作者非常感谢达莉亚塞拉女士和EMI沙龙先生在实验室中他们的专业和优秀的技术工作。

动物按照关于在研究中使用的动物的ARVO分辨率处理。该研究方案经实验动物研究机构委员会（批准号022-4598-2; 021211）。 1.准备手术＃1 <ol><li>权衡和麻醉13日龄新西兰白兔用盐酸氯胺酮100毫克/毫升（100毫克/千克）和盐酸赛拉嗪25毫克/毫升（12.5毫克/千克）的肌肉注射。这些剂量使用，由于手术过程的长度。由于缺乏疼痛反射保证麻醉的适当水平。 </li><li>使用无菌棉签，申请眼药膏的少量非操作眼以防止角膜干燥。在操作过程中应用0.9％生理盐水滴在操作眼睛角膜，以防止角膜干燥。 </li><li>在局部麻醉后每只眼睛执行三个轴向长度测量（oxybuprocain使用超声A-扫描仪Ë盐酸盐0.4％），然后平均的测量。垂直应用探头角膜中央。 </li></ol> 2.手术＃1 - 预交联步骤<ol><li>使用眼科手术镊子和剪刀眼科手术显微镜下，进行手术的眼科镊子和剪刀360°结膜peritomy。 </li><li>使用有角度的钳或肌肉挂钩确定的四个眼外直肌和2-0编织丝非针刺缝合隔离。 注:丝线缝合将与所希望的方向移动的地球帮助。眼球被分成四个直肌肌肉（四象限）之间的象限。 <ol><li>标记用皮肤标记物两个区在每个象限中，一个在赤道巩膜和一个在巩膜。这些是在照射区域。 </li></ol></li><li>准备3毫升水5ml注射器含0.1％右旋糖酐自由，核黄素-5- phosphat即注射器连接到＆G泪道插管或地下25锥形hydrodelineator。 </li><li>制备的照射装置，其包括连接到一个斜面向下定制光纤的UV A（370纳米）的光源。继能源动力和校准测量（见下文说明），将设备设置为57毫瓦/平方厘米。 注意:校准由制造商进行。这种方法提供了57焦耳/ cm 2的总UVA光剂量。 （在0.2 平方厘米57毫瓦/平方厘米2是使用11.4毫瓦/ 0.2 平方厘米200秒11.4毫瓦/ 0.2厘米2的每0.2 2.2Ĵ厘米2点的累积负载） </li></ol> 3.手术＃1 - 交联<ol><li>通过在选定的象限的相反方向拉动丝线移动眼球待处理（ 例如，上拉和鼻治疗下颞象限时）。执行使用2-0编织丝非针刺缝合眼球运动，我solates每个直肌。 </li><li>应用含有0.1％游离葡聚糖核黄素-5-磷酸盐到照射开始前的照射区20秒光敏剂溶液。 </li><li>使用斜面向下定制光纤200秒的照射时间照射的照射区。测量每个区0.2平方厘米以4mm的半径的区域。 </li><li>应用在200秒照射周期含有0.1％游离葡聚糖核黄素-5-磷酸盐到辐照区每20秒光敏剂溶液。 注:执行照射和两名外科医生同时核黄素的下降。 </li><li>重复步骤3.1-3.4为四个象限眼球的。 </li></ol> 4.手术＃1 - 后交联步骤<ol><li>刮眼睛周围的皮毛。采用手术眼科剪修剪眼睑边缘，然后轻轻地缝合上，用4-0丝线编织象牙色下眼睑 - 3/8圈重诗切割 - 13毫米长度C-3针（tarsorrhaphy）。在步骤（氯霉素5％）的端部适用于盖缘眼药膏少量。 </li></ol> 5.手术＃1 - 术后护理<ol><li>保持加热灯下动物恢复过程中。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><li>将动物在干净的笼子新的床上用品，直到其完全恢复。恢复后，将动物将被返回到动物房。返回动物对他的母亲，只有当清醒。 </li><li>在第一个48小时，检查有关的一般情况和疾病或感染的迹象的动物。辖镇痛按部1如果需要观察兔行为的变化辖镇痛。 （安乃近降至50毫克/ kg，每6小时）。 </li></ol> 6.手术＃2 <ol><li>之后一下55天urgery＃1，称重并用氯胺酮盐酸盐100毫克/毫升（100毫克/千克）和赛拉嗪盐酸盐25毫克/毫升（12.5毫克/千克）的肌内注射麻醉兔。 </li><li>使用眼科手术显微镜下手术眼科剪，去掉tarsorrhaphy。在表面麻醉下使用超声A-扫描器（盐酸奥布卡因0.4％）后，每一只眼睛执行三个轴向长度测量，然后平均的测量。垂直应用探头角膜中央。 </li><li>安乐死腹腔苯巴比妥钠（200毫克/ 1.5 kg体重）的兔子。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

我们使用具有核黄素和UVA辐照交联技术中存在的预防轴性近视的体内研究的第一个在兔模型。虽然不同的实验室动物可以在这种类型的研究中使用，我们选择了兔大多是由于眼睛的大小，并在巩膜表面上执行交联的需要。 我们发现，暴露兔子巩膜和缝合上下眼睑是具有挑战性的程序。我们建议修整眼睑边缘，并使用在我们的协议中提到的缝线，以确保眼睑被牢?...

近视是最常见的折射病症。在美国和欧洲近视的患病率被报告为30％左右，而在亚洲国家，它会影响到一般人群1,2的60％。近视的进展出现在多达近视患者的50％，通常在约-0.5屈光度超过两年的时间间隔3的速率。近视实行的医疗费用相当高，包括费用眼镜，隐形眼镜和屈光手术以及与青光眼，白内障，视网膜脱离和视力损伤4-6的增加健康风险的成本。 在近视的动物实验中，视力降低是由眼睑缝合7-10，封堵器放置在离眼睛和角膜纹身11短距离感应。但是，对于在这些研究中，以发生人工近视，遮挡过程必须执行在非常年幼的动物，因为没有视力剥夺实验卡尔灭蝇灯出对成人标本证明是成功的。 之一的高度近视的重要特征是与巩膜的渐进变薄，可能是巩膜的病理变化，由于视觉剥夺12或后正视的干扰反馈机制由于巩膜的一些代谢紊乱，如在Ehlers-当洛综合征13。最终，双方机制导致了拉伸和巩膜，视网膜和脉络膜变薄，由于近视巩膜的结构异常，如胶原减少纤维直径14,15和骚乱在原纤维16。 几项研究已经表明，受损的胶原交联是在近视巩膜17-18的减弱过程的一个重要因素。 Wollensak 等 19-21应用光敏剂核黄素和紫外线A（UVA）照射引起的胶原交联（370纳米）并指出在猪和人巩膜的刚性的显著，157％的增加在体外 19和增加了465％，在兔子巩膜刚性体内 （杨氏模量）20。 交联也对兔子巩膜长期效果体内 :4个月后，3天后的刚性增加了320.4％，277.6％和8个月（杨氏模量）22后502％。 治疗尝试阻止近视进展已经发表23-26但这些方法的成功是有争议的。防止进行性近视的没有有效的手段已被发现的日期。 近视的病因仍有争议，及其治疗提出了挑战。这些发现的基础上，它是假设，巩膜交联剂可以作为近视进展的基础巩膜处理的装置。这项研究的目的是检查巩膜胶原crosslinki纳克通过视轴闭塞诱导的轴性近视的发展的影响。

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			<td height="23" style="height:23px;width:244px;">Ultrasonic biometer</td>
			<td>Allergan-Humphrey</td>
			<td style="width:119px;">820-519</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Skin marker</td>
			<td style="width:137px;">Devon</td>
			<td style="width:119px;">4237101664X</td>
			<td></td>
		</tr>
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scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

近视的个体，特别是那些有严重的近视，是在白内障，青光眼，视网膜脱离和脉络膜视网膜异常高于正常危险。此外，病理性近视是视力障碍和失明1-3常见的不可逆的原因。我们的研究表明巩膜交联使用核黄素和紫外线的辐射对轴性近视的兔模型发展的影响。眼球的眼轴长度是由新西兰13岁天（男性和女性）白兔A扫描超声检测。眼睛然后行360°结膜peritomy巩膜交，其次是tarsorrhaphy。轴向伸长被缝合的右眼眼睑（tarsorrhaphy）13日龄新西兰兔诱导。眼睛被分成象限，每个象限有两个巩膜照射区域，每个区域具有0.2平方厘米的面积和4毫米的半径。交联是通过滴加0.1％进行葡聚糖 - 自由核黄素-5-磷酸盐到紫外线-A照射前照射区20秒，并且在200秒的照射时间每20秒。 UVA辐射（370纳米），在57毫瓦/平方厘米（总UVA光剂量，57焦耳/平方厘米）垂直于巩膜应用。 Tarsorrhaphies进行55天遭到移除，随后重复轴向长度的测量。这项研究表明，核黄素和紫外线的辐射交联巩膜有效防止阻塞引起的轴向伸长在兔模型。

图1和图 2图示表明两组的轴向长度的测量。组1家兔接受巩膜交联和tarsorrhaphy上右眼，而左眼上未（ 图1）进行操作。而左眼上未（ 图2）操作的第2组兔的右眼接受仅peritomy和tarsorrhaphy。 在第1组，其接受巩膜交联和tarsorrhaphy，平均在右眼轴向长度测量眼睑缝合前10....

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

我们证明与核黄素和UVA巩膜交联的上一个轴向伸长兔眼的影响。轴向伸长率13日龄新西兰兔缝合的右眼眼睑（tarsorrhaphy）（男性和女性）引起的。

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

scleral-cross-linking-using-riboflavin-ultraviolet-radiation-for

Research

JoVE Journal

Medicine

8.8K 次观看.  Rabin Medical Center, Beilinson Campus. 我们证明与核黄素和UVA巩膜交联的上一个轴向伸长兔眼的影响。轴向伸长率13日龄新西兰兔缝合的右眼眼睑（tarsorrhaphy）（男性和女性）引起的。 

视频: Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

在小鼠模型体内生物发光成像乳腺癌脑转移的肿瘤乏氧动力学

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

科研

医学

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.

基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

蛛网膜下腔出血急性和后遗症的研究兔血分流型号:技术问题

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

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.

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 ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...