Name: a simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe
Uploaded: 2012-06-12T13:00:00
Description: Watch this Scientific Journal Video about A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe at JoVE.com

Эта работа была поддержана частично за счет грантов Regione Венето (Ricerca Sanitaria Finalizzata n.292/2008 и Ricerca Sanitaria Finalizzata 2009). Авторы выражают благодарность д-р C. Griffoni на 2009/2010 сводные данные по использованию человеческих глобусы глаз.

1. В местах иссечения роговицы от глазных глобус <ol><li> Включите ламинарного потока воздуха шкаф примерно 15 минут перед использованием. Очистите капот ламинарного потока с использованием 70% изопропилового спирта. Положите на индивидуальную защитную одежду, таких как хирургически?...

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	<li>Fay, A., Goldman, L., Ausiello, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diseases+of+the+visual+system.">Diseases of the visual system.</a> Cecil Medicine. , (2007).</li><li>Parekh, M., Megaw, R., Ray-Chaudhuri, A., Ahmad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patents+in+Limbal+Stem+Cell+Biology.">Patents in Limbal Stem Cell Biology.</a> Recent patents on Regenerative Medicine. 1, 207-212 (2011).</li><li>Pharm, U. 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+Eye:+The+physiology+of+human+perception.">The Eye: The physiology of human perception.</a> Britannica educational publishing. 36, 19-27 (2011).</li><li>Lang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ophthalmology: A pocket textbook atlas. , 115-117 .</li><li>Liesbeth, P. e. l. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture:+The+method+of+choice+for+preservation+of+human+donor+corneas.">Organ Culture: The method of choice for preservation of human donor corneas.</a> Br. J. Ophthalmol. 81, 523-525 (1997).</li><li>Meyer, J. S., Howden, S. E., Wallace, K. A., Verhoeven, A. D., Wright, L. S., Capowski, E. E., Pinilla, I., Martin, J. M., Tian, S., Stewart, R., Pattnaik, B., Thomson, J., Gamm, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. , (2011).</li><li>Trifunovic, D., Karali, M., Camposampiero, D., Ponzin, D., Banfi, S., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+RNA+expression+atlas+of+retinitis+pigmentosa+genes+in+human+and+mouse+retinas.">A high-resolution RNA expression atlas of retinitis pigmentosa genes in human and mouse retinas.</a> Invest. Ophthalmol. Vis. Sci. 49, 2330-2336 (2008).</li><li>Mora, P., Montanini, L., Ferrari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retina. 30, 1555 (2010).</li><li>Claybon, A., Bishop, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Mouse+Eye+for+a+Whole+Mount+of+the+Retinal+Pigment+Epithelium.">Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium.</a> J. Vis. Exp. (48), e2563 (2011).</li><li>Skeie, J. M., Tsang, S. H., Mahajan, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evisceration+of+Mouse+Vitreous+and+Retina+for+Proteomic+Analyses.">Evisceration of Mouse Vitreous and Retina for Proteomic Analyses.</a> J. Vis. Exp. (50), e2795 (2011).</li></ol>

И, правильное удаление и надлежащую сохранность роговицы и сетчатки тканей являются критическими как незначительные дефекты, такие как повреждение эндотелия или большим количеством складок Десцеметова может привести к недостаточности трансплантата роговицы в то время как температ?...

Материалы для удаления роговицы и сетчатки. <table border="1"><tbody><tr><td> Описание товара </td><td> Размеры </td><td> Компания / Институт </td></tr><tr><td> Охраняемая одноразовый скальпель </td><td> Размер лезвия 15 </td><td> Swann-Morton </td></tr><tr><td> Стерильные бинты </td><td> 5 см х 5 см, 8 слоистые, 5 шт </td><td> Artsana </td></tr><tr><td> Стерильные одноразовые медицинские полотенце </td><td> 35 X 50 см </td><td> U.Jet </td></tr><tr><td> Стерильные ножницы </td><td> Лезвия 24 мм / длина. 95 мм изогнутые, тупые </td><td> e.janach </td></tr><tr><td> Стерильные пинцеты </td><td> Нержавеющая сталь -100 мм 11 X 2 правит 0,70 мм зубы </td><td> e.janach </td></tr><tr><td> Роговицы когтей - одноразовые медицинские устройства </td><td></td><td> NIIOS (Hippocratech) </td></tr&gt; <tr><td></td><td> Подготовка </td><td></td></tr><tr><td> PBS </td><td> 100 мл PBS [10 раз] в 900 мл д / ш (дистиллированная вода) </td><td> Sigma-Aldrich </td></tr><tr><td> Na-тиосульфата </td><td> 1 г Na-тиосульфата в 1 л PBS [1x] </td><td> Sigma-Aldrich </td></tr><tr><td> I-PVP </td><td> 5 г I-PVP в 1 л D / W </td><td> Sigma-Aldrich </td></tr></tbody></table> Таблица 2. Таблице описаны материалы, используемые для удаления роговицы и сетчатки и компании они получены от. Материалы для носителя (2000 мл). <table border="1"><tbody><tr><td> Компоненты </td><td> Поставщик </td><td> Номер по каталогу </td><td> Концентрация </td><td> Количество </td></tr><tr><td> MEM(1X) жидкости </td><td> Invitrogen </td><td> 32360-034 </td><td></td><td> 1900 мл </td></tr><tr><td> Натрия пируват </td><td> Invitrogen </td><td> 11360-039 </td><td> 1 мм (10 мл / л) </td><td> 20 мл </td></tr><tr><td> L-глютамин </td><td> Invitrogen </td><td> 25030-032 </td><td> 2 мм (10 мл / л) </td><td> 20 мл </td></tr><tr><td> Антибиотикам / противогрибковым </td><td> Sigma-Aldrich </td><td> A5955-20ML </td><td> 10 мл / л </td><td> 20 мл </td></tr><tr><td> Новорожденные телячьей сыворотки </td><td> Invitrogen </td><td> 26010-74 </td><td> 2% (20 мл / л) </td><td> 40 мл </td></tr></tbody></table> Таблица 3. Материалы для носителя. Подготовка носителя Добавить все ингредиенты при помощи специальных концентрациях, как указано выше в банке и хорошо перемешать. Фильтр ихиспользование размером пор 0,2 мкм фильтр (Millipore, Милан, Италия) с помощью перистальтического насоса. Сохранение среды в бутылках при комнатной температуре. Материалы для транспортной среды (2000 мл). <table border="1"><tbody><tr><td> Компоненты </td><td> Поставщик </td><td> Номер по каталогу </td><td> Концентрация </td><td> Количество </td></tr><tr><td> MEM (1X) жидкости </td><td> Invitrogen </td><td> 32360-034 </td><td></td><td> 1800 мл </td></tr><tr><td> Натрия пируват </td><td> Invitrogen </td><td> 11360-039 </td><td> 1 мм (10 мл / л) </td><td> 20 мл </td></tr><tr><td> L-глютамин </td><td> Invitrogen </td><td> 25030-032 </td><td> 2 мм (10 мл / л) </td><td> 20 мл </td></tr><tr><td> Новорожденные телячьей сыворотки </td><td> Invitrogen </td><td> 26010-74 </td><td> 2% (20 мл / л) </tг&gt; <td> 40 мл </td></tr><tr><td> Декстрана T500 </td><td> Pharmacosmos </td><td> 551005004007 </td><td> 6% (60 г / л) </td><td> 120 г / л </td></tr></tbody></table> Таблица 4. Материал для транспортной среды. Подготовка транспортной среды Добавить декстран 6% в ~ 1,5 литра MEM и оставить его на ночь. Добавить остальные ингредиенты в средствах массовой информации и с помощью расширения 0,2 мкм фильтр (Millipore, Милан, Италия) с помощью вакуумного насоса. Сохранение среды в бутылках при комнатной температуре.

a simplified technique for in situ excision of cornea and evisceration of retinal tissue from human ocular globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

Watch this Scientific Journal Video about A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe at JoVE.com

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

В статье описывается упрощенная техника акцизного роговицы и сетчатки потрошить тканей глазной шар трупа человека доноров. Техника, описанная здесь поможет акцизного хорошее качество ткани использовать для трансплантации, хирургическому или исследовательских целей, не повреждая другие ткани глазного шара.

Упрощенная Техника<em&gt; В месте</em&gt; Удаление роговицы и сетчатки Evisceration ткани человека глобус Глазные

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

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A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

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