نشكر مختبر أعضاء جيفري Adijanto ، بانزون تينا ، لى رونغ ، وان تشين تشانغ Congxiao ، زهاو جينغ تشى كوني ، ضياء عويس ، Strunnikova ناتاليا للمساعدة في تشخيص هذه الثقافات الخلية. شكر خاص الى زهاو جينغ تشى كوني ، وتينا بانزون لمساعدتهم في الحفاظ على مخزونات كبيرة من الثقافات الخلية. وأيد هذا العمل من قبل المعاهد الوطنية للبرنامج البحوث الصحية الجماعية.

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN{gamma}+Regulates+Retinal+Pigment+Epithelial+Fluid+Transport.">IFN{gamma} Regulates Retinal Pigment Epithelial Fluid Transport.</a> Am J Physiol Cell Physiol. 297, C1452-C1465 (2009).</li><li>Li, R., Maminishkis, A., Wang, F. E., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDGF-C+and+-D+induced+proliferation/migration+of+human+RPE+is+abolished+by+inflammatory+cytokines.">PDGF-C and -D induced proliferation/migration of human RPE is abolished by inflammatory cytokines.</a> Invest Ophthalmol Vis Sci. 48, 5722-5732 (2007).</li><li>Maminishkis, A., Jalickee, S., Blaug, S. A., Rymer, J., Yerxa, B. R., Peterson, W. M., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+P2Y(2)+receptor+agonist+INS37217+stimulates+RPE+fluid+transport+in+vitro+and+retinal+reattachment+in+rat.">The P2Y(2) receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat.</a> Invest Ophthalmol Vis Sci. 43, 3555-3566 (2002).</li><li>Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204/211+alters+epithelial+physiology.">MicroRNA-204/211 alters epithelial physiology.</a> FASEB J. 24, (2010).</li><li>Adijanto, J., Banzon, T., Jalickee, S., Wang, N. S., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2-induced+ion+and+fluid+transport+in+human+retinal+pigment+epithelium.">CO2-induced ion and fluid transport in human retinal pigment epithelium.</a> J Gen Physiol. 133, 603-622 (2009).</li><li>Li, R., Maminishkis, A., Zahn, G., Vossmeyer, D., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+mediates+attachment,+migration,+and+proliferation+in+human+retinal+pigment+epithelium:+relevance+for+proliferative+retinal+disease.">Integrin alpha5beta1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.</a> Invest Ophthalmol Vis Sci. 50, 5988-5996 (2009).</li><li>Peterson, W. M., Meggyesy, C., Yu, K., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+ATP+activates+calcium+signaling,+ion,+and+fluid+transport+in+retinal+pigment+epithelium.">Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium.</a> J Neurosci. 17, 2324-2337 (1997).</li><li>Voloboueva, L. A., Liu, J., Suh, J. H., Ames, B. N., Miller, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R)-alpha-lipoic+acid+protects+retinal+pigment+epithelial+cells+from+oxidative+damage.">R)-alpha-lipoic acid protects retinal pigment epithelial cells from oxidative damage.</a> Invest Ophthalmol Vis Sci. 46, 4302-4310 (2005).</li></ol>

الإعلان عن أي تضارب في المصالح.

في تجارب الحاضر ، ونحن تصف تعديلات إضافية من التقنيات التي تم نشرها مسبقا لدينا 3 تهدف إلى تبسيط الخطوات اللازمة لإنتاج متعددة الثقافات بما يتفق الأولية hfRPE مع ارتفاع عدد الخلايا المتاحة في العين. تم اختبار صارم كل تغيير في الإجراء الأصلي في علم وظائف الأعضاء وتجارب متعددة البيولوجيا الجزيئية للتأكد من أن التغييرات لم يعرض القطع الاثرية والتي يجري اختبارها باستمرار من قبل مختبرات أخرى كثيرة باستخدام هذه الخلايا. أخيرا ، أجريت تجارب إضافية إلى مقارنة في نتائج المختبر للاضطرابات مماثلة في النماذج الحيوانية.

الصكوك اللازمة لبروتوكول تشريح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ستيريو المجهر -- أي التكبير المتاحة مع x250 العمل اللازمة لتشريح </li><li style=";text-align:right;direction:rtl"> تشريح طبق -- على سبيل المثال 100 × 20mm كيمبل # 23062 (واحد لكل تشريح) أي مصدر </li><li style=";text-align:right;direction:rtl"> Sylgard 184 -- WPI القط. # * SYLG184 * لتحضير طبق تشريح اتبع التعليمات شملت 184 Sylgard الاختلاط ، صب السائل الاستومر مختلطة في طبق بتري لتشكيل 5 - 8MM طبقة. الاستومر تسمح لعلاج لمدة لا تقل 24hrs. يمكن تعقيم الطبق مع الاستومر الشفاء باستخدام الايثانول 70 ٪ وإعادة استخدامها مرات عدة (~ 100 مرات) </li><li style=";text-align:right;direction:rtl"> 27 G من الإبر PrecissionGlide دينار بحريني 1 ¼ مدة (5 في تشريح) أي مصدر </li><li style=";text-align:right;direction:rtl"> HBSS 1X محلول يحتوي على أملاح الكالسيوم والمغنيسيوم -- GIBCO القط # 14025 500ml (جيد للتشريح متعددة). </li><li style=";text-align:right;direction:rtl"> مقص القزحية كربيد مسننة النصل ، منحني -- FST القط # 14559-11 (واحد). </li><li style=";text-align:right;direction:rtl"> الشبكية مقص -- Katena القط # K4 - 5300 (واحد). </li><li style=";text-align:right;direction:rtl"> ملقط -- على سبيل المثال دومون الملقط # 5 مصقول مع نصائح من القط WPI # 500085 (2 قطعة). </li><li style=";text-align:right;direction:rtl"> Sideport سكين -- ألكون ، 1MM اضح المعالم ، شطبة مزدوجة ، القط الزاوية # 8065921540. </li><li style=";text-align:right;direction:rtl"> 15ml أنبوب الطرد المركزي -- (واحد في العين) أي مصدر </li><li style=";text-align:right;direction:rtl"> ماصة باستير الزجاج -- (واحد لكل تشريح) أي مصدر </li><li style=";text-align:right;direction:rtl"> 12 لوحة جيدا (واحد لكل تشريح) أي مصدر </li><li style=";text-align:right;direction:rtl"> Primaria 25cm 2 قارورة -- (2-3 في العين) أي مصدر </li></ol>

experimental models for study of retinal pigment epithelial physiology and pathophysiology

وضعنا الداخلي ثقافة الخلية التي يمكن أن تنتج كميات كبيرة من monolayers متموجة من الجنين البشري الأساسي الثقافات صباغ الشبكية (hfRPE) ظهارة مع الخصائص المورفولوجية والفسيولوجية والجينية لRPE الإنسان الأصلية. هذه الثقافات الخلية hfRPE المعرض تصبغ الثقيلة ، والمجهر الإلكتروني تظهر واسعة الزغيبات الصغيرة الغشاء القمي. وقد تم تحديد المجمعات صلي وسم المناعي مع مختلف البروتينات مفرق ضيق. قطبية الظهارية وظيفة تلك الثقافات الأولية استنساخه بسهولة شبها درس سابقا نماذج من الثدييات RPE الأصلية ، بما في ذلك الإنسان. وتم تمديد هذه النتائج من خلال تطوير التدخلات العلاجية في النماذج الحيوانية العديد من أمراض العين البشرية. وقد ركزنا على استراتيجيات لإزالة تراكم السوائل غير طبيعي في الشبكية أو في الفضاء تحت الشبكية. الفضاء خارج الخلية تحت الشبكية يفصل قطاعات مبصرة الخارجي والغشاء القمي للRPE وهذا أمر مهم لصيانة التجهيزات والمعدات الشبكية ، ومجموعة كاملة من RPE / الشبكية التفاعلات.

Watch this Scientific Journal Video about Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology at JoVE.com

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

نحن نقدم طريقة لاستنساخه monolayers زراعة الأجنة البشرية متموجة الخلايا الظهارية في شبكية العين صباغ (hfRPE) الذي يحمل مورفولوجيا الخلايا ، علم وظائف الأعضاء ، قطبية ، والبروتين وأنماط التعبير الجيني للأنسجة الأم الكبار. وقد تم تمديد هذا العمل إلى نموذج حيواني من عدة أمراض العيون.

نماذج تجريبية لدراسة فسيولوجيا صبغات الشبكية الظهارية والفيزيولوجيا المرضية

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

experimental-models-for-study-retinal-pigment-epithelial-physiology

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.2K Views.  National Institutes of Health. نحن نقدم طريقة لاستنساخه monolayers زراعة الأجنة البشرية متموجة الخلايا الظهارية في شبكية العين صباغ (hfRPE) الذي يحمل مورفولوجيا الخلايا ، علم وظائف الأعضاء ، قطبية ، والبروتين وأنماط التعبير الجيني للأنسجة الأم الكبار. وقد تم تمديد هذا العمل إلى نموذج حيواني من عدة أ...

Video: Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

تشريح العين ماوس لجبل الجامعة للصبغات الظهارة الشبكية

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

التلاعب الجيني للمخيخي الحبيبة الخلايا العصبية<em&gt; في المختبر</em&gt; و<em&gt; في فيفو</em&gt; لدراسة مورفولوجيا الخلايا العصبية والهجرة

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

فحوصات حاسة الشم لنماذج من الماوس مرض الاعصاب

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

و<em&gt; في المختبر</em&gt; نموذج لدراسة الفيزيولوجيا المرضية الخلوي في الخلية حثل المادة البيضاء الكروي

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

الحوفي-النهج تحت الشبكية حقن الفيروسية المتجهات للعلاج الجيني في الفئران الشبكية الظهارة الصبغية

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

منخفض الكثافة الابتدائية الحصين العصبية الثقافة

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

وضع البروتوكول المكرر لزرع الخلايا الظهارية الصبغية الشبكية البشرية ترانس-سكليرال سوبريتينال في عيون الفئران

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

نماذج تجريبية لدراسة الحماية العصبية من الوضعية الحمضية ضد نقص التروية الدماغية

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

إعداد المقطعين الماهيينيات في شبكية العين للكيمياء المناعية

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of the mouse neuroretina&#160;with intact retinal pigment epithelium (RPE). Depending on the research question, retinas can be isolated from wild-type animals or from disease models, to study, for instance, diabetic retinopathy or hereditary retinal degeneration. Eyes from early postnatal day 2&#8211;9 animals are enucleated under aseptic conditions. They are partially digested in proteinase K to allow for a detachment of the choroid from the RPE. Under the stereoscope, a small incision is made in the cornea creating two edges from where the choroid and sclera can be gently peeled off from the RPE and neuroretina. The lens is then removed, and the eyecup is cut in four points to give it a four-wedged shape resembling a clover leaf. The tissue is finally transferred in a hanging drop into a cell culture insert holding a polycarbonate culturing membrane. The cultures are then maintained in R16 medium, without serum or antibiotics, under entirely defined conditions, with a medium change every second day.
The procedure described enables the isolation of the retina and the preservation of its normal physiological and histotypic context for culturing periods of at least 2 weeks. These features make organotypic retinal explant cultures an excellent model with high predictive value, for studies into retinal development, disease mechanisms, and electrophysiology, while also enabling pharmacological screening.

The protocol describes organotypic explants of mouse neuroretina, cultivated together with its retinal pigment epithelium (RPE), in R16 defined medium, free of serum and antibiotics. This method is relatively simple to perform, less expensive, and time-consuming when compared to in vivo experiments, and can be adapted to numerous experimental applications.

على المدى الطويل، زراعة خالية من المصل من النباتات الشبكية الماوس Organotypic مع ظهارة صبغة الشبكية سليمة

In ophthalmic research, there is a strong need for in vitro models of the neuroretina. Here, we present a detailed protocol for organotypic culturing of ...