The authors have no acknowledgements.

Bu protokoller, bizim kurumun insan araştırma etik komitesinin yönergeleri izleyin. 1. Geleneksel Kornea Kollajen CXL (C-CXL) Hastanın 1. Hazırlık <ol><li> 5 gün ameliyat öncesi,% 1 pilokarpin iki kez tedavi gözünde bir gün düşer koydu. </li><li> Operasyon odasında, aseptik koşullarda, onun / onun sırtında hasta yatıyor. </li><li> Topikal anestezi gibi oksibuprokain% 0.4 yönetin. </li><li> Göz ve iki kez antiseptik iyot ile göz çevresindeki deri temizleyin. </li><li> Gözü açık tutmak için bir kapak spekulumu kullanın. </li></ol> 2. Epitel Kaldırma <ol><li> Bir daire kornea işaretleyici ile korneanın merkezi 9.0 mm işaretleyin. </li><li> Künt spatula kullanarak mekanik debridman ile kornea epitel merkezi 7,0 9,0 mm çıkarın. </li></ol> 3. Riboflavin Uygulaması <ol><li> % 0.1 th% 20 Dextran&#39;ın ile riboflavin Uygula<html> e kornea 20 dakika boyunca her dakika. </li></ol> 4. UVA Işınlama <ol><li> 3 mW / cm2 (5.4 J / cm2 yüzey doz) ışınımda ve 30 dakika boyunca çalışma mesafesi, bir 5 cm &#39;de bir 370 nm dalga boyu UVA ışığı ile kornea ışın tedavisi. </li></ol><img alt="figür 1" src="/files/ftp_upload/53119/53119fig1.jpg" /> Şekil 1:., C-cxl UVA ışıma kornea 3 mW / cm2 (5.4 J / cm2 yüzey doz) ışınımda ve 30 dakika boyunca 5 cm&#39;lik çalışma mesafesi bir 370 nm dalga boyu UVA ışığı ile ışınlandırılır. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol start="2"><li> Işınlama sırasında korneaya her 5 dakikada riboflavin damla uygulanır. </li><li> Işınlama sırasında, topikal anestezi (oksibuprokain% 0.4), gerekirse ilave edin. </li></ol>Cerrahi &quot;&gt; 5. Sonu <ol><li> Put antibiyotikli damlalar (tobramisin% 0.3) ve işletilen göz içine yapay gözyaşı (hyaluronate 0.18% düşer). </li><li> Re-epitelizasyon tamamlanana kadar ameliyat sonunda yumuşak bir bandaj kontakt lens yerleştirin. Yeniden epitelizasyon genellikle 3 gün sürer. </li><li> Parasetamol (500 mg), ve kodein (30 mg), 6 hap Günde olarak analjezikler öngörülmesi-. </li><li> Kornea reepitelizasyon sonra steroid topikal tedavi (topikal deksametazon 1 mg / ml) başlatmak ve 3-4 hafta devam eder. Ayrıca, 1 ay boyunca suni gözyaşları günde 4 kez kullanın. </li></ol> 2. Hızlandırılmış Kornea Kollajen CXL (A-CXL) Hastanın 1. Hazırlık <ol><li> 5 gün ameliyat öncesi,% 1 pilokarpin iki kez tedavi gözünde bir gün düşer koydu. </li><li> Operasyon odasında, aseptik koşullarda, onun / onun sırtında hasta yatıyor. </li><li> Topikal anestezi gibi oksibuprokain% 0.4 yönetin. </li><li> E temizleyinSiz ve iki kez antiseptik iyot ile göz çevresindeki deri. </li><li> Gözü açık tutmak için bir kapak spekulumu kullanın. </li></ol> 2. Epitel Kaldırma <ol><li> Bir daire kornea işaretleyici ile korneanın merkezi 9.0 mm işaretleyin </li><li> Künt spatula kullanarak mekanik debridman ile kornea epitel merkezi 7,0 9,0 mm çıkarın. </li></ol> 3. Riboflavin Uygulaması <ol><li> 10 dakika boyunca kornea üzerinde Dextran&#39;ın olmadan her 2 dakikada bir% 0.1 riboflavin uygulayın. </li></ol> 4. UVA Işınlama <ol><li> 30 mW / cm2 (5.4 J / cm2 yüzey dozu) ve 3 dakika boyunca 5 cm&#39;lik çalışma mesafesinde bir radyasyonunun bir 370 nm dalga boyu UVA ışığı ile kornea ışın tedavisi. </li><li> (Oxybuproca &amp; # 239;% 0.4 NE) Işınlama sırasında, topikal anestezi eklemek gerekirse. </li></ol> Cerrahi 5. Sonu <ol><li> Antibiyotikli damlalar (tobramisin% 0.3) ve yapay gözyaşları (hyaluronate 0 damla yerleştirin<html> Ameliyat göz içine 0,18%). </li><li> Re-epitelizasyon tamamlanana kadar ameliyat sonunda yumuşak bir bandaj kontakt lens yerleştirin. Yeniden epitelizasyon genellikle 3 gün sürer. </li><li> Parasetamol (500 mg), ve kodein (30 mg), 6 hap Günde olarak analjezikler öngörülmesi-. </li><li> Kornea reepitelizasyon sonra steroid topikal tedavi (topikal deksametazon 1 mg / ml) başlatmak ve 3-4 hafta devam eder. Ayrıca, 1 ay boyunca suni gözyaşları günde 4 kez kullanın. </li></ol> 3. İyontoforez (I-CXL) Hastanın 1. Hazırlık <ol><li> 5 gün ameliyat öncesi,% 1 pilokarpin iki kez tedavi gözünde bir gün düşer koydu. </li><li> Operasyon odasında, aseptik koşullarda, onun / onun sırtında hasta yatıyor. </li><li> Topikal anestezi gibi oksibuprokain% 0.4 yönetin. </li><li> Göz ve iki kez antiseptik iyot ile göz çevresindeki deri temizleyin. </li><li> Gözü açık tutmak için bir kapak spekulumu kullanın. </li></ol><html> _step &quot;&gt; 2. İyontoforez Cihazı yerleştirin. <ol><li> Operatif alan altında alnına yapışkan pasif elektrot uygulayın. </li><li> Açık göze, aktif elektrot, bir emme halkası uygulayın. Emme bırakmadan önce korneanın çevresine emme halkası ortalayın. </li></ol><img alt="Şekil 2," src="/files/ftp_upload/53119/53119fig2.jpg" /> Şekil 2. İyontoforez cihazı. Pasif elektrot ameliyat alanının altındaki alnına ve aktif elektrot, bir emme halkası üzerinde uygulanan açık göze uygulanır. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> 3. Riboflavin Uygulaması <ol><li> 0,1 hypoosmolar% Dekstran olmayan riboflavin emme halka doldurun. </li></ol><html> jpg &quot;/&gt; I-CXL Şekil 3. Riboflavin uygulaması. Emme halkası Dextran&#39;ın olmadan hypoosmolar% 0.1 riboflavin ile doldurulur. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <ol start="2"><li> 0.2 mA elektrik akımı başlayın ve giderek 5 dakika (Şekil 4) toplam iyontoforez süre için 1,0 mA artar. </li></ol><img alt="Şekil 4," src="/files/ftp_upload/53119/53119fig4.jpg" /> Nüfuz riboflavin için Şekil 4. İyontoforez cihazı. Elektrik akımı başlangıçta 0,2 mA ve yavaş yavaş 1.0 mA yükselmiştir. Toplam iyontoforez süresi 5 dakikadır. <a href="https://www.jove.com/files/ftp_upload/53119/53119fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> ep &quot;&gt; 4. UVA Işınlama <ol><li> 10 mW / cm2 (5.4 J / cm2 yüzey dozu) ve 9 dakika boyunca 5 cm&#39;lik çalışma mesafesinde bir radyasyonunun bir 370 nm dalga boyu UVA ışığı ile kornea ışın tedavisi. </li><li> (Oxybuproca &amp; # 239;% 0.4 NE) Işınlama sırasında, topikal anestezi eklemek gerekirse. </li></ol> Cerrahi 5. Sonu <ol><li> Antibiyotik damla (tobramisin% 0.3) ve işletilen göz içine yapay gözyaşı (hyaluronate 0.18% düşer) yerleştirin. </li><li> Parasetamol (500 mg), ve kodein (30 mg), 6 hap Günde olarak analjezikler öngörülmesi-. </li><li> Ameliyattan sonra, steroidler ile topikal tedavi (topikal deksametazon 1mg / ml) başlatmak ve 3-4 hafta devam eder. Ayrıca, 1 ay boyunca suni gözyaşları günde 4 kez kullanın. </li></ol>

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+safety+of+transcorneal+iontophoresis+of+tobramycin+in+the+rabbit.">Pharmacokinetics and safety of transcorneal iontophoresis of tobramycin in the rabbit.</a> Invest Ophthalmol Vis Sci. 29 (9), 1397-1401 (1998).</li><li>Vinciguerra, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+iontophoresis+corneal+collagen+cross-linking+for+progressive+keratoconus:+initial+clinical+outcomes.">Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes.</a> J Refract Surg. 30 (11), 746-753 (2014).</li><li>Caporossi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboflavin-UVA-induced+corneal+collagen+cross-linking+in+pediatric+patients.">Riboflavin-UVA-induced corneal collagen cross-linking in pediatric patients.</a> Cornea. 31 (3), 227-231 (2012).</li><li>Buzzonetti, L., Petrocelli, G., Valente, P., Larossi, G., Ardia, R., Petroni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+transepithelial+corneal+cross-linking+to+halt+keratoconus+in+pediatric+cases:+15-month+follow-up.">Iontophoretic transepithelial corneal cross-linking to halt keratoconus in pediatric cases: 15-month follow-up.</a> Cornea. 34 (5), 512-515 (2015).</li><li>Baiocchi, S., Mazzotta, C., Cerretani, D., Caporossi, T., Caporossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+crosslinking:+riboflavin+concentration+in+corneal+stroma+exposed+with+and+without+epithelium.">Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium.</a> J Cataract Refract Surg. 35 (5), 893-899 (2009).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histological+changes+after+corneal+crosslinking+with+and+without+epithelial+debridement.">Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.</a> J Cataract Refract Surg. 35 (3), 540-546 (2009).</li><li>Soeters, N., Wisse, R. P., Godefrooij, D. A., Imhof, S. M., Tahzib, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+versus+epithelium-off+corneal+cross-linking+for+the+treatment+of+progressive+keratoconus:+a+randomized+controlled+trial.">Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial.</a> Am J Ophthalmol. 159 (5), 821-828 (2015).</li></ol>

The authors have nothing to disclose.

UVA ışınlama ve riboflavin kullanılarak CXL keratokonus ilerlemesini durdurma standart tedavi yöntemidir. Riboflavin UVA 3 ile ışınlanması kimyasal kovalent bağlar (çapraz bağlar) oluşturan bir ışığa olan. Kornea olarak, bu olay, kornea sertliğini arttırmak kolajen fibrilleri arasındaki çapraz bağlantılar oluşturur. Bu fenomen, tarif edilmiş olmasına rağmen, şimdiye kadar, kornea içi çapraz bağların doğrudan bir kanıt yoktur. Bununla birlikte, bazı çalışmalar, böylece C-...

Keratokonus genellikle kornea şeklinin değiştirilmesi ile sonuçlanan genel popülasyonda 1 2,000 içinde 1 bildirilen ikili ve ilerleyici korneal ektazi ve böylece görme 2 azaldı. Keratokonus erken ergenlik genellikle mevcut ve hastalık genellikle stabilize eğilimi zaman ilerleme hastanın yaşamı boyunca değişken olabilir, ancak yaşamın dördüncü on üçüncü dek ilerler. Keratokonus ilerlemesini durdurarak, çapraz bağlama erteleme veya önlemek keratoplasti amaçlamaktadır. Bugüne kadar, klinik çalışmalarda kanıtlanmış ilerici keratokonus tek etkili ve güvenli bir tedavi sertliğini arttırmak ve dolayısıyla keratokonus ilerlemesini durdurmak 3-8 amaçlayan geleneksel kornea kollajen çapraz bağlama (C-CXL) protokoldür. Çalışma süresini ve enfeksiyöz keratit veya stromal pus 9 olarak C-CXL diğer olası risk faktörlerini azaltmak amacıyla, birçok gelişmiş protokoller var isetanımlanmıştır. İlk olarak, hızlandırılmış cxl (A-cxl) &#39;de, UVA daha yüksek bir ışık şiddeti azaltılmış zaman üzerinde 10 kornea teslim edilir. İkincisi, epitel debridmanı için gerekliliğini önlemek için, transepitelyal yaklaşımlar kullanılmıştır. Geleneksel protokole 11 oranla Ne yazık ki, onlar başarıyı sınırlıdır. CXL sırasında kornea riboflavin teslimat için en son transepitelyal yöntemi iyontoforez (I-CXL), ancak bu tedavinin titiz değerlendirme henüz 12 yapılmamıştır. İyontoforez küçük bir elektrik akımı, bir doku içinde bir iyonize ilacın nüfuz etmesini artırmak için uygulandığı bir invasif olmayan bir tekniktir. Iyontoforez ile CXL olarak, riboflavin epitel içinden kornea nüfuz iyonize edilir. Vivo konfokal mikroskopi (IVCM) bu tür keratokonus 13 gibi hastalıklarda anormal kornealar hücresel değişiklikler vurgulayabilirsiniz kornea görüntüleme yöntemidir. Gerçekten de, IVCMalt taban sinir ağına yoğunluğu, belirli bir azalma ile Keratokonusta korneanın tüm katmanları değişiklikler gösterdi ve stromal 13-15 keratositlerde gelmiştir. Ayrıca, IVCM Cı-cxl sonrası 16 korneanın mikro analiz için son derece uygun olduğu kanıtlanmıştır. Kornea sınır çizgisi 300 mikron 17,18 derinlikte C-CXL sonra hiperreflektif (OKT AS) ön segment optik koherens tomografi görülen hat 1 ay olarak tarif edilir. IVCM aşağıdaki C-CXL 300 mikron derinliğinde kornea keratositlerin yokluğu da dahil olmak üzere kornea yapısal değişiklikler hakkında bilgi sağlar. Bu acellular bölgenin derinliği, yanı sıra korneal stroma içinde sınır hattının derinliği, OCT 1 AS de CXL tedavisine 19 ve kornea sınır hattı derinliği ölçümü etkin derinliği ile ilişkili görünmektedir OKT AS üzerinde ortaya CXL sonra ay verimli bir klinik olarak öne sürülmüştürcxl etkinliği 18 değerlendirilmesi için yöntem. Bu çalışmada OCT ve konfokal mikroskopi AS tarafından kornea stromal sınır çizgisinin ölçümü kullanılarak üç farklı (geleneksel, hızlandırılmış ve iyontoforez) kornea kollajen çapraz bağlanma protokollerin etkinliğini araştırmak. Biz ayrıca niceliksel üç tedavilerden sonra kornea mikro değişiklikleri analiz etmek IVCM kullanılır.

<table>
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td>Riboflavin&#160;&#160;&#160;&#160;&#160;&#160;&#160; Product number</td>
			<td></td>
		</tr>
		<tr>
			<td>C-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 468465-6</td>
			<td></td>
		</tr>
		<tr>
			<td>A-CXL</td>
			<td>Avedro Inc, Waltham, Massachusetts</td>
			<td>VibeX&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 520-01863-006</td>
			<td></td>
		</tr>
		<tr>
			<td>I-CXL</td>
			<td>Sooft SPA, Montegiorgio, Italy</td>
			<td>Ricrolin+&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 975481-6</td>
			<td>Passive electrode: PROTENS ELITE 4848LE/ Active electrode: IONTOFOR CXL</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>UVA Machine</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 3 mW/cm2 30 min</td>
		</tr>
		<tr>
			<td>KXL System</td>
			<td></td>
			<td></td>
			<td>UVA: 30 mW/cm2 10 min</td>
		</tr>
		<tr>
			<td>X-Vega</td>
			<td></td>
			<td></td>
			<td>UVA: 10 mW/cm2 9 min</td>
		</tr>
	</tbody>
</table>

three different protocols of corneal collagen crosslinking in keratoconus: conventional, accelerated and iontophoresis

Keratokonus ikili ve ilerleyici korneal ektazi olduğunu. Ilerlemesini yavaşlatmak amacıyla, kornea kollajen çapraz bağlama (CXL) kısa etkili bir tedavi seçeneği olarak kullanıma girmiştir. Biyolojik ve kimyasal bilimleri, çapraz reaktif moleküller arasında oluşan yeni kimyasal bağlar anlamına gelir. Bu nedenle, kornea kollajen cxl amacı sentetik kornea stromada kolajen fibrilleri arasındaki çapraz bağların oluşumunu artırmaktır. Geleneksel CXL (C-CXL) protokolünün etkinliği zaten çeşitli klinik çalışmalarda gösterilmiştir olmasına rağmen, prosedür ve kornea epitel çıkarılması süresindeki iyileştirmeler yarar olabilir. Bu nedenle, iki yeni ve optimize cxl protokolleri tutarlı bir değerlendirme sağlamak üzere, üç tedavi cxl bir geçirmiş keratokonus hasta araştırılmıştır: iyontoforez (I-cxl), CXL (A-cxl), ve konvansiyonel CXL (hızlandırılmış C-CXL). A-CXL 6 süresi, daha hızlı CXL prosedürü uHala bir epitel çıkarılması da dahil olmak üzere on zaman yüksek UVA ışınımı şarkı ama. İyontoforez küçük bir elektrik akımı kornea boyunca riboflavin penetrasyonunu artırmak tatbik edildiği bir transepitelyal invasif olmayan bir tekniktir. (OKT AS) ve in vivo konfokal mikroskopi (IVCM) ön segment optik koherens tomografi kullanarak, tedavi penetrasyon derinliği konusunda, geleneksel CXL protokolü ilerici keratokonus tedavisinde standart olmaya devam sonucuna varıldı. Hızlandırılmış CXL ince korneası tedavisi için, hızlı, etkili ve güvenli bir alternatif gibi görünüyor. Iyontoforez kullanımı hala araştırılmaktadır ve daha dikkatli ele alınmalıdır.

<html> Kornea sınır çizgisi (73.6 SD) 301,6 um ortalama derinlikte vakaların% 92&#39;sinde OCT AS görünür oldu <img alt="Şekil 5," src="/files/ftp_upload/53119/53119fig5.jpg" /> C-CXL sonra Şekil 5. Demarkasyon hattı. (OKT AS) Yüksek çözünürlüklü kornea ön segment optik koherens tomografi taraması, geleneksel kornea kollajen çapraz sonra 358 mikron (beyaz ok), 1 ay ortalama derinliği kornea stromal sınır hattı...

Watch this Scientific Journal Video about Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis at JoVE.com

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Keratokonus Kornea Kollajen çapraz bağlama Üç Farklı Protokoller: Konvansiyonel, Hızlandırılmış ve İyontoforez

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

three-different-protocols-corneal-collagen-crosslinking-keratoconus

Research

JoVE Journal

Medicine

19.8K Görüntüleme.  Quinze-Vingts National Ophthalmology Hospital. Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Video: Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

İnsan Pankreas Adacıklar için boyama Protokoller

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Tıp

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Fare Kornea Neovaskülarizasyonunun bir Alkali-yanık Yaralanma Modeli

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Rat Model of Ventricular Fibrillation and Resuscitation by Conventional Closed-chest Technique

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic components of cardiopulmonary resuscitation in humans is herein described. The model was developed in 1988 and has been used in approximately 70 peer-reviewed publications examining a myriad of resuscitation aspects including its physiology and pathophysiology, determinants of resuscitability, pharmacologic interventions, and even the effects of cell therapies. The model featured in this presentation includes: (1) vascular catheterization to measure aortic and right atrial pressures, to measure cardiac output by thermodilution, and to electrically induce ventricular fibrillation; and (2) tracheal intubation for positive pressure ventilation with oxygen enriched gas and assessment of the end-tidal CO2. A typical sequence of intervention entails: (1) electrical induction of ventricular fibrillation, (2) chest compression using a mechanical piston device concomitantly with positive pressure ventilation delivering oxygen-enriched gas, (3) electrical shocks to terminate ventricular fibrillation and reestablish cardiac activity, (4) assessment of post-resuscitation hemodynamic and metabolic function, and (5) assessment of survival and recovery of organ function. A robust inventory of measurements is available that includes &#8211; but is not limited to &#8211; hemodynamic, metabolic, and tissue measurements. The model has been highly effective in developing new resuscitation concepts and examining novel therapeutic interventions before their testing in larger and translationally more relevant animal models of cardiac arrest and resuscitation.

This article describes a rat model of electrically-induced ventricular fibrillation and resuscitation by chest compression, ventilation, and delivery of electrical shocks that simulates an episode of sudden cardiac arrest and conventional cardiopulmonary resuscitation. The model enables gathering insights on the pathophysiology of cardiac arrest and exploration of new resuscitation strategies.

Konvansiyonel Kapalı göğüs Tekniği ile Rat Ventriküler Fibrilasyon Modeli ve Resüsitasyon

A rat model of electrically-induced ventricular fibrillation followed by cardiac resuscitation using a closed chest technique that incorporates the basic ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Farelerde Kalp Kapak Hastalıkları Kapsamlı Fenotipik Karakterizasyonu için ekokardiyografik Yaklaşımlar ve Protokoller

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Farelerde Hızlandırılmış Ateroskleroz Indüksiyonu: "Tel-Yaralanma" Modeli

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

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.

Kornea epitel aşınma oküler Burr ile bir Model olarak kornea için yara iyileşme

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 ...

Simultaneous Laryngopharyngeal and Conventional Esophageal pH Monitoring

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, hoarseness or asthma, which can be caused by laryngopharyngeal reflux (LPR). Due to their multifactorial origin, those symptoms can be a great diagnostic and therapeutic challenge. Esophageal pH-monitoring is commonly used to determine abnormal esophageal acid exposure and confirm the diagnosis of GERD. However, for better evaluation of acid exposure above the upper esophageal sphincter, a new laryngopharyngeal pH measurement system is now available and may lead to more reliable results in patients with predominantly extraesophageal symptoms. This article aims to present a standardized protocol for simultaneous pH measurement using esophageal and laryngopharyngeal pH probes in order to obtain acid exposure scores from both measurements.

Laryngopharyngeal pH monitoring has been specifically designed to measure acid exposure above the upper esophageal sphincter and complements diagnostic evaluation in patients that present with mainly extraesophageal reflux symptoms. Patients with suspected laryngopharyngeal reflux (LPR) were evaluated using distal esophageal and laryngopharyngeal pH testing simultaneously.

Eşzamanlı Laringofaringeal ve Konvansiyonel Özofagus pH İzleme

In addition to typical reflux symptoms, many patients with gastroesophageal reflux disease (GERD) present with extraesophageal symptoms such as cough, ...

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Karaciğer Rejenerasyon Stratejisi Olarak Üç Boyutlu Kollajen Matris İskele İmplantasyonu

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Kornea Epitelinin Mekanik ve Kimyasal Yaralanmaları için Ex Vivo ve In Vivo Hayvan Modelleri

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Descemet'in Sadece Mühendislik Fibroblast Büyüme Faktörü 1 Tarafından Uyarılan Hızlandırılmış İyileşme ile Sıyırılmasının İnsan Kornea Organ Kültürü Modeli

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...