青光眼在本实验室的研究是国家青光眼研究从美国卫生署助理基金会的支持。

<ol><li>准备使用移液器拉马的玻璃吸液管，连接到1ml注射器，并用封口膜密封连接。退回到吸液管的尖端解决方案2毫升，并准备注射。 </li><li>麻醉大鼠腹腔注射氯胺酮（80mg/kg）和甲苯噻嗪（8mg/kg）（体积比为2:1）。 </li><li>应用一个前玻璃体内注射局部麻醉剂对大鼠的眼睛，下降0.5％alcaine。 </li><li>大鼠的位置，并揭露鼻地区优越的眼睛。 </li><li>使用30号针头穿刺杆扁平疣水平优于鼻巩膜。避免接触眼部肌肉和血管。 </li><li>给眼球轻微的压力，打通后房型从穿刺孔除去少量的玻璃体。 </li><li>在45 °角巩膜通过插入玻璃吸管提示通过穿刺孔进入玻璃体。注入后房2溶液，并保持在大约几秒钟，然后轻轻取出针。避免接触镜头及人工血管。 </li><li>玻璃体内注射后，适用于眼科Tobrex软膏对大鼠的眼睛，以防止感染。 </li></ol>

<ol>
	<li>Chiu, K., Lam, T. T., Ying Ku, W. W., Caprioli, J., Kwong Kwong, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain+and+N-methyl-d-aspartate+(NMDA)-induced+excitotoxicity+in+rat+retinas.">Calpain and N-methyl-d-aspartate (NMDA)-induced excitotoxicity in rat retinas.</a> Brain Research. 1046, 207-215 (2005).</li><li>Fisher, D., Pavlidis, M., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataractogenic+lens+injury+prevents+traumatic+ganglion+cell+death+and+promotes+axonal+regeneration+both+in+vivo+and+in+culture.">Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.</a> Investigative Ophthalmology &amp; Visual Science. 41, 3943-3954 (2000).</li><li>Ji, J. -. Z., Elyaman, W., Yip, H. K., Lee, V. W. H., Yick, L. -. W., Hugon, J., So, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTF+promotes+survival+of+retinal+ganglion+cells+after+induction+of+ocular+hypertension+in+rats:+the+possible+involvement+of+STAT3+pathway.">CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway.</a> European Journal of Neuroscience. 19, 265-272 (2004).</li><li>Lam, T. T., Abler, A. S., Kwong, J. M. K., Tso, M. O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)-Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)-Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li></ol>

根据动物研究在教学和科研使用活的动物（CULATR）香港大学和协会在视觉与眼科语句研究委员会批准的使用协议处理动物眼科及视觉研究中使用动物。

良好的玻璃体内注射液是用于建立动物模型以及直接人工处理。首先，注射部位不应该破坏视网膜结构。二，出血，应避免使用，以减少感染的风险。第三，镜片应不变，以避免外伤性白内障。最重要的是正常的眼部结构，以减少中断。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Operating Microscope </td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>OME</td>
<td>&nbsp;</td>
<tr>
<td>Glass Pipettes </td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Puller </td>
<td>&nbsp;</td>
<td>David Kopf Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1ml syringes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>single use</td>
</tr>
<tr>
<td>30G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>single use</td>
</tr>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Aldasan</td>
<td>&nbsp;</td>
<td>anesthetic</td>
</tr>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Aldasan</td>
<td>&nbsp;</td>
<td>anesthetic</td>
</tr>
<tr>
<td>Alcaine</td>
<td>&nbsp;</td>
<td>Alcon-Couvreur</td>
<td>&nbsp;</td>
<td>0.5% proparacaine hydrochloride</td>
</tr>
<tr>
<td>Tobrex</td>
<td>&nbsp;</td>
<td>Alcon-Couvreur</td>
<td>&nbsp;</td>
<td>ointment, 3% tobramyxin.</td>
</tr>		</tbody>
		</table>
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intravitreous injection for establishing ocular diseases model

玻璃体内注射液是一种广泛使用的技术在视觉科学的研究。它可用于建立与眼部疾病或局部治疗的直接应用的动物模型。本视频介绍了如何使用简单和便宜的工具来完成的玻璃体内注射过程。使用1毫升注射器，而不是一个hemilton注射器，使用。实用技巧，如何作出适当的注射针头，用玻璃移液管以完美的技巧，以及如何轻松地连接用玻璃吸管将注射器针头紧紧地握在一起。 要进行一个很好的的玻璃体内注射，有三个方面来观察：1）注射部位不应该破坏视网膜结构; 2）出血应避免，以减少感染的风险; 3）镜头应不变，以避免外伤性白内障。简单地说，最重要的一点是正常的眼部结构，以减少中断。为了避免中断视网膜，被选为优越的鼠眼的鼻地区。另外，在标准杆的平面，这是从鼠眼的角膜缘地区约1.5 mm针的穿刺点。轻轻推少量的玻璃体通过穿刺孔，以减少注射前的眼压。喷射角度45度，这是不太可能导致在大鼠眼外伤性白内障，从而避免了从透镜因素相关的并发症和影响力。在这次行动中，有没有切割，无出血，结膜和眼部肌肉。快速和轻微的损伤，一个成功的玻璃体内注射可以在几分钟内完成。 在这个特殊的协议概述注入玻璃体内注射的具体。然而，这里介绍的方法和材料也可以用于其他注射给药程序的小型哺乳动物的脑，脊髓或其他器官。

Watch this Scientific Journal Video about Intravitreous Injection for Establishing Ocular Diseases Model at JoVE.com

Intravitreous Injection for Establishing Ocular Diseases Model

玻璃体内注射液是一种视觉科学的研究，广泛用于眼部疾病或局部治疗的直接应用的技术。该视频展示了玻璃体内注射用1ml注射器与玻璃吸管的协议。有用的技巧，如何避免大出血和晶状体损伤。

玻璃体内注射眼部疾病模型建立

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

intravitreous-injection-for-establishing-ocular-diseases-model

Research

JoVE Journal

Biology

36.5K 次观看.  The University of Hong Kong - HKU. 玻璃体内注射液是一种视觉科学的研究，广泛用于眼部疾病或局部治疗的直接应用的技术。该视频展示了玻璃体内注射用1ml注射器与玻璃吸管的协议。有用的技巧，如何避免大出血和晶状体损伤。

视频: Intravitreous Injection for Establishing Ocular Diseases Model

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

激光诱导SD大鼠慢性高眼压高压模型

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

科研

生物学

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

专访：蛋白质折叠与神经退行性疾病的研究

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

斑马鱼脑室注射液

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

眼部微血管血管生成前维沃胆球状芽分析

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Establishing Human Lung Organoids and Proximal Differentiation to Generate Mature Airway Organoids

The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. We describe a defined protocol to derive human lung organoids from adult stem cells in the lung tissue and induce proximal differentiation to generate mature airway organoids. The lung organoids are then consecutively expanded for over 1 year with high stability, while the differentiated airway organoids are used to morphologically and functionally simulate human airway epithelium to a near-physiological level. Thus, we establish a robust organoid model of the human airway epithelium. The long-term expansion of lung organoids and differentiated airway organoids generates a stable and renewable source, enabling scientists to reconstruct and expand the human airway epithelial cells in culture dishes. The human lung organoid system provides a unique and physiologically active in vitro model for various applications, including studying virus-host interaction, drug testing, and disease modeling.

The protocol presents a method to derive human lung organoids from primary lung tissues, expand the lung organoids and induce proximal differentiation to generate 3D and 2D airway organoids that faithfully phenocopy the human airway epithelium.

建立人肺类器官和近端分化以产生成熟的气道类器官

The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

从人眼高效、一致地生成视网膜色素上皮/脉络膜平面安装，用于组织学分析

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...