저자는 토스카나 지역 (POR CREO FESR 2007-2013, 반도 유니코 R &amp; 2012 S)에 의해 투자 FORTE 프로젝트, 부분적으로 연구 활동을 지원하는 유럽 연합 (EU) FP7 ECHORD ++ 실험 LA-장미, 그리고 부여 된 FP7 BiophotonicPlus 프로젝트 &quot;라이트&quot;감사드립니다 토스카 지역별.

이 연구는 병원 윤리위원회의 전향 적으로 승인 하였다; 동의서를 얻었다. 이 연구는 헬싱키 선언의 원칙을 준수했다. 1. 기증자 내피 준비 <ol><li> 실온에서 지역 안구 은행에 의해 제조 된 기증자의 각막을 사용합니다. </li><li> 수술실에서의 배달 컨테이너에서 기증자의 각막을 당겨 각막 전송에 사용되는 조직의 보존과 영양에 대한 솔루션을 구별. </li><li> 액체가 흘러 변수 압력을 만들 수있는에 인공 전방, 이상 기증자 corneo - 공막 림을 놓고; 조직 유지 헤드와 각막을 커버합니다. </li><li> 각막 보존 및 영양 액체로 가득 주사기에 3 방향 커넥터를 통해 인공 전방 (AAC)를 연결합니다. </li><li> 장소에 안전하게 고정을 유지하기 위해 압축 링을 사용합니다. </li><li> SYR를 사용하여최적 압력 (1.7 참조​​)에 도달 할 때까지 조직 보존 용액으로 전방을 채우 인게은 전방 내 압력을 유지하기에 접속 AAC. 커넥터를 닫습니다. </li><li> 손가락으로 전방 내부의 압력을 테스트합니다. (범위 60 ~ 70 mmHg로에서) 오른쪽 내부 압력에 도달 할 때까지 내부 압력을 변경합니다. 3 방향 커넥터습니다. </li><li> 빛 간섭 Thomography OCT ()와 도너 각막의 두께를 측정한다. 악기 광학 앞에 고정 된 위치에 그 각막과 전방을 유지하고 전체 두께 10월 수집을. </li></ol> 기부자 내피 2. 펨토초 레이저 준비 <ol><li> 펨토초 레이저 장치에서 기증자의 각막의 압평을 수행합니다. 후방 사이드 컷, 전체 층상 및 전방 사이드 컷 : 세 이후의 상처와 기증자의 조직을 잘라 femtosec 레이저를 사용합니다. 세 인하에 대해 다음 매개 변수를 설정합니다.<ol><li> 전체 라멜라 컷 (래스터 패턴, 아웃 시작)의 경우, 항상 95 μm의 감산 (1.8에 기재된 바와 같이 OCT를 사용하여 측정) 공여자 각막의 얇은 포인트에 대응하는 절단 깊이를 설정한다. 범위 (작업 깊이에 따라 다름) 0.8-0.9 μJ의 펄스 에너지를 설정합니다. 8.7 mm의 직경을 설정합니다. 2 μm의 접선 장소의 분리를 설정합니다. 2 μm의 반경 자리 분리를 설정합니다. </li><li> 전방 사이드 컷의 경우, 이전의 전체 라멜라 컷 30 μm의 깊은 후방 깊이를 설정합니다. 2.10 μJ에 펄스 에너지를 설정; 8.6 mm의 직경; 층 분리 동안 3 μm의 3 μm의 자리 분리. </li><li> 후방 사이드 컷의 경우, 후방 측의 전방 깊이가 전체 라멜라 컷보다 전방으로 30 μm의 컷 찾습니다. 900 μm의 후방 깊이를 설정합니다. 2.10 μJ에 펄스 에너지를 설정; 8.3 mm의 직경; 2 μm의 자리 분리; 2 μm의 층 분리. </li></ol></lI&gt; </ol> 3.받는 사람 눈 준비 <ol><li> 수술 환자를 준비합니다. 블레이드를 사전 교정 4.0 mm의 12시 한 4.00 mm의 각막 절개를합니다. 2시에 배치 1.2 mm의 사전 교정 블레이드 하나 윤부 천자를 확인합니다. 30 ° 자상 칼과 6시 방향에서 다른 윤부 천자를 확인합니다. </li><li> 2시 천자를 통해, 환자의 전방에 전방 메인테이너를 삽입합니다. </li><li> 전용 후크 한 8.2 mm 직경의 원형 descemetorexis을 수행합니다. </li><li> 후방 기질에서 데스 메 막을와 내피를 벗겨과 데스 메 후크와 조직을 제거합니다. </li></ol> 4. 발색단 준비 <ol><li> 1.5 ml의 microcentrifuge 관에서 인도 사이 아닌 그린 분말 1 mg을 넣어. </li><li> 멸균 9 mg을 추가 (ICG 수용액은 W / W 10 %). </li><li> 수동 금속 교반기 ICG 가루와 물을 혼합한다. </lI&gt; </ol> 기부자 내피 5. 염색 <ol><li> 분사 장치 표면과 접촉하는 내부면이 되네요 인젝터의 넓은 부분에 도너 내피을 넣어. </li><li> 주걱을 사용하여, 그 주변 부분에, 발색 용액 도너 내피의 내측 얼룩. 제대로 스테인드 조직은 균일 한 녹색 색상을 가지고있다. 단계 5.3을 시작하기 전에 3 분을 기다립니다. </li><li> 되네요 - 인젝터의 앞쪽 부분에 기증자의 내피 세포를 당겨, 압연. </li></ol> 6. 기증자 내피 삽입 <ol><li> 잡고 내피 lenticule에 대한 외상성 동축 집게를 사용하여 접힌 기증자의 내피 세포를 삽입합니다. </li><li> 전방 메인테이너를 제거합니다. </li><li> 각막 절개와 나일론 10.0 단일 스티치와 천자 (3.1 단계에서 수행)를 봉합. </li><li> 공여 조직을 전개하고 수신자 각막에 대해 그것을 누르는 기포 주입한다. 공기 방울 미리보기 다운로드합니다letely 전방 공간을 채우기. </li></ol> 7. 레이저 용접 <ol><li> 후크 또는 주걱으로 각막의 외부로부터 공기 기포 이동 내부로부터, 수신자 각막의 중앙에 도너 lenticule 배치. </li><li> 개구 수 (NA) 0.22과 같이 300 μm의 코어 직경 멸균 광섬유 갖춘 810 nm에서 발광 다이오드 레이저를 사용한다. </li><li> 안구 외측 섬유 팁을 유지하고, 투명한 각막 조직을 통해 염색 내피 향해 레이저 광을 전달한다. 광섬유 팁은 비접촉 구성이다. </li><li> 단일 지점 방출 모드, 70 ~ 80 밀리 초 펄스 폭, 펄스 당 35 ~ 40 엠제이 : 레이저에 대해 다음 설정을 사용합니다. 그는 느가에 빔을 목표로합니다. </li><li> 기증자 내피 세포의 염색 주변에 하나의 레이저 스팟을 제공합니다. 순차적으로 스폿 배달 : 최종 형태는 도너 lenticule의 주위에 스폿의 링이다. 두 adja 사이의 거리센트 명소 센터 스폿 직경의 두 배입니다. </li><li> 토 브라 마이신 0.3 % 및 0.1 % 덱사메타손 안과 현탁액과 함께, 환자의 눈에 콘택트 렌즈를 적용. </li></ol>

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The authors declare that they have no competing financial interests.

&quot;모든 레이저&quot;내피 이식은 원래의 접근 최소 침습 각막 이식하는 것입니다. 프로토콜 내에서 설명하는 모든 절차는 멸균 장갑, 가운, 마스크와 모자의 사용으로 수술하는 동안 일반적인 관행이다 위생과 살균 과정을 관찰, 수술 실에서 실시 하였다. ICG 용액은 곧 도너 내피 염색에서의 도장 전에, 수술실에서 제조 하였다. ICG 분말, 물 및 염색 용액을 제조하는 데 사용?...

본 연구에서 우리는 공여 조직 및 수신자 베드 상을 용접 근적외선 레이저 다이오드를 제조하는 펨토초 레이저의 사용을 기반 각막 내피에 원래 방식을 제시한다. 공여자 각막의 수술 측정 올바르게 공여 조직 치수를 설계하는 것이 필요하다. 각막 내피는 내피 1,2- 질병 치료에 각막을 교체하는 최근에 제안되었다. 이 기술의 가장 큰 장점은 수술, 이식 거부의 감소 위험과 눈의 무결성을 보존하는 동안 각막 감소 마취를 관통에 대하여, 빠른 시력 회복이다. 주요 위험 인자는 수술 후 기증자 lenticule 탈구이다. 표준 기법이 기포 주입에 의해 유지되는 최종 위치에 도너 내피를 삽입함으로써 수행된다 : 어떤 봉합사 때문에, 생 물리적 및 기계적 치수 체인지의 사용되지내피 세포의 cteristics. 또한, 시력 회복으로 인해 이식 두꺼운 조직에 주로 있기 때문에 기증자와받는 사람의 조직 사이의 불일치 제한 할 수 있습니다. 여기에서 우리는 이러한 주요 문제를 극복 할 수있는 내피 각막 이식술을 수행의 절차를 제시한다. 도너 내피는 레이저 용접 기술을 사용함으로써 최종 위치에 확보 할 수있다. 이 제어 및 지역화 광열 과정이다 : 그것은 기증자 /받는 사람 인터페이스에서 유도 될 수있다. 그것은 지난 10 년 동안 공부하고 각막과 내피 3-5의 이식에 제안되었다. 근적외선 (파장 810 ㎚) 저전력 레이저 다이오드에 의해 방출은 상처 부위에서 생체 조직을 향해 전달된다. 각막이 파장에 자연적으로 투명하다 : 레이저 광을 흡수하여이 조직을 만들기 위해서는, 발색단 그것을 염색하는 것이 필요하다. 제안 된 염료이다 멸균 포화인도 사이 아닌 그린 (ICG)의 D 수용액. 우리는 각막 조직이 제대로이 ICG 제제로 염색 할 때, 810 nm의 6에서 흡수 피크를 나타내고 있음을 보여 주었다. 또한, ICG 널리 임상 진단에 사용되며 안전성은 이미 인간의 과목에서 입증되었다. 각막 염색은 다이오드 레이저 광 에너지를 흡수하여 생성 된 주요 효과는 용접 부위의 온도 상승을 제어한다. 아니 열 효과는 흠없는 조직에서 유도되지 않습니다. 온도의 향상은 냉각시 상처 벽 즉각적인 closuring와, 간질 콜라겐 가역적 열 변성을 유도한다. 이 레이저 용착 효과 첫째 백내장 및 각막 7,8 9,10 관통에서 입증되었다. 우리는이 논문에서 제시되는 최적화 방법은 각막 내피 세포의 응용 프로그램에 대한 연구되고있다. 제안 된 수술에서, 하나의 레이저 반점 (밀리의 지속 수십) ARE는 스폿 사이즈 (직경 ㎛의 수백) 내에 국부적 광열 효과를 초래 조직에 전달 : 유도 효과는 도너 / 호스트 인터페이스에 국한 콜라겐 광응고술 이루어진 하드 레이저 용접이다. 용접 부위 콜라겐 변성의 결과는 표준 기술에 따라서 (바늘)을 얻을 수 없다 봉합 효과를 제공하고, 도너와 호스트 조직 사이에 강한 접착력이다. 조직은 짧은 후속 (1 개월)에 그의 천연 거리는은 도너 및 / 숙주 조직과의 밀착성은 치료상의 초기 단계에 설치된 용접에 의해 향상된다. 두꺼운 공여 조직 이식 즉 내피 각막의 다른 주요 위험을 피하기 위해, intrasurgical 광 간섭 단층 OCT ()이 사용되어 정확한 절단 프로파일이 설계 될 수 있도록 상업 디바이스, 도너 각막의 두께를 측정femtosec 레이저. 제안 된 &quot;모든 레이저&quot;내피 이식 따라서이 최소 침습 수술의 임상 결과를 개선하는 것 같다.

<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;모든 레이저&quot;수술은 최소 침습 각막 이식을 수행하기 위해 제안된다. 대하여 표준 내피 이식, 각막 두께 측정 공여 조직을 염색 첨가 레이저 광 전달의 단계들 : 절차 (도 1 참조)을 수행하기 쉽다. 달성 이점은 주로 몇 분의 증가 수술 시간을 보상한다. 공여자 각막 두께와 도너 lenticule 치수를 지정하는 데 사용 펨토초 레이저를 측정하기 위해 수술 중의 OCT 사용 도너 / 호스트 ?...

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.

&quot;모든 레이저&quot;내피 세포 이식

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 조회수.  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.

비디오: 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.

경쟁 손쥐 골수 이식 모델에서 기증자 세포 Engraftment를 확인하려면 수량 실시간 PCR을 사용하여

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

연구

의학

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.

쥐 이소성 심장 이식 기술

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.

활성 포르토 대정맥 션트를 사용하여 돼지 간 조달 및 동소 이식 이식의 기술

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.

동종 유도 혈관 거부 및 이식 동맥 경화 마우스 모델

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.

쥐 신장 이식 기술

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.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

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.

UV-Vis및 말라리아 방지 프리마퀸의 육안 측정을 위한 최적화된 Griess 반응

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.

쥐 간 이식 모형에 있는 혈역학적인 변경을 시뮬레이션하여 기관 허혈-재관류 상해

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 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.

내피 세포 트랜스사이토시스 분석법(Endothelial Cell Transcytosis assay)은 내부 혈액-망막 장벽 투과성을 평가하기 위한 시험관 내 모델로서 분석합니다.

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