저자는 기술 지원 및 도움을 실험 양 리사 오츠카 감사드립니다. 이 연구는 부분적으로 교육부, 과학, 스포츠와 문화에 의해 지원되었다 부여 - 에이드의 젊은 과학자 (B), 2010, 22700476, 그리고 KT에 대한 Suzuken 기념 재단은 2010 년이 연구가의 연구 개발에 의해 지원되었다위한 생활 물질, MEXT의 차세대 슈퍼 컴퓨터 프로젝트의 개발 및 사용의 일부, 그리고 일본 표준시 ERATO Suematsu 가스 생물학 프로젝트에 의해 일부의 차세대 통합 시뮬레이션.

1. 플루오레신 Isothiocyanate 이성질체 I (FITC)와 적혈구에 레이블 우리가 사용하는 모든 실험 프로토콜은 의학 게이오 대학 학부 동물 보호위원회에 의해 승인되었다. <ol><li> 기증자 마우스를 마취하고, 제작 후 celiotomy 절개는 헤파린의 소량이 들어있는 1-ML 주사기와 하부 대정 맥에서 온 혈액을 철회. 마우스가 즉시 나트륨 pentobarbital 사출 (100 밀리그램 / kg IP)에 의해 euthanized되었다. </li><li> 스윙 - 양동이 로터의 400g에서 약 5 분 동안 원심 분리하여 PBS (산도 7.4)로 두 번 전체 피를 씻으십시오. </li><li> 100 MM 나 2 HPO 4 5 ML에 FITC의 10 MG를 해산하고 0.22-μm의 모공을 가진 멤브레인로 필터링합니다. </li><li> 시험관 3 밀리미터 포도당의 0.15 ML, 180 MM 100 mm의 저질 2 PO 4, 1.5 ML 2 HPO 4 나의 0.25 ML로 FITC 솔루션 1 ML을 섞는다. R의 0.2 ML 추가기원전 펠렛과 믹싱을 위해 튜브를 누릅니다. </li><li> 4 ° C에서 2 시간 동안 튜브를 유지하고 두 번 PBS의 스테인드 RBCs 씻는다. </li><li> 슬라이드 잔디에 RBC 현탁액 소량을 넣고 RBCs 건강보고 있는지 (즉, 그들의 모양과 크기가 정상이라는)과 형광 강도가 충분한지. </li><li> 산도 7.4의 PBS의 0.9 ML에서 RBCs의 0.1 ML을 일시 중지 및 4에서 정학을 유지 ° C를 주사까지. </li></ol> 2. 산소에 민감한 염료의 작성 <ol><li> 산도 7.4의 PBS의 10 ML에 BSA 500 MG를 해산. </li><li> 경찰에서 30 밀리그램을 추가 (II)-meso-tetra (4 carboxyphenyl) BSA 솔루션에 porphine (경찰에서 TCPP)과 하룻밤 저어. </li><li> (선택 사항) 무료로 경찰에서 TCPP에서 분리하는 중력 흐름 크로마 토그래피 또는 스핀 컬럼을 사용하여 BSA 바인딩된 경찰에서 TCPP 압축을 풉니다. </li><li> 경찰에서 TCPP 소화 되 다만 제거하고 0.22-μm의 모공을 가진 멤브레인 필터 표면에 뜨는를 필터링 솔루션을 원심 분리기. &lt;/ 리&gt; <li> -20 ° C.에있는 튜브에 저장 1-ML aliquots 반복 냉동 해동 사이클을 피하십시오. </li></ol> 3. 동물 준비 <ol><li> microcirculation의 현미경 관찰을 위해서 구멍이 넘는 30 - 음 - 네모 커버 유리 구멍 직경 20 mm, 그리고 테이프로 플라스틱 접시를 준비합니다. </li><li> 마우스를 마취, 피부 모피를 제거하고, 준비. heparinized PBS 가득한 10 cm 폴리에틸렌 (PE 10) 카테터에 연결된 30 G 바늘을 사용하여 약물 주입에 대한 헛된 꼬리에 카테터를 놓습니다. </li><li> 정중 절개 후, 플라스틱 접시에 간장의 주요 lobule을 연장하고, 흉골 드러누움에 마우스를 놓습니다. </li><li> 호흡과 함께 간 이동 및 건조로부터 지키는 억제하는 간장 엽의 가장자리 주변의 부엌 랩 (3mm X 8mm)과 기와 함께 작은 전표를 준비합니다. </li><li> 현미경으로 간장 microcirculation을 (전송 라이트 포함) 관찰 및 확인 그 Blood 흐름은 적어도 15 분 동안보기의 분야에서 더 스테이 시스가 없습니다. </li><li> 천천히 혈액 흐름 관찰이나 PO 2 측정을위한 경찰에서 TCPP 솔루션의 0.2 ML에 대한 fluorescently 분류 RBCs의 0.2 ML 투입. FITC-라벨 RBCs의이 금액은 시각 영역의 순환에있는 모든 RBCs의 1 / 50에 설명할 것입니다. </li></ol> 4. 혈액 흐름 시각화 <ol><li> 대역 통과 필터 (450-490 nm의) 1 통과했다 수은 램프 표시등으로 irradiating하여 FITC를 자극. </li><li> CCD 카메라로 형광 이미지를 기록합니다. </li></ol> 5. PO 2 측정 PD-TCPP phosphorescence는 상대적으로 약한이며, 높은 감도 탐지기로 감지되어야한다. 모든 실험은 어두운 방에서 실시해야합니다. <ol><li> 경찰에서 TCPP의 흡수 피크는 410 nm의와 532 nm의에있다, 그래서 두 번째 고조파 파장 532 nm의은 여기 권장합니다. YAG 펄스 레이저 2 :이 파장은 ND에 의해 생성하실 수 있습니다. </li><li> 거꾸로 현미경의 해당 포트에 YAG 레이저, 그리고 초점 평면의 중앙 위치로 빔 스폿을 조정 : ND에서 광선을 먹이. 공간적 해상도는 핀홀을 통해 빔을 전달하여 변경 레이저 스폿 크기에 따라 달라집니다. </li><li> phosphorescence을 감지하기 위해 긴 패스 필터 (&gt; 620 nm의)와 현미경의 다른 포트에 photomultiplier 튜브 (PMT)을 첨부합니다. PMT는 특히 700 nm의 주위에, 빨간색 파장에 민감한 여야합니다. </li><li> phosphorescence 신호 (500 점, 200 kHz에서에서 샘플을) 샘플과 부패에 지수 함수 맞추하여 부패의 지속 시간을 계산합니다. </li><li> 교정 PO 2 150 mmHg과 0 mmHg로 경찰에서 TCPP 솔루션의 샘플의 두 세트를 준비합니다. 산소 부재를 생산하는 맛보고 dithionite 나트륨에게 볼륨의 1 %를 추가합니다. 7.4에서 시료의 산도와 온도 유지교정하는 동안 37 ° C에서 진짜야. </li><li> 스턴 Volmer 상수 k는 Q와 산소가없는 발광의 감쇠 시간이에게 방정식의 τ 0 (1) 3 수정합니다. 37 ° C와 7.4의 산도에 경찰에서 TCPP 솔루션과 우리의 케이스에서, k는 Q가 374 mmHg -1들 -1이며, τ 0 0.74 MS입니다. 이러한 측정 파라미터는 장비 (예 : 레이저, 검출기, 기타 광학 기기) 및 신호 처리 방법에 의존하므로 교정은 각 측정 시스템 4 필요합니다. τ 0 / τ = ​​1 + K Q • τ 0 • PO 2 (1) </li><li> 현미경 스테이지에있는 접시에 마우스를 설정 간장 microcirculation을 관찰하고, 레이저 스폿의 포인트로 대상 microvessel의 위치를​​ 조정합니다. </li></ol> 6. 간염 모델 <ol><li> 하룻밤 물에 무료로 이용할 수있는 빠른 생쥐. </li><li> 준비20 DMSO의 아세트 아미노펜 (APAP)의 ML 솔루션 당 밀리그램. </li><li> 한 ml/100 g BW (예, 200 밀리그램 / kg BW)에 intraperitonealy 그것을 주사. 주사 후, 마우스는 음식과 물을 5 ~ 무료로 액세스할 수있는 권한을 부여하실 수 있습니다. </li><li> 24시간 주입 후에 마우스를 마취하고 중앙 분리대를 절개하여 간장을 폭로. 간염이 달성되면 pericentral 지역의 괴사가 육안으로 쉽게 볼 수 있습니다. </li></ol> 7. 대표 결과 간장 microcirculation의 예제 이미지는 () 전송 - 밝은 이미지 그리고 (b) 형광 이미지입니다 그림 1에 표시됩니다. 개인 FITC-라벨 적혈구는 비디오 영화에서 관찰되며, 포털 venules, sinusoids, 그리고 중앙 venules은 혈액 흐름 방향으로 인정받고 있습니다. 그림 2에 표시된 바와 같이, x100 목표 렌즈를 통해 중앙 venule을 irradiating 레이저 스폿은 approxim였다직경 10 μm의 ately. 간장 microcirculation의 Intravascular PO 2 수컷 C57BL / 6 마우스로 측정하고, 그림 3 (가) PO 2와 포털 및 중앙 venules의 혈관 직경 사이의 관계를 보여줍니다. sinusoids 통과하면서 중앙 venules으로 포털에서 PO 2 그라디언트 효율적으로 해당 RBCs 릴리스 산소를 나타냅니다. 그림 3 (B)와 같이 평균 PO 2는 포털 혈관, sinusoids에서 48.2 mmHg, 그리고 중앙 venules에서 38.9 mmHg에서 59.8 mmHg였다. 우리가 APAP intraperitonealy을 주입하여 급성 간염을 생산하면, intravascular PO 2가 현저히 간장 microcirculation의 각 부분에서 상승했습니다 : P sinusoids 및 중앙 정맥 (그림 3 (C))에 포털 정맥, P &lt;0.01에서 &lt;0.05. <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="그림 1" /> 그림 1. 낮은 배율 전mages는 (a) 전송 가벼운 및 (b) 형광과 간장 microcirculation을 보여주고 있습니다. <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="그림 2" /> 그림 2. PO 2 측정을위한 타겟이 중앙 venule을 irradiating 레이저 스폿의 이미지. <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="그림 3" /> 건강한 제어 생쥐와 아세트 아미노펜 유발 간장 손상과 생쥐의 간장 microcirculation의 그림 3. PO 2 구배. () PO 2 대 포털 venules (PV)와 중앙 venules (CV)에 대한 혈관 직경을 보여줍니다, 그리고 (b) sinusoids (S)를 통해 PV에서 이력서에 산소 그라데이션을 보여줍니다. PV와 CV 사이 포이 차이는 hepatocytes 또는 기타 parenchymal 세포의 산소 소비를 반영합니다. (C) APAP 행정부에 의해 유도된 간염 조건, PO 2는 상당히 PV, S, 그리고 CV 및 PO 2 상승되었음을 보여줍니다기울기는 간장 산소 소비가 손상되었음을 나타냅니다 감소.

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우리는 공개 할게 없다.

조직에 산소를 전달하는 것은 microcirculation의 필수적인 역할이며, 논문의 많은 microvessel 해부학 및 레올로지 6에 관련된 질병을 설명합니다. 간 혈액 흐름이 동맥과 정맥 혈액의 조합입니다. 간장에 혈액 흐름의 약 30 %가 포털 정맥에 의해 제공 간장 동맥 및 기타 70 %에 의해 제공된 잘 산소를 혈액이며, 비장과 위장관 7 저조한 산소를 혈액 정맥 유출입니다. 이러한 지방간, 충격, 또...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> PD (II) meso-Tetra (4 carboxyphenyl) porphine </td><td> 프론티어 과학 주식 회사 </td><td> PdT790 </td></tr><tr><td> 플루오레신 isothiocyanate 이성질체 전 </td><td> 시그마 - 올드 리치 (주) </td><td> F7250 </td></tr><tr><td> 아세트 아미노펜 </td><td> 시그마 - 올드 리치 (주) </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> 장비의 명칭 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> photomultiplier 튜브 </td><td> 하마 마츠 photonics KK </td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

간장 산소 소비가 상대적으로 높은이지만 간장 혈액 공급의 약 70 %가 저조한 위장관과 비장에서 파생된 포털 정맥 혈액을 산소 때문에 산소 공급과 간장의 수요 사이에 상당한 차이가있다. 산소는 sinusoids 통해 중앙 venule에 포털 정맥의 터미널 지점에서 흐르는 혈액에 의해 hepatocytes에 전달하고, 이것은 간장 lobules의 산소 그라디언트를 만들 수 있습니다. 산소 기울기는 업스트림 및 다운 스트림 간장 microcirculation의 효소 표현하지만, 간장 microcirculation의 산소 소비를 측정하기위한 기술의 부족 간장의 산소 대사에 관련된 메커니즘의 해설을 연기했습니다 관련된 중요한 물리적 매개 변수입니다. 따라서 우리는 간장 microcirculation을 시각화하기 위해 FITC-라벨이 적혈구를 사용하고있다 microvessels에서 산소의 부분 압력을 측정하기 위해 레이저를 이용한 phosphorimetry를 사용했습니다. Nonconta중부 표준시과 지속적인 광학 측정은 혈액 흐름 판매율, 선박 직경 및 간장에서의 산소 소비량에 관한 산소 그라디언트를 정할 수 있습니다. 급성 간염 모델에서 우리는 pericentral 영역에 hepatocyte 괴사가 산소 압력을 최대 이동 및 효소 발현에 영향을 미칠 수있다는 의미, 우리는 포털 및 중앙 venules 모두에서 증가 산소 압력지만 sinusoids의 감소 산소 그라데이션을 관찰 생쥐에 아세트 아미노펜을 투여하여 만든 periportal 영역 인치 결론적으로 측정 간장 혈류 역학 및 산소 소비를위한 광학 방법은 간장 질환에 관련된 메커니즘을 밝힐 수 있습니다.

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

광학 시스템은 FITC-라벨이 적혈구와 간장 microcirculation을 시각화하고 레이저를 이용한 phosphorimetry으로 microvessels에서 산소의 부분 압력을 측정하기 위하여 개발되었습니다. 이 방법은 microvascular 구조, 직경, 혈류 속도 및 산소 장력을 분석하여 생리 및 병리 학적 메커니즘을 조사하는 데 사용할 수 있습니다.

급성 간염 모델 신청 : 시각화와 간장 Microcirculation의 혈류와 산소 소비 분석

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K 조회수.  Keio University. 광학 시스템은 FITC-라벨이 적혈구와 간장 microcirculation을 시각화하고 레이저를 이용한 phosphorimetry으로 microvessels에서 산소의 부분 압력을 측정하기 위하여 개발되었습니다. 이 방법은 microvascular 구조, 직경, 혈류 속도 및 산소 장력을 분석하여 생리 및 병리 학적 메커니즘을 조사하는 데 사용할 수 있습니다.

비디오: Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Murine 발광 생물 간장 Tumour 모델

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

연구

의학

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

이소성 보조 쥐 간 이식 급성 간 장애에 포털 정맥 Arterialization를 유량 조절

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

지주막 하 출혈의 급성과 늦은 후유증의 연구에 대한 토끼 피 션트 모델 : 기술적 측면

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

실험 척수 부상에서 척수 혈액 흐름의 평가를위한 향상된 초음파 이미징 대비

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

치료를 위해 혈액 망막 장벽 위반 및 잠재적 인 마약을 평가하는 급성 망막 모델

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

의 최적화 분석<em&gt; 생체</em&gt; 및<em&gt; 체외</em&gt; 간 지방증

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

Ameroid 압축을 사용 하 여 돼지에서 만성 심장 허 혈 모델

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

세포 외 유출 분석기를 사용 하 여 기본 교양된 마우스 신생아 Cardiomyocytes에 산소 소비 속도 분석

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.