我们的研究是由美国国立卫生研究院，国家眼科研究所院内研究计划的支持。

所有的设备和试剂使用无菌。批准该协议是由动物护理和使用聂/ NIH委员会（ACUC）（动物研究协议NEI - 553），并根据美国国立卫生研究院的准则和法规执行。 1。产生血管生成因子含有颗粒在这一部分中，包含利益的血管生成因子的颗粒准备。 <ol><li>设为10％（W / V），硫糖铝，用PBS液。贮存在室温下。 </li><li>设为12％（W ​​/ V）聚甲基丙烯酸羟乙酯用无水乙醇。贮存在室温下。 </li><li>设为1μg/μL的碱性成纤维细胞生长因子的解决方案。储存在-80 ° C。 </li><li>为了使50颗粒，混合聚甲基丙烯酸羟乙酯，5μL加入1μl硫糖铝，碱性成纤维细胞生长因子原液和4μL，涡旋彻底。 </li><li>在培养皿中放置了一块干净的封口膜，暴露在紫外光下，在组织培养罩15分钟。拖放一个颗粒的封口膜0.2μL的溶液混合。因此，10μL混合液应产生50颗粒。让在室温下干燥1-2小时的颗粒。干燥后的颗粒可以保存在4℃数天，-80 ° C的几个月。 </li><li>用法后，撬，拿起一双镊子提示颗粒。要格外小心，不要打破他们或使他们的春天离。 </li></ol> 2。执行鼠标角膜口袋检测在这一部分，我们将演示如何使一个在小鼠角膜的口袋里，和如何插入一个颗粒的口袋。颗粒中的血管生成因子将逐步释放到角膜周边地区（图1）。在这个演示中，我们使用8个星期的女性C57/Bl6小鼠。 <ol><li>鼠标深深系统与氯胺酮（75毫克/公斤）和甲苯噻嗪（经口5mg/kg，腹腔注射）的混合麻醉。这将保持30-60分钟麻醉状态下鼠标。局部麻醉剂（0.5％丙美卡因盐酸）适用于角膜。 5分钟后，一只眼睛在解剖显微镜下修复。 </li><li>一个非常温和的削减是在角膜1.2 -1.4毫米冯格雷夫白内障刀角膜缘（如图1所示）中。要特别小心，不要穿过角膜。 </li><li>一个口袋下的角膜上皮细胞层是由横向插入到中间的角膜刀，并延长刀对角膜缘仔细（图1）。口袋大小，大到足以容纳一个血管生成因子含有颗粒。要非常小心，不要通过角膜穿刺。 </li><li>插入的钳入囊中慢慢对颗粒。尝试尽可能地使颗粒作为平。 </li><li>颗粒植入后，外用眼科抗生素眼膏涂于眼。仪器与动物之间70％的酒精垫被消灭。 </li><li>移动鼠标到一个温暖，干燥的地区，监视它，直到它能够保持直立的姿势，然后返回其家笼。全身性镇痛药（如丁丙诺啡，2毫克/公斤，腹腔注射）和外用抗生素软膏（如庆大霉素）2天，每天两次手术后，鼠标为3天密切监察。预计愈合手术后的最初48小时内完成。口袋里的眼睛，将审查下一个裂隙灯biomicro之间的颗粒植入后4-10天的范围。 </li></ol> 3。代表性的成果<ol><li>颗粒植入后7天，鼠标是全身麻醉和局部2.1中所述。 </li><li>诱导的血管生成因子在解剖显微镜下观察角膜的血液船只。在与车辆治疗角膜，没有血管生长可观察到（图2A，车辆治疗角膜）。强大的血管生成与碱性成纤维细胞生长因子治疗角膜，可以看出（图2B）。血管生长，可以评估使用的最大长度（从正常的角膜缘船只的新船的顶部），血管和总血管领域（图2B，用红色虚线在碱性成纤维细胞生长因子的线划定治疗角膜）。其他定量方法也被描述2,5。 </li></ol><img src="/files/ftp_upload/3077/3077fig1.jpg" alt="图1" /> 图1。一个正常的角膜H＆E染色。存在于正常的角膜无血管，B。请上皮下面的角膜，C层的口袋。装入口袋中嵌入一个颗粒，D 。血管生成因子被释放颗粒，正常的角膜缘血管生长新的血管。 <img src="/files/ftp_upload/3077/3077fig2.jpg" alt="图2" /> 5天之后，IM代表结果图2。种植沉淀。角膜与只包含车辆的颗粒。没有新的血液，在角膜血管的形成。只有预先存在的正常的角膜缘血管可见，B 。一个颗粒含碱性成纤维细胞生长因子与角膜。强大的新血管（点内衬）的增长从预先存在的正常的角膜缘血管。

<ol>
	<li>Cao, Y., Linden, P., Shima, D., Browne, F., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+angiogenic+activity+and+hypoxia+induction+of+heterodimers+of+placenta+growth+factor/vascular+endothelial+growth+factor.">In vivo angiogenic activity and hypoxia induction of heterodimers of placenta growth factor/vascular endothelial growth factor.</a> The Journal of clinical investigation. 98, 2507-2511 (1996).</li><li>Rogers, M. S., Birsner, A. E., D'Amato, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+micropocket+angiogenesis+assay.">The mouse cornea micropocket angiogenesis assay.</a> Nature. 2, 2545-2550 (2007).</li><li>Poche, R. A., Saik, J. E., West, J. L., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+cornea+as+a+transplantation+site+for+live+imaging+of+engineered+tissue+constructs.">The mouse cornea as a transplantation site for live imaging of engineered tissue constructs.</a> Cold Spring Harbor protocols. , 5416-54 (2010).</li><li>Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+is+dispensable+for+blood+vessel+growth+but+critical+for+their+survival,+and+VEGF-B+targeting+inhibits+pathological+angiogenesis.">VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis.</a> Proceedings of the National Academy of Sciences of the United States of America. , 106-6152 (2009).</li><li>Tong, S., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose+response+of+angiogenesis+to+basic+fibroblast+growth+factor+in+rat+corneal+pocket+assay:+I.+Experimental+characterizations.">Dose response of angiogenesis to basic fibroblast growth factor in rat corneal pocket assay: I. Experimental characterizations.</a> Microvascular research. 75, 10-15 (2008).</li><li>Muthukkaruppan, V., Auerbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+the+mouse+cornea.">Angiogenesis in the mouse cornea.</a> Science. 205, 1416-1418 (1979).</li><li>Ziche, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+assay+for+angiogenesis.">Corneal assay for angiogenesis.</a> Methods in molecular medicine. 46, 131-142 (2001).</li><li>Ziche, M., Morbidelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+pocket+assay.">The corneal pocket assay.</a> Methods in molecular biology. 467, 319-329 (2009).</li></ol>

没有利益冲突的声明。

鼠标角膜口袋检测模型是在1979年首次描述Muthukkaruppan VR和奥尔巴赫R 6。 2,3,7,8不同的实验室已经公布了详细的协议。我们的实验室已修改和 4多年来，在我们的研究使用这种方法。鼠标角膜口袋里的分析是一个很大的重复性相对简单的模型。这种模式的另一个优点是，由于角膜通常缺乏血管在这个实验中，有背景的最低金额。因此，这种模式通常用于研究不同的因素和分子的血管?...

<table border="1"><tbody><tr><th>设备</th><th>公司</th><th>目录编号</th></tr><tr><td>解剖显微镜</td><td>世界精密仪器</td><td> PZMIV - BS </td></tr><tr><td>冯格雷夫白内障刀（1.5x25mm刀片，平） </td><td>安布勒手术</td><td> 3401E </td></tr><tr><td>杜蒙＃5镊子</td><td>精细科学的工具</td><td> 11252-50 </td></tr><tr><td>裂隙灯</td><td>蔡司</td><td> SL - M 30 </td></tr></tbody></table> 表1设备<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录＃ </th><th>评论</th></tr><tr><td>碱性成纤维细胞生长因子（碱性成纤维细胞生长因子） </td><td> PEPROTECH INC。 </td><td> 100 - 18B </td><td></td></tr><tr><td>硫糖铝（蔗糖octasulfate铝复合） </td><td>西格玛</td><td> S0652 </td><td> 10％的PBS </td></tr><tr><td>聚甲基丙烯酸羟乙酯（聚（甲基丙烯酸2 - 羟乙基）） </td><td>西格玛</td><td> P3932 </td><td> 12％乙醇</td></tr></tbody></table> 表2。试剂和材料

a mouse model of the cornea pocket assay for angiogenesis study

一个正常的角膜是明确的血管组织。然而，血管可诱发，在强有力的血管生成因子1的角膜成长和生存。这种独特的角膜口袋检测最常用的血管生成研究模型之一。角膜细胞组成的多层次。因此，可以嵌入颗粒含有的血管生成因子在角膜上，探讨其血管生成作用 2,3利益。在这里，我们提供了一步一步示范如何（一）产生血管生成因子含颗粒（二）嵌入到角膜颗粒（三）分析利益的血管生成因子诱导的血管生成。由于碱性成纤维细胞生长因子（bFGF）是已知的最有力的血管生成因子之一，它是用来诱发角膜新生血管形成。

Watch this Scientific Journal Video about A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study at JoVE.com

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

角膜是独特的，因为它没有血管组织。然而，强大的血管的生长和生存，可以在角膜上，通过强有力的血管生成因子诱导。因此，角膜可以与我们提供一个有价值的工具，对血管生成的研究。此协议演示了如何执行角膜口袋检测，以及如何评估使用这个模型的血管生成因子诱导的血管生成的小鼠模型。

一个小鼠模型角膜血管生成研究掌上含量

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

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Research

JoVE Journal

Medicine

15.9K 次观看.  National Eye Institute. 角膜是独特的，因为它没有血管组织。然而，强大的血管的生长和生存，可以在角膜上，通过强有力的血管生成因子诱导。因此，角膜可以与我们提供一个有价值的工具，对血管生成的研究。此协议演示了如何执行角膜口袋检测，以及如何评估使用这个模型的血管生成因子诱导的血管生成的小鼠模型。

视频: A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

科研

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

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 ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...