Yazarlar Toskana Bölgesi&#39;nin (POR Creo FESR 2007-2013 Bando Unico R &amp; S 2012) tarafından finanse FORTE Projesi, kısmen araştırma faaliyetleri desteklenen AB 7.ÇP ECHORD ++ Deney YG-ROSES ve verilen FP7 BiophotonicPlus Projesi &quot;LITE&quot; teşekkür etmek istiyorum Toskana Bölgesi tarafından.

Çalışma hastanenin Etik Kurul onayı ile prospektif yapılmıştır; bilgilendirilmiş onam alındı. Çalışma Helsinki Bildirgesi&#39;nin ilkelerine bağlılık oldu. 1. Donör Endotel Hazırlık <ol><li> Oda sıcaklığında yerel göz banka tarafından hazırlanan bir donör korneanın kullanın. </li><li> Ameliyat odasında, kendi dağıtım konteynerinden donör kornea çekin ve kornea taşıma için kullanılan doku korunması ve beslenmesi için çözüm ayrı ayarlayabilirsiniz. </li><li> Bir sıvı akmaya ve değişken basınç oluşturabilir içine yapay ön kamara üzerinde donör corneo-skleral rim yerleştirin; Doku tutucu kafa ile kornea kapağı. </li><li> Kornea korunması ve beslenme sıvı ile dolu bir şırınga 3-yollu konnektörü aracılığıyla yapay ön kamara (AAC) bağlayın. </li><li> Yerine sıkıca tutucu tutmak için sıkıştırma halkasını kullanın. </li><li> SYR kullanınOptimum basıncı (1.7 bakınız) ulaşana kadar doku koruma solüsyonu ile ön odasını doldurun: inge intrakamaral basıncı korumak için AAC bağlı. Konektörü kapatın. </li><li> Bir parmak ile ön kamara içindeki basıncı test edin. (Aralık 60-70 mmHg olarak) sağ iç basıncı ulaşana kadar iç basıncını değiştirir. 3-yollu konnektörü kapatın. </li><li> Optik koherens Thomography (OKT) ile donör kornea kalınlığını ölçün. Enstrüman optik önünde sabit bir pozisyonda olduğunu kornea ile ön odasını koruyun ve ardından tam bir kalınlıkta-Ekim satın almak. </li></ol> Donör Endotelyumunun 2. Femtosecond Lazer Hazırlık <ol><li> Femtosaniye lazer aygıtının altında donör kornea aplanasyon gerçekleştirin. Arka tarafı kesme, tam lamellar ve ön yan kesim: Üç müteakip kesim ile donör doku kesmek için femtosec lazer kullanın. Üç kesimler için aşağıdaki parametreleri ayarlayın.<ol><li> Tam lameller kesim (raster desen, işe başlamak) için, daima 95 mikron çıkarılarak, (1.8 anlatıldığı gibi OKT ile ölçülen) donör kornea ince noktasına karşılık gelen kesik derinliğini ayarlayın. Aralık (çalışma derinliğine bağlı) 0.8-0.9 μJ darbe enerjisini ayarlayın. 8.7 mm çap ayarlayın. 2 um teğet nokta ayrımı ayarlayın. 2 um radyal nokta ayrımı ayarlayın. </li><li> Ön yan kesim, önceki tam lameller kesim 30 mikron derin arka derinliğini ayarlayın. 2.10 μJ nabız enerjisi ayarlayın; 8,6 mm çapı; tabaka ayrılması sırasında 3 um 3&#39;e um nokta ayırma. </li><li> Posterior yan kesim için, arka tarafı ön derinliği tam lameller kesim daha öne 30 mikron kesti bulun. 900 mikron posterior derinliğini ayarlayın. 2.10 μJ nabız enerjisi ayarlayın; 8.3 mm çap; 2 um nokta ayırma; 2 um tabaka ayrışması. </li></ol></li&gt; </ol> 3. Alıcı Göz Hazırlık <ol><li> Ameliyat için hastayı hazırlayın. Bıçak önceden ayarlandı 4,0 mm 12:00 azından bir 4.00 mm kornea kesi yapmak. 02:00 yerleştirilen bir 1,2 mm önceden ayarlandı bıçağı ile tek limbal parasentez, emin olun. 30 ° bıçak bıçak ile 06:00 başka biri limbal parasentez olun. </li><li> 02:00 parasentez ile, hastanın ön kamarada bir ön kamara sürdürücü yerleştirin. </li><li> Özel kancalı biri 8.2 mm çapında dairesel descemetorexis gerçekleştirin. </li><li> Posterior stromadan Descemet membranı ve endotel soyun ve bir Descemet kanca ile dokuları çıkarın. </li></ol> 4. Kromofor Hazırlık <ol><li> 1.5 ml mikrosantrifüj tüp içinde İndosiyanin Yeşil tozu 1 mg koyun. </li><li> Steril su, 9 mg ekleyin (ICG su çözeltisi ağırlık / ağırlık% 10). </li><li> Manuel metal karıştırıcı ile ICG tozu ve su karışımı. </li&gt; </ol> Donör Endotelyumunun 5. boyanması <ol><li> Enjektör yüzeyi ile temas halinde, iç tarafı bir Busin enjektör daha geniş kısmına donör endoteli koyun. </li><li> Bir spatula kullanarak, kendi periferik kısmında, kromofor çözeltisi ile donör endoteli iç tarafını Leke. Doğru lekeli doku homojen yeşilimsi bir renge sahiptir. Adım 5.3 başlamadan önce 3 dakika bekleyin. </li><li> Busin-enjektörün ön kısmına donör endotel çekin devirdi. </li></ol> 6. Donör endoteline takma <ol><li> Kavrayın ve endotel lenticule için atravmatik koaksiyel forseps kullanarak katlanmış donör endotelyumu yerleştirin. </li><li> Ön kamara sürdürücü çıkarın. </li><li> Korneal kesi ve Naylon 10.0 tek Stich ile parasentez (adım 3.1 gerçekleştirilen) dikin. </li><li> Donör doku açılmak ve alıcı kornea karşı basın bir hava kabarcığı enjekte edilir. hava kabarcığı zorunlu olmalıletely ön kamara boşluğu doldurmak. </li></ol> 7. Lazer Kaynak <ol><li> Bir kanca ile veya bir spatula ile korneanın dış hava kabarcığı hareket içinden, alıcı kornea merkezinde donör lenticule yerleştirin. </li><li> Sayısal açıklık (NA) olarak 0.22 ile 300 mikron çekirdek çapı steril fiber optik ile donatılmış 810 nm&#39;de yayan bir diyot lazer, kullanın. </li><li> Göz küresi dışında lif ucu tutun ve şeffaf kornea dokusunun aracılığıyla, lekeli endotele doğru lazer ışığı sağlar. fiber ucu temassız konfigürasyonundadır. </li><li> Tek nokta emisyon modu, 70-80 msn darbe süresi, darbe başına 35-40 mJ: lazer için aşağıdaki ayarları kullanın. He-Ne ışın hedefleyen. </li><li> Donör endotel lekeli çevresine tek lazer noktalar sunun. Sırayla noktalar sunun: nihai boy donör lenticule çevresinin noktalar bir halkasıdır. İki adja arasındaki mesafeyüzde noktalar merkezi nokta çapı iki katıdır. </li><li> % 0.3 tobramisin ve% 0.1 deksametazon oftalmik süspansiyon ile birlikte, hasta, göz ile ilgili bir kontakt lens uygulanır. </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, F., 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., 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=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, F., 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, L., 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, L., 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, L., 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=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

&quot;Tüm lazer&quot; endotel nakli orijinal bir yaklaşım minimal invaziv kornea nakli etmektir. Protokolü kapsamında açıklanan tüm prosedürler gibi steril eldiven, önlük, maske ve kapak kullanımı gibi ameliyatlar sırasında ortak uygulamaları, hijyenik ve sterilizasyon işlemi gözlemleyerek, cerrahi odasında yapıldı. ICG çözümü yakında donör endotelyumu boyama onun uygulama öncesi, ameliyat odasında hazırlanmıştır. ICG toz, su ve boyama çözeltisi hazırlamak...

Bu çalışmada, donör doku ve alıcı yatağın üzerine kaynak yapılması için bir kızıl ötesine yakın diyot lazer hazırlamak için femto saniye lazer kullanımına dayalı endotelyal keratoplastinin özgün bir yaklaşım sunmaktayız. Donör kornea intraoperatif ölçümü doğru donör doku boyutlarını tasarlamak için gereklidir. Endotel keratoplasti endotel hastalık 1,2 tedavisinde penetran keratoplasti yerine son yıllarda öne sürülmüştür. Bu tekniğin önemli avantajı, cerrahi, aşı reddi bir azalma riski ve göz bütünlüğünün korunması sırasında keratoplastinin indirgenmiş anestezi nüfuz ile ilgili olarak, daha hızlı bir görsel bir geri kazanılmasıdır. temel risk faktörü postoperatif donör lenticule çıkık olduğunu. standart teknik bir hava kabarcığı enjekte tarafından yapılmaktadır nihai pozisyonunda donör endoteli takarak gerçekleştirilir: hayır dikiş, çünkü mekanik biyofizik ve boyutsal chara ve kullanılanendotel cteristics. Ayrıca, görme keskinliği kurtarma nedeniyle nakledilen kalın dokuya temel nedeni donör ve alıcı dokular arasında bir uyumsuzluk nedeniyle sınırlı olabilir. Burada o ana sorunların üstesinden gelebilir endotel keratoplasti yerine getirirken bir prosedür mevcut. Donör endotel lazer kaynak tekniği ile, son pozisyonda sağlanabilir. Bu kontrollü ve lokal fototermal süreçtir: bu donör / alıcı ara yüzeyinde indüklenebilir. Bu son on yıl içinde okudu ve keratoplasti ve endotele 3-5 nakli öne sürülmüştür. kızıl ötesine yakın ışığı (dalga uzunluğu: 810 nm), bir düşük güç diyot lazer tarafından yayılan yara bölgesinde biyolojik doku doğru sevk edilir. Kornea bu dalga boyunda doğal saydamdır: lazer ışığı absorbe Bu doku yapmak için, bir kromofor ile lekelenmek gereklidir. Önerilen boya steril doyur olanIndosiyanin Green (ICG) d su çözeltisi. Biz kornea dokusu düzgün bu ICG hazırlığı ile boyanmış olduğunda, bu 810 nm 6&#39;da absorpsiyon pik gösterir gösterdi. Ayrıca, yaygın olarak ICG Klinik teşhis olarak kullanılır ve güvenlik hali hazırda insan deneklerde gösterilmiştir. lekeli kornea diyot lazer ışığı enerjisini emer ve ana sonuçlanan etkisi kaynak yerinde kontrollü bir sıcaklık artışı olduğunu. Resim termal etkiler boyanmamış dokularda indüklenir. Sıcaklık artışı soğutma üzerine sarılmış duvarlar acil closuring olan stromal kollajen tersine çevrilebilir bir termal denatürasyon indükler. Bu lazer kaynak etkisi öncelikle katarakt ameliyatı 7,8 ve keratoplasti 9,10 delici gösterilmiştir. Bu yazıda sunuyoruz optimize edilmiş bir yaklaşım endotelyal keratoplasti uygulama için çalışılmıştır. Önerilen cerrahisinde tek lazer noktalar (msn süren onlarca) are nokta boyutunda (çapı mikron birkaç yüz) içinde lokalize bir fototermal etkisi ile sonuçlanan doku teslim: kaynaklı etkinin donör / ana arayüzünde sınırlı kollajen fotokoagülasyon oluşan sert bir lazer kaynak olduğunu. kaynaklı yerinde kollajen denatürasyon sonucu, böylece standart tekniklere (dikiş) ile elde etmek imkansız bir sütür etki sağlayarak, donör ve host dokular arasında güçlü bir yapışma olduğunu. Doku kısa takip (1 ay) onun doğal bir görünüm kavuşur ve donör / konak dokuları arasında yapışma iyileşme evresinin çok erken dönemde sağlanan kaynakla geliştirildi. Kalın donör doku nakli, yani endotelyal keratoplasti diğer ana riskini önlemek için, intrasurgical optik koherens tomografi (OCT) kullanılır: Doğru kesim profili ile dizayn edilebilir, böylece ticari bir cihaz, donör kornea kalınlığını ölçerfemtosec lazer. Önerilen &quot;tüm lazer&quot; endotel nakli dolayısıyla bu minimal invaziv cerrahi klinik sonuçlarını geliştirmek gibi görünüyor.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pulsion Medical Systems, Germany</td>
			<td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td>
			<td>Alternative product: IC-GREEN, Akorn&#160; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td>
		</tr>
		<tr>
			<td>Femtosecond Laser&#160;</td>
			<td>Abbott Medical Optics,&#160; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td>
			<td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Coherence Tomography (OCT)</td>
			<td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td>
			<td>Visante</td>
			<td></td>
		</tr>
		<tr>
			<td>Diode Laser</td>
			<td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td>
			<td>Mod. WELD 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Anterior Chamber</td>
			<td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td>
			<td>&#160;Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_anterior_chamber.html)</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution for tissue preservation and nutrition&#160;&#160;</td>
			<td>&#160;AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&#242; - PD 
			&#160;ITALY</td>
			<td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&#176;-c-eb/carry-c-eb)</td>
			<td></td>
		</tr>
	</tbody>
</table>

an &#8220;all-laser&#8221; endothelial transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

&quot;Tüm lazer&quot; cerrahi işlem minimal invaziv kornea nakli gerçekleştirmek için önerilmiştir. saygı ile standart endotel nakli, kornea kalınlığı ölçümü donör doku boyama ve eklenen lazer ışığı teslim sadece birkaç adım için: prosedürü (Şekil 1) gerçekleştirmek kolaydır. elde edilen avantajlar büyük ölçüde birkaç dakika artan bir cerrahi zamanı telafi eder. Donör kornea kalınlığı ve donör lenticule boyutlarını özelleştirmek için kullanılan femtosani...

Watch this Scientific Journal Video about An â€œAll-laserâ€ Endothelial Transplant at JoVE.com

An &amp;#8220;All-laser&amp;#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

Bir &quot;All-lazer&quot; Endotelyal Nakli

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Research

JoVE Journal

Medicine

An &#8220;All-laser&#8221; Endothelial Transplant

9.3K Görüntüleme.  Italian National Research Council. An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

Video: An &#8220;All-laser&#8221; Endothelial Transplant

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Rekabetçi bir mürin Kemik İliği Transplantasyon Modelinde Donör Hücre Aşı belirleme Kantitatif Gerçek-zamanlı PCR kullanarak

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Tıp

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Mürin Heterotopik Kalp Nakli Tekniği

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

Active Porto-Caval Shunt kullanarak Domuz Karaciğer Tedarik ve Ortotopik Nakli Tekniği

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Aloimmün kaynaklı Vasküler Reddi ve Nakli arterioskleroz Fare Modeli

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Murin Böbrek nakli Tekniği

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

Anti-sıtma Primaquine UV-Vis ve Çıplak Göz Tayini için Optimize Griess Reaksiyon

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Sıçan Karaciğer Nakli Modelinde Hemodinamik Değişiklikleri Simüle Ederek Organ İskemi-Reperfüzyon Yaralanması

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

TBase - an Integrated Electronic Health Record and Research Database for Kidney Transplant Recipients

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory values, medical reports, radiology and pathology data) via standardized interfaces with manual data entry during routine treatment (e.g., clinical notes, medication list, and transplantation data). By this means, a comprehensive database for KTR is created with benefits for routine clinical care and research. It enables both easy everyday clinical use and quick access for research questions with highest data quality. This is achieved by the concept of data validation in clinical routine in which clinical users and patients have to rely on correct data for treatment and medication plans and thereby validate and correct the clinical data in their daily practice. This EHR is tailored for the needs of transplant outpatient care and proved its clinical utility for more than 20 years at Charit&#233; - Universit&#228;tsmedizin Berlin. It facilitates efficient routine work with well-structured, comprehensive long-term data and allows their easy use for clinical research. To this point, its functionality covers automated transmission of routine data via standardized interfaces from different hospital information systems, availability of transplant-specific data, a medication list with an integrated check for drug-drug interactions, and semi-automated generation of medical reports among others. Key elements of the latest reengineering are a robust privacy-by-design concept, modularity, and hence portability into other clinical contexts as well as usability and platform independence enabled by HTML5 (Hypertext Markup Language) based responsive web design. This allows fast and easy scalability into other disease areas and other university hospitals. The comprehensive long-term datasets are the basis for the investigation of Machine Learning algorithms, and the modular structure allows to rapidly implement these into clinical care. Patient reported data and telemedicine services are integrated into TBase in order to meet future needs of the patients. These novel features aim to improve clinical care as well as to create new research options and therapeutic interventions.

TBase combines an electronic health record with an innovative research database for kidney transplant recipients. TBase is built upon an in-memory database platform, connected to different hospital systems, and used for regular outpatient care. It automatically integrates all relevant clinical data including transplantation-specific data creating a unique research database.

TBase - Böbrek Nakli Alıcıları için Entegre Elektronik Sağlık Kaydı ve Araştırma Veritabanı

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

İç Kan-Retinal Bariyer Geçirgenliğini Değerlendirmek için In Vitro Model Olarak Endotel Hücre Transsitozu Testi

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...