隆大学眼库眼科部，德国获得。 1.实验准备<ol><li>制备96％的乙醇，并在不同的管中的磷酸缓冲盐水（PBS）。制备在含有2％牛血清白蛋白（BSA）的PBS的推荐稀释的第一抗体，并保持在冰上的所有解决方案。准备每个样品100微升。 </li><li>清洁所有乐器和用锋利的手术刀。重复相同的手术刀切割后，改变手术刀。 </li><li>获得1-2 COLIBRI钳，直微解剖剪刀，＃10手术刀或眼科手术刀微羽毛，和四＆G针。 </li><li>包裹铝箔周围聚苯乙烯板或使用软木板上贴上组织。一个双目立体显微镜下工作。如果需要一个层流下工作无菌。 </li></ol> 2.准备巩膜<ol><li>轻轻握住灯泡和角巩膜部分进行打孔环锯术，然后旋转环锯。仔细而均匀地旋转环锯在表面上进行同样切割的圆形。使用15.5毫米大小的环钻。与切弯剪剩下的附件。取出角巩膜环锯术。这将导致一个向前打开灯泡。 <ol><li>把巩膜与开放的部分向上棉签。取出视网膜和葡萄膜，使用镊子COLIBRI拉断从巩膜两个层（视网膜和色素膜）。不要离开在内巩膜色素葡萄膜。用弯剪去除乳头视网膜和葡萄膜。删除REM癌宁结膜，眼外肌，和从表面的巩膜榫的胶囊。 </li></ol></li><li>通过使用直型剪刀和镊子COLIBRI中取出所需要的尺寸巩膜样本。取2厘米左右从不同的地点2大小的巩膜样本。由于规模小，轻轻握住巩膜切出所需的尺寸采用了直板型剪刀广场。角巩膜环钻限定巩膜样品的前缘。 <ol><li>避免与镊子为那里抢到的组织是不适合激光共聚焦显微镜分析区域反复抓。制备所需数目取决于实验的类型的样品（ 例如 ，前方与后方，比较图1）和抗体的使用量。 </li></ol></li><li>把样品在1.5ml管中供以后使用。固定在1.5ml 96％乙醇的样品15分钟，然后把它们洗干净三次，每次5分钟在摇床1.5毫升PBS中。如果冷冻组织合作，进行快速冷冻之前这一步。 </li><li>使用新鲜的组织。但是，如果这是不可能的，卡扣冷冻在液氮巩膜并保持在-20℃以备后用。如果冷冻组织，使用前解冻工作。避免反复冻融循环。 </li><li>保持在PBS中含有1.5毫升5％的BSA在室温下2小时各样品。此步骤导致这对层叠有帮助的样品的膨胀，也可以防止非特异性结合。 </li><li>样品已经膨胀后，准备巩膜广场层压。显微镜， 例如 ，一个双目立体显微镜下工作。贴上后巩膜样本以聚苯乙烯膜包裹在铝箔或使用＆G针状软木板上。 </li><li>弯针的边缘，使它们不与在显微镜下的视野干扰。 </li><li>层压全层巩膜SAMPLES。 <ol><li>首先，使用抱钳COLIBRI前巩膜边缘。然后小心地切开使用＃10解剖刀，所述眼科手术刀微羽毛，或刮刀刀片浅表巩膜的薄层。水平握住手术刀和底层层薄薄地切表面层。 </li><li>从解剖巩膜广场薄薄一层巩膜。这种方法是比得上标准外科手术技术来小梁切除术过程中制备的翼片和应导致30-80微米厚的层（比较图2）。 </li></ol></li><li>通过， 例如 ，50微升PBS中使用移液管加入少量的PBS于组织防止巩膜的干燥。 </li><li>把切层为100微升PBS在96孔板和标签两个方向（向extern和实习生）和层（肤浅和深刻的）， 例如具有防水颜色。 </li><li>重复步骤2.7-2.9，直到组织充分地被碾压。&lt;/ LI&gt; </ol> 3.进行免疫组化<ol><li>加入100μl所需初级抗体的进入推荐稀释。这里， 例如使用光盘31（单克隆小鼠抗人）和LYVE1抗体（兔抗人），二者以1:1的稀释度:100在PBS（含有2％BSA）并在4℃下孵育过夜。来改变在96孔板的培养基，保持吸管到孔的壁上并取出流体。 </li><li>第二天，洗涤样品三次，每次5分钟，用200-300微升的PBS上的振动筛。添加100微升相应的次级抗体;这里使用的山羊抗小鼠异硫氰酸荧光素（FITC）和山羊抗兔花青染料3（Cy3标记），并孵育在室温下将样品1-2小时。稀释二级抗体1:300在含有2％山羊血清的PBS中。 </li><li>再次冲洗样本三次，每次在200-300微升PBS中的5分钟的振动筛。 </li><li>进行核染色， 如: </EM&gt;。 4&#39;，6-二脒基-2-苯基吲哚（DAPI）（稀释1:2000，在每100微升孔孵育在室温下10分钟），然后再洗涤2次在200-300微升PBS中在摇动器。 </li><li>转移样品到显微镜载玻片，将它们嵌入在荧光封固剂1-2滴，加盖玻片，由覆盖有透明清漆的边缘密封它，并储存在4℃以备后用或者直接检查用共焦的幻灯片显微镜。 </li><li>使用共聚焦显微镜（或等同物）来分析样品。使用所需的放大倍率， 例如 10-40X的放大倍率。 </li><li>为了验证样品的厚度，测量用的Z堆叠共聚焦显微镜。可能的巩膜部分的量取决​​于巩膜的位置，随着厚度的不同之平展和向后（比较介绍）。 </li></ol>

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

层压人类巩膜是这个组织的执行共聚焦显微镜的方法。在这个过程中的一个关键步骤是使用乙醇代替福尔马林用于固定组织。根据我们的经验，使用乙醇代替福尔马林固定时获得更好的结果。钝解剖刀加剧过程，应避免使用。同样地，应避免巩膜的干涸，因为它的复杂性的方法，降低了图像的质量。 对于免疫组织化学染色，可以使用比这里施加的那些其他抗体。在一般情况?...

巩膜是覆盖眼，它是由致密结缔组织的刚性外层。它有助于保护眼内的结构和保持眼压。因此，巩膜是明确的目标至关重要。它不含淋巴管1,2，从而形成它与自由淋巴-内眼3-7之间的外自由淋巴边界。它也提供了眼外肌附着位点，从而与筋共享解剖相似之处。 因为巩膜主要由I型胶原的致密束和具有型胶原III，IV，V，VI，VIII族 8,9和弹性10,11的较小的数字，这个组织是不容易使用用于免疫组织化学。 解剖学，巩膜可以分为三个主要层:（1）浅血管巩膜外层，实测结膜和眼球筋膜囊下方并朝向侧面和第面对轨道眼睛E回; （2）巩膜间质，巩膜的主要部分;及（3）的薄层fusca的，这是直接位于葡萄膜上方的薄的着色层。我们对巩膜解剖知识主要来源于20 世纪上半叶。在那个时候，研究人员研究了血管的解剖结构主要是利用墨汁注射12和血管铸造13-15。后来，它血管造影研究16-19进行了研究。 自那时以来，旧的技术进行了改进，新的问题被开发出来，使我们能够补充先前的解剖知识。例如，它仅仅是约十年，因为我们有过这样可靠淋巴标记淋巴血管内皮特定透明质酸受体1（LYVE1）20或podoplanin 21。共聚焦显微镜提供了新的可能性，研究不同的TI的解剖学特征眼睛的ssues。它允许将用于分化细胞的标记物或用于细胞有关的血管和其它解剖结构的本地化多个污渍。它提供了一个概述当样品是尺寸较大的，使我们能够通过样品扫描中搜索一个特定的细胞类型的时。同的Z-Stack技术，共聚焦显微镜可以用于样品达100-200微米。巩膜的不同之处的肌肉插入后面0.3毫米和在后极11 1毫米的厚度。由于其厚度和不透明度两者，巩膜不适合用传统的方法共聚焦显微镜。 为了解决这个问题，巩膜组织进行层叠，以允许他们的共聚焦显微镜分析。这项技术是在人类巩膜更好地了解双方的生理和病理情况下非常有用。

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

巩膜是致密结缔组织覆盖和保护眼睛。它主要由致密的胶原束（I型，III，IV，V，VI和VII）的。由于它的自身荧光，不透明，和厚度，它没有被发现适合于共焦显微镜。另一种方法的一个在这里提出，其使用石蜡包埋用于免疫组织化学福尔马林固定巩膜，具有技术挑战，预热抗原修复组织时尤其如此。由于巩膜在两个细胞和血管相对较差，使用较大的组织样本进行了探讨，以帮助防止俯瞰细胞，并了解他们有关的船只及其他解剖部位的定位。以允许共焦显微镜下放大的组织样本的分析，进行层压技术来创建从巩膜薄层。继CD31血管和淋巴管内皮hyalu的结果分析罗南受体1（LYVE1）阳性细胞，获得针对该批准科学检查，该方法的优点和局限性进行了讨论。

在这里进行的实验的代表，也有因使用这种特殊层压技术的衍生论证好处。在第一个实验中示出的巩膜外血管丛的三种代表性的照片（ 图3）的不同的网络。这些船只是积极为CD31。 第二个实验中显示了免疫细胞，在巩膜和它们对CD31阳性血管关系的特定LYVE1 +细胞。这里是在10X放大率用的Z-Stack技术穿过组织进行扫描...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

使用层压技术来执行人类巩膜共聚焦显微镜


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K 次观看.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

视频: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

角膜共聚焦显微镜:一种新型的非侵入性技术来量化小纤维病变周围神经病变

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

科研

医学

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

利用经颅磁刺激，以研究人类的神经肌肉系统

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

基于探测器的激光共聚焦激光显微内镜对泌尿道的技术

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

蒸汽腔体建设用于公开小鼠酒精在所有三个学期人类发展的等效

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;:单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

一个算法的开发与高位脊髓损伤使用遥测装置进行自主神经异常反射的动物的综合研究

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

气道局部麻醉清醒用于内镜插管使用喷雾为你去技术在高氧流量

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

使用层压技术来执行人类巩膜共聚焦显微镜

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