وأيد هذا العمل جزئيا عن طريق المنح من فينيتو Regione (من Ricerca Sanitaria Finalizzata n.292/2008 ومن Ricerca Sanitaria Finalizzata 2009). الكتاب أشكر الدكتور جيم Griffoni للبيانات 2009/2010 ملخص عن استخدام الكرات العين البشرية.

<ol>
	<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>

الكتاب ليس لديهم تعارض في المصالح.

كلا، الختان الصحيح والسليم لحفظ الأنسجة القرنية والشبكية حاسمة كما وجود بعض العيوب البسيطة مثل تلف بطانة الأوعية الدموية أو عدد كبير من طيات descemet ويمكن أن يؤدي إلى فشل الكسب غير المشروع للقرنية في حين التعديلات درجة الحرارة أو سوء يمكن ان تعرض للخطر سلامة أنسجة الشبكية. والهدف من هذه الورقة هو لاظهار كيف يمكن أن تكون معزولة الأنسجة القرنية والشبكية على النحو الأمثل، من دون إحداث أضرار أو التعديلات التي يمكن أن تضر استعمالها للزراعة أو لأغراض البحوث. الخلية الحد الأدنى المطلوب كثافة (&gt; 2200 خلية / ملم 2)، مورفولوجيا جيدة مثل حجم الخلية، وهيكل، انحطاط منخفض من الحدود بين الخلايا وعدم وجود ضمور جنبا إلى جنب مع الشفافية جيدة وسمك مناسب للقرنية هي السمات المميزة التي تجعل من أنسجة مناسبة لل زرع. إذا تم استخدام شفرة مشرط تقريبا أو تستخدم أدوات أخرى بشكل مكثف، فإنه لا يضر فقط في القرنية، ولكن أيضا في الجسم الزجاجي الذي يجعل furtالختان لها من الأنسجة الأخرى أكثر صعوبة (الشكل 4). الشبكية هي عبارة عن نسيج شفاف وبالتالي يصعب تحديد إذا كان إطلاق السائل الزجاجي مع ذلك، وهذا يجعل من عادة الختان من أنسجة الشبكية أكثر تعقيدا. إذا رفعه مع الجسم الزجاجي، ونوعية الأنسجة الشبكية أو الحمض النووي الريبي المستخرج تكون سيئة للغاية بسبب التلوث مع أجزاء العين الأخرى. وبالتالي، تقنية مناسبة لقرنية العين 1 المكوس أو شبكية العين من المهم جدا لاسترداد الأنسجة عالية الجودة. بياناتنا السابقة من خلال العامين الماضيين (الجدول 1) تشير إلى أن ما يقرب من 60٪ من القرنيات التي نجمعها من الكرات العين هي مناسبة لأنواع مختلفة من الزرع. وهذا هو دليل آخر على أن تقنية استئصال القرنية وصفها في هذه الورقة هي وسيلة جيدة لجمع أنسجة القرنية عالية الجودة من الكرات بصري. قد القرنيات، بالطبع، أن تكون غير صالحة للزراعة بسبب غيرها من المعلمات قاوك كما شذوذ اللحمية، والتلوث، وكثافة البطانية / المورفولوجيا ومدى ملاءمتها للمانحين والصدمات الميكانيكية / المادية الناجمة مع الحفاظ على القرنية. ومع ذلك، إذا المعلمات أعلاه على ما يرام، والتقنية التي وصفناها هنا يسمح لاستخدامها استرداد من الأنسجة ذات نوعية جيدة للزرع في المرضى الذين يعانون من أمراض سطح العين. في حين أن المعلومات عن سلب من أنسجة الشبكية من النماذج الحيوانية متاحة على نطاق واسع 11،12، على حد علمنا أن هذه هي المرة الأولى التي وصفت مثل هذا الأسلوب لعزل أنسجة الشبكية من الكرات العين البشرية. في الواقع، والبحث في الأدب، لم تسفر عن أي نتيجة على أوراق مناقشة هذا الموضوع. نحن نعتقد أنه إذا كان يتم تشريح الإنسان العالم خارج العين كما هو موضح، وعلماء البحوث سوف تكون قادرة على أنسجة من نوعية جيدة لاستخدامها في مشاريع أبحاثهم. بنوك العيون كثيرة في مختلف أنحاء العالم من المرجح أن تلقي المصابيح العين (ولكن أيضا القرنيات) التي لا Suitable للزرع. في حالتنا، يمكن أن ما يقرب من 40٪ من القرنيات (والمصابيح العين) التي نجمعها وعملية لا يمكن زرع (نتيجة لمجموعة واسعة من الأسباب)، ولكن ما زال من الممكن استخدامها لأغراض البحث العلمي. تقنية وصفها في هذه الورقة لا تسمح لجمع الأنسجة ذات جودة عالية. وقد تجلى هذا في الآونة الأخيرة من قبل مجموعتنا عند تحليل الحمض النووي الريبي مع T &lt;24hrs المستخرجة من أنسجة الشبكية التي تم حصادها باستخدام تقنية الموضحة في هذه الدراسة 10. نعتقد يمكن أن تستخدم نفس الأسلوب لعزل الأنسجة الشبكية لأغراض كثيرة مختلفة أخرى مثل (ط) للدراسات transcriptome الشبكية، (الثاني) لدراسة وتطوير واستخدام فعل الإنسان الخلايا الجذعية المحفزة في مجال الطب شخصية 8، (ثالثا ) لوضع أطلس RNA التعبير للجينات التهاب الشبكية الصباغي في شبكية العين البشرية 9، (رابعا) لتحسين طريقة استخراج الحمض النووي الريبي، (V) دراسات الخلايا الجذعية لتجديد أنسجة الشبكية، الخ. هذه التقنية ثيكون سوء مفيد بالنسبة لأولئك الذين يريدون أن يمارسن الختان أو تلك التجارب ولكن لديها معلومات محدودة عن طريقة لاستخدام القرنيات البشرية أو شبكية العين.

المواد اللازمة لاستئصال القرنية والشبكية. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> وصف المنتج </td><td> الأبعاد </td><td> شركة / معهد </td></tr><tr><td> حراسة المتاح مشرط </td><td> حجم شفرة 15 </td><td> سوان، مورتون </td></tr><tr><td> ضمادات معقمة </td><td> 5 سم × 5 سم، 8 الطبقات، 5 قطع </td><td> Artsana </td></tr><tr><td> منشفة معقمة الطبية القابل للتصرف </td><td> 35 X 50 سم </td><td> U.Jet </td></tr><tr><td> معقم مقص </td><td> ريش 24 مم / الطول الاجمالي. 95 مم المنحني، كليل </td><td> e.janach </td></tr><tr><td> ملقط معقم </td><td> الفولاذ المقاوم للصدأ 11 ملم -100 X 2 يحكمها 0،70 ملم أسنان </td><td> e.janach </td></tr><tr><td> القرنية مخلب - الجهاز الطبي المتاح </td><td></td><td> NIIOS (Hippocratech) </td></tr&gt; <tr><td></td><td> الاستعدادات </td><td></td></tr><tr><td> برنامج تلفزيوني </td><td> 100 مل PBS [10X] في 900 مل (الماء المقطر) د / ث </td><td> سيغما الدريخ </td></tr><tr><td> NA-ثيوكبريتات </td><td> 1 جرام الصوديوم، ثيوكبريتات في 1 لتر من برنامج تلفزيوني [1X] </td><td> سيغما الدريخ </td></tr><tr><td> I-PVP </td><td> 5 جم I-PVP في 1 د لتر / ث </td><td> سيغما الدريخ </td></tr></tbody></table> الجدول 2. يصف الجدول المواد التي تستخدم في استئصال القرنية والشبكية والشركة تلقيها من. مواد وسيطة تخزين (2000 مل). <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> مكونات </td><td> المزود </td><td> فهرس العدد </td><td> تركيز </td><td> كمية </td></tr><tr><td> MEM(1X) السائل </td><td> إينفيتروجن </td><td> 32360-034 </td><td></td><td> 1900 مل </td></tr><tr><td> الصوديوم البيروفات </td><td> إينفيتروجن </td><td> 11360-039 </td><td> 1MM (10ML / لتر) </td><td> 20 مل </td></tr><tr><td> L-الجلوتامين </td><td> إينفيتروجن </td><td> 25030-032 </td><td> 2mm في (10ML / لتر) </td><td> 20 مل </td></tr><tr><td> مضاد حيوي / مضاد فطري </td><td> سيغما الدريخ </td><td> A5955-20ML </td><td> 10ML / لتر </td><td> 20 مل </td></tr><tr><td> الوليد العجل المصل </td><td> إينفيتروجن </td><td> 26010-74 </td><td> 2٪ (20 مل / لتر) </td><td> 40 مل </td></tr></tbody></table> الجدول 3. مواد وسيطة تخزين. إعداد وسائط تخزين إضافة جميع المكونات باستخدام تركيزات محددة على النحو المبين أعلاه في وعاء وتخلط جيدا. تصفية لهمباستخدام حجم المسام من فلتر ميكرون 0.2 (ميليبور، ميلانو، ايطاليا) مع مساعدة من مضخة تمعجية. الحفاظ على المتوسط ​​في زجاجات على RT. المواد اللازمة لنقل متوسطة (2000 مل). <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> مكونات </td><td> المزود </td><td> فهرس العدد </td><td> تركيز </td><td> كمية </td></tr><tr><td> MEM (1X) السائل </td><td> إينفيتروجن </td><td> 32360-034 </td><td></td><td> 1800 مل </td></tr><tr><td> الصوديوم البيروفات </td><td> إينفيتروجن </td><td> 11360-039 </td><td> 1MM (10 مل / لتر) </td><td> 20ml </td></tr><tr><td> L-الجلوتامين </td><td> إينفيتروجن </td><td> 25030-032 </td><td> 2mm في (10 مل / لتر) </td><td> 20ml </td></tr><tr><td> الوليد العجل المصل </td><td> إينفيتروجن </td><td> 26010-74 </td><td> 2٪ (20 مل / لتر) </tد&gt; <td> 40 مل </td></tr><tr><td> ديكستران T500 </td><td> Pharmacosmos </td><td> 551005004007 </td><td> 6٪ (60 جم ​​/ لتر) </td><td> 120 غرام / لتر </td></tr></tbody></table> الجدول 4. المواد لمتوسطة وسائل النقل. إعداد المتوسطة النقل إضافة ديكستران 6٪ في لتر من 1.5 ~ MEM وترك الأمر بين عشية وضحاها. إضافة باقي المكونات في وسائل الاعلام وتصفيتها باستخدام 0.2 ميكرون فلتر (ميليبور، ميلانو، ايطاليا) باستخدام مضخة فراغ. الحفاظ على المتوسط ​​في زجاجات على RT.

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

وتصف الورقة تقنية مبسطة لاستئصال القرنية وسلب أنسجة شبكية العين من الكرة الأرضية من الجهات المانحة جثي الإنسان. ووصف هذه التقنية هنا ستساعد على استئصال الأنسجة ذات نوعية جيدة لاستخدامها في زراعة الأعضاء، وأغراض الجراحية أو البحث دون الإضرار الأنسجة الأخرى من الكرة الأرضية العين.

أسلوب مبسط لل<em&gt; في الموقع</em&gt; استئصال القرنية ونزع الأحشاء من نسيج شبكي من الكرة الأرضية العين البشرية

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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Nanyang Technological University

Research

JoVE Journal

Medicine

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

20.8K Views.  Fondazione Banca Degli Occhi del Veneto O.N.L.U.S.. وتصف الورقة تقنية مبسطة لاستئصال القرنية وسلب أنسجة شبكية العين من الكرة الأرضية من الجهات المانحة جثي الإنسان. ووصف هذه التقنية هنا ستساعد على استئصال الأنسجة ذات نوعية جيدة لاستخدامها في زراعة الأعضاء، وأغراض الجراحية أو البحث دون الإضرار الأنسجة الأخرى من ?...

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

Technique to Collect Fungiform (Taste) Papillae from Human Tongue

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.

Technique to Collect Fungiform Taste Papillae from Human Tongue

Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue.

تقنية لجمع كمئي (التذوق) النتوئات الإنسان من اللسان

The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

أسلوب مبسط لإنتاج نموذج الجرح الدماغية

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

متعدد القطب تسجيلات صفيف من داء الصرع بعد الجراحة القشرية الأنسجة البشرية

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

عزل واستئصال الفئران الأبهر. تقنية متعددة الاستخدامات في دراسة أمراض القلب والأوعية الدموية

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

إنشاء الإنسان الظهارية Enteroids وColonoids من الأنسجة كامل وخزعة

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

العمل مع الأنسجة البشرية لبالحركة أبحاث السرطان

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

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.

سحجه الظهارة القرنية مع لدغ العين كنموذج للقرنية التئام الجروح

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 ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

تأثير تركيبات المسيل للدموع الاصطناعية على النشاط الأيضي للخلايا الظهارية القرنية البشرية بعد التعرض للديسيكيشن

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

توصيف رواية الإنسان Organotypic تقنية ثقافة الشبكية

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

نظام تحريض الشبكية Organoid لاستخلاص أنسجة الشبكية ثلاثية الأبعاد من الخلايا الجذعية البشرية متعددة القدرات

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...