The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

참고 :이 절차에 사용 된 모든 동물 비전 연구 협회와 안과 문 안과 및 비전 연구에서 동물의 사용에 대한뿐만 아니라 세인트 루이스 대학의 동물 감독위원회에 의해 아래로 설정 한 지침에 따라 처리됩니다. 참고 : 모든 수술 장비 및 솔루션은 눈의 세균 감염을 제한하기 위해 수술 전에 소독한다. 그것은 동물이 과정에서 약간의 통증을 경험 수행하면서, 우리는 진통제를 사용하지 않는다는 것을 주목해야한다. 모든 진통제는 소염이고 각막 이식 염증 반응을 수반하기 때문에, 항염증제의 사용은 각막 이식 실패에 관여하는 요인이 무엇인지를 결정하는 능력을 손상 때문에 이것에 대한 이유이다. 1. 마취 <ol style="margin-left: 40px;"><li> 케타민 ((86)의 IP 주사하여 전신 마취 하 기증자와받는 사람 마우스를 놓습니다.98 ㎎ / ㎏)과 자일 라진 (xylazine) (13.04 ㎎ / ㎏). </li><li> 일반적으로 시간에 30 분 걸리는 절차 전반에 걸쳐 마취받는 사람 마우스를 유지합니다. 결과적으로, 지속적으로 의식을 회복의 징후가 마우스를 모니터링 할 수 있습니다. </li><li> 동물 건조를 방지하기 위해 마취 후에 수술을하지 않습니다 눈에 puralube 연고를 적용합니다. </li></ol> 2. 각막 이식술 <ol style="margin-left: 40px;"><li> 기증자의 각막 버튼을 얻었다. <ol><li> 동물이 완전히 마취되면, 1 % tropicamide와 2.5 % 페닐에 염산염의 눈에 몇 방울의 투여에 의한 적절한 산동을 얻을 수 있습니다. </li><li> 수평 튼튼한 이동 지원에 배치 보드에 공여 동물의 머리를 놓습니다. 눈 전체 동작 걸쳐 수평 위치에 있도록 목 걸쳐 테이프의 스트립을 고정 헤드. </li><li> 아웃, 선단 메틸 블루로 염색 하였다 2mm 직경 트레를 사용하여,중앙 각막 이식 사이트 라인. </li><li> 날카로운 블레이드로, 각막을 침투 도너 내피 기본 렌즈의 손상의 가능성을 줄일를 깊게하는 전방 내로 주입 결과 Healon. </li><li> vannas 가위와 기증자 이식을 절제하고 사용까지 행크스 &#39;균형 염 용액을 포함하는 접시에 놓습니다. </li><li> 공여자 이식편을 제거 한 후, CO 2 흡입 통해 도너 마우스를 안락사. </li></ol></li><li> 이식 침대를 준비. <ol><li> 수신자 2.1.2 통해 2.1.1에 설명 된 것과 동일한 단계를 반복한다. </li><li> 1.5 mm 직경 트레를 사용하여 수신자 그래프트 사이트를 개요. </li><li> 날카로운 블레이드와, 각막을 침투 기본 렌즈에 손상의 가능성을 줄일를 심화하기 위해 전방에 결과 Healon를 주입. </li><li> vannas 가위를 사용하여받는 사람의 설명 중심 각막 버튼을 제거하고 폐기합니다. </li></ol></li><li> 봉합이식 <ol><li> 받는 사람의 각막에 침대 이식을 통해 기증자의 각막을 놓습니다. 적절한 결과 Healon 렌즈와 직접 접촉에 의한 손상으로부터 기증자의 내피 세포를 보호하기 위해 기증자의 각막 아래에 있는지 확인합니다. </li><li> 슈퍼 뾰족한 microforceps를 사용하여 한 묶어,받는 사람의 편에 전체 두께의 90 % 깊이와 기증자를 통해, 기증자 측에 11-0 나일론 봉합사의 첫 번째 바이트를 배치합니다. </li><li> 각막 제자리에 고정되면, 각막 8~10 총 봉합사를 가지며 공여 각막 안전하게 줄 지어 수신자 각막 이식 침대에 부착되도록 midcardinal 단속 봉합을 수행한다. </li></ol></li><li> 전방 심화 <ol><li> 전방에 HBSS 또는 공기 방울을 주입하여 전방 깊어 부드럽게 셀룰로오스 스폰지로 누출 각막 이식의 무결성을 확인합니다. 주 : 전방을 개질 할 수없는 경우 (c)의 높은 확률이 존재이식 된 각막의 미래 평가가 매우 어려운 것 또한 잠재적 기증자의 각막 내피 세포의 기능 장애 때문에 이식 실패로 이어질 것입니다 ataract. </li></ol></li><li> 최종 평가 <ol><li> 동공은 원형이며, 전방의 깊이가 정상이 있는지 결정하기 위해 눈을 관찰한다. 참고 :이 조리개를 봉합 중에 손상되었음을 나타냅니다 따라서 이식은 기술적 인 문제로 간주됩니다 둥근 눈동자가 아닌 경우. </li><li> 눈에 항생제 연고를 적용합니다. 옵션 : 7-0 실크 봉합사로 눈 뚜껑을 닫습니다. </li><li> 그들이 완전히 깨어 후 개별적으로 수술 후 이틀 최소를 수용 할 때까지 쥐를 관찰한다. </li></ol></li></ol> 3. 봉합 제거 <ol style="margin-left: 40px;"><li> 뚜껑 봉합사가 사용되는 이러한 경우에, 전술 한 바와 같이 마우스를 마취하고 48 시간에 뚜껑 봉합사를 제거. </li><li> 수술 후 7 일째에 쥐를 마취. sutur 제거ES 각막 이식을 확보. 봉합사를 제거하고, 동물이 완전히 깨어 나면, 케이지로 복귀. </li></ol> 4. 임상 평가 <ol style="margin-left: 40px;"><li> (전방에있는 혈액) 백내장 (렌즈의 흐림), 전방 출혈을 포함하는 절차 적 합병증의 지시 사항에 대해 눈, 적절한 깊이, 또는 각막의 중요한 불투명이 아닌 전방을 검사합니다. &quot;손상&quot;이러한 합병증을 보여 그 사람들을 고려하고 CO 2 흡입을 안락사. <ol><li> unanesthetized 쥐에 모든 검사를 수행합니다. 다른 한편으로는 눈에 더 잘 볼 수 있도록 눈을 proptose 할 수 있도록 한 손 따라서 마우스를 억제하여 마우스를 잡습니다. 관찰이 완료되면, 그 케이지에 동물을 반환합니다. </li></ol></li><li> 치료 그룹에 익숙하지 않은 관찰자는 각막 이식의 흔적을 각막을 2 ~ 3 회 이식 평가했다거부 에피소드 또는 각막 이식 실패. 수술 현미경이 관찰을위한 수평 세극등 생체 현미경 하나를 사용하십시오. <ol><li> 다음과 같이 규모가 정의 5. 10-0의 규모를 사용하여 불투명도마다 각막을 평가 : <ol><li> 불투명의 흔적이없는 사람들 각막 0의 점수를 할당합니다. </li><li> 최소한의 표면 불투명도를 보여 그 각막 1의 점수를 할당합니다. </li><li> 온화하고 깊은 불투명도를 표시하지만 기본 동공과 홍채가 여전히 뚜렷한이다 각막 2의 점수를 할당합니다. </li><li> 홍채는 동공 마진 제외 상세히 볼 수없는 특징 간질 불투명도를 표시 각막 3의 점수를 할당한다. </li><li> 조밀 한 기질 불투명도를 표시하고 어떤 기본 구조를 볼 수 없습니다 경우 각막 4의 점수를 할당합니다. </li><li> 완전히 가려 동공과 홍채와 완전한 불투명도 및 집중적 인 간질 부종을 표시 각막 5의 점수를 할당합니다. </li></ol></li><li><html> 또한, 혈관의 침윤 (혈관 신생)이를 위해 1 내지 8의 평가 척도를 이용하는 정도마다 각막 평가 4 동일한 사분면 이루어진으로 각막을 확인하고 이들 사분면 각각에서 혈관의 양을 결정 그 사분면의 광범위한 혈관에 대한 2 0 (선박)에서 그 의지의 범위를 점수. 최종 신생 혈관 점수를 계산하기 위해 각 사분면에서 개별 점수를 추가합니다. </li><li> 그들은 오주 포인트 최대 시간 동안 두 개의 연속적인 관찰 3의 점수가있는 경우로 급성 거부 각막을 분류. </li><li> 누구의 각막 오주에서 분명했다 쥐를 분류하지만받은 말기 각막 이식 거부 반응이있는 것으로,&gt; 사십오일 연속 2 시점 3의 점수, 생착을 게시 시간에 혼탁을 개발한다. 이식편 생존을 분석하는 카플란 - 마이어 생존 곡선을 사용한다. </li></ol></li></ol> 모델 5. 조작 에서 왼쪽 : 40px; &quot;&gt; <li> 비장에서 단일 세포의 준비. <ol><li> 하나의 세포를 준비하려면, 먼저 기증자 마우스를 안락사. 그런 다음, 비장을 제거합니다. </li><li> 셀 스트레이너에 비장을 배치하고는 3 ML의 주사기에서 주사기 플런저 방해. </li><li> 행크스 균형 소금 용액 10 mL의 세포에 resuspend을 씻으십시오. </li><li> 세포 현탁액 10 μl를 제거하고 0.4 % 트리 판의 10 μL에 파란색을 추가하고 혼합한다. 중앙 그리드의 세포를 혈구에 그를 추가하고 계산합니다. 튜브의 셀의 수는 세포 수 × 104 × 2 (트립 판 블루 희석 계수) (10) (튜브의 부피) ×이다. </li></ol></li><li> 전방에 주입. <ol><li> 전술 한 바와 같이 쥐를 마취. </li><li> 해부 현미경을 사용하여 주사를 수행합니다. 각각의 전방 내 주입의 경우, 10 6 0.005 ml의 볼륨에 비장 세포와 33 G 바늘 장착 된 0.25 ML의 마이크로 리터 주사기를 사용합니다. 참고 : 다른 조작모델의 특정 요인 동소 각막 이식 수술 후 재생 될 수 역할을 결정하거나 길항제 또는 작용제로서 작용 시약으로 동물을 처리함으로써 수행 될 수있다. </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD95+ligand+(FasL)-induced+apoptosis+is+necessary+for+corneal+allograft+survival.">CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival.</a> J Clin Invest. 99, 396-402 (1997).</li><li>Yamagami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Fas-Fas+ligand+interactions+in+the+immunorejection+of+allogeneic+mouse+corneal+transplants.">Role of Fas-Fas ligand interactions in the immunorejection of allogeneic mouse corneal transplants.</a> Transplantation. 64, 1107-1111 (1997).</li><li>Stuart, P. M., Pan, F., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fas/Fas+ligand+interactions+regulate+neovascularization+in+the+cornea.">Fas/Fas ligand interactions regulate neovascularization in the cornea.</a> Invest. Ophthalmmol. Vis. Sci. 44, 93-98 (2003).</li><li>Stuart, P. M., Yin, X. T., Pan, F., Haskova, Z., Plambeck, S., Ferguson, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+matrix+metalloproteinases+activity+prolong+corneal+allograft+acceptance+by+increasing+FasL+expression.">Inhibitors of matrix metalloproteinases activity prolong corneal allograft acceptance by increasing FasL expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1169-1173 (2004).</li><li>Joo, C. -. K., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+mediated+responses+in+a+murine+model+of+orthotopic+corneal+transplantation.">T-cell mediated responses in a murine model of orthotopic corneal transplantation.</a> Invest.Ophthalmol. Vis. Sci. 36, 1530-1540 (1995).</li><li>Sonoda, Y., Sano, Y., Ksander, B., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cell-mediated+immune+responses+elicited+by+orthotopic+corneal+allografts+in+mice.">Characterization of cell-mediated immune responses elicited by orthotopic corneal allografts in mice.</a> Invest. Ophthalmol. Vis. Sci. 36, 427-434 (1995).</li><li>Sano, Y., Osawa, H., Sotozono, C., 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=Cytokine+expression+during+orthotopiccorneal+allograft+rejection+in+mice.">Cytokine expression during orthotopiccorneal allograft rejection in mice.</a> Invest. Ophthalmol. Vis. Sci. 39, 1953-1957 (1998).</li><li>Haskova, Z., Usui, N., Ferguson, T. A., Pepose, J. S., Stuart, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+cells+are+critical+in+corneal+but+not+skin+allograft+rejection.">CD4+ T cells are critical in corneal but not skin allograft rejection.</a> Transplantation. 69, 483-488 (2000).</li><li>Tan, Y., 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=Immunological+disruption+of+antiangiogenic+signals+by+recruited+allospecific+T+cells+leads+to+corneal+allograft+rejection.">Immunological disruption of antiangiogenic signals by recruited allospecific T cells leads to corneal allograft rejection.</a> J. Immunol. 188, 5962-5969 (2012).</li><li>Dana, M. R., Yamada, J., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+interleukin-1+receptor+antagonist+promotes+corneal+transplant+survival.">Topical interleukin-1 receptor antagonist promotes corneal transplant survival.</a> Transplantation. 63, 1501-1507 (1997).</li><li>Cunnusamy, K., Chen, P. W., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17A-dependent+CD4+CD25++regulatory+T+cells+promote+immune+privilege+of+corneal+allografts.">IL-17A-dependent CD4+CD25+ regulatory T cells promote immune privilege of corneal allografts.</a> J. Immunol. 186, 6737-6745 (2011).</li><li>Fu, H., 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=Arginine+depletion+as+a+mechanism+for+the+immune+privilege+of+corneal+allografts.">Arginine depletion as a mechanism for the immune privilege of corneal allografts.</a> Eur. J. Immunol. 41, 2997-3005 (2011).</li><li>Medina, C. A., Rowe, A. M., Yun, H., Knickelbein, J. E., Lathrop, K. L., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azithromycin+treatment+increases+survival+of+high-risk+corneal+allotransplants.Cornea.">Azithromycin treatment increases survival of high-risk corneal allotransplants.Cornea.</a> , 32-658 (2013).</li><li>Cho, Y. K., Zhang, X., Uehara, H., Young, J. R., Archer, B., Ambati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Endothelial+Growth+Factor+Receptor+1+morpholino+increases+graft+survival+in+a+murine+penetrating+keratoplasty.">Vascular Endothelial Growth Factor Receptor 1 morpholino increases graft survival in a murine penetrating keratoplasty.</a> Invest. Ophthalmol. Vis. Sci. 53, 8458-8471 (2012).</li><li>Kim, H. K., Choi, J. A., Uehara, H., Zhang, X., Ambati, B. K., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, 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+graft+rejection.">Corneal graft rejection.</a> Surv. 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The authors have no competing financial interests.

여기에 설명 된 각막 이식의 쥐 모델 요인이 가장 모두 거부 15,17,18,20, 26 ~ 30 각막의 수용 21-25과 관련된 무엇을 예측하는 모델에서 각막 이식 거부 반응을 연구하기 위해 연구자 수 있습니다 동종 이식. 환자를 치료하거나 예방하거나 거부 (31)에 어느 국소 또는 전신적 스테로이드 치료를 부여하는 각막 이식, 달리이 모델은 일반적으로 치료의 부재에서 거부 동종 관련된 ?...

각막 이식은 인간에서 수행 이식의 가장 성공적이고 일반적인 유형 중 하나입니다. 이 수술을 행하는 이유는 부상 한 감염성 질환, 또는 비 - 감염성 질환 각막이 다른 형태의 결과이다. 미국의 안구 은행 협회 숫자는 46,000 이상이 (: restoresight.org/eye_banks/eye_banks.html에서 웹 사이트 참조) 2011 년에 수행 된 것을 나타냅니다. 성공의 표시는 동종 각막 이식 1 년 고장율 10-15%과 성공이 70 % 3-8 초과 5 년 범위 것입니다. 많은 연구가 도시 한 바와 같이, 각막 동종 이식의 성공은 직접 눈 면역 특권 사이트 것과 관련되어있다. 면역 특권 사이트와 각막 상태에 책임 요소는 suppres 각막 의해 생성 각막 모두 혈액 및 림프 혈관 부족, 항원 제시 세포의 상대적인 부재, 요소를 포함면역 이펙터 funtions에게 9-15이야, MHC의 낮은 발현은 16 항원 및 FasL의 17 ~ 20의 발현. 그러나 성공이 이식을 걸리기 이러한 요인에도 불구하고, 그들은 거절 3-7를 받아야 않습니다. 따라서,이 거절을 매개 이러한 메커니즘을 이해뿐만 아니라 거부를 방지하기 위해 다양한 치료법을 테스트하는 것은 매우 중요하다. 이를 위해, 우리는 여기에 통제 된 실험 환경에서 각막 이식을 연구하기 위해 20 년 이상 사용되었습니다 각막 이식의 쥐 모델을 설명합니다. 이식 응답은 궁극적가 이식 된 조직이 실패 또는 성공 여부를 확인할 수 콘서트에서 일하는 많은 다른 요소들을 포함하기 때문에, 어느 하나의 시험 관내 모델에서 이들 요소의 중요성을 이해하는 것은 불가능하다. 따라서, 그대로 동​​물을 사용하는 연구가 성공 또는 failu 중 하나에 중요한 요인이 무엇인지를 결정해야한다이식 조직의 재. 동물의 다른 종 각막 이식을 연구하는 데 사용되었지만, 다른 종을 사용하는 것에 비해, 뮤린 모델은 몇 가지 장점을 갖는다. 첫 번째는 특정 유전자를 발현 또는 기능 이식의 더 나은 연구 할 수있는 특정 면역 인자의 발현을 부족한 유전자를 대상으로 한 마우스의 여러 변종의 존재입니다. 또한, 동물의 많은 다른 종의 존재하지 않는 마우스에 대한 특정 많은 시약 (요인을 중화 재조합 요인과 항체 모두)이 있습니다. 때문에 이들 인자의 존재,이 모델은 급성 각막 동종 이식 반응 15 17,18,20 -29 관련된 관련 요소를 식별하는 데 광범위하게 사용되어왔다. 또한, 각막 이식과 관련된 많은 요인은 다른 조직 이식 기능적 것으로 알려져있다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zeiss Surgical Microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Rebuilt</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe</td>
			<td>BD</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mL Syringe</td>
			<td>BD</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL Syringe</td>
			<td>BD</td>
			<td>309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vannus Scissors</td>
			<td style="width:71px;">Stortz</td>
			<td style="width:119px;">E-3387</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">11-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">717939M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trephine 2.0mm</td>
			<td style="width:71px;">Katena</td>
			<td style="width:119px;">K 2-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trephine 1.5 mm</td>
			<td style="width:71px;">Katena</td>
			<td style="width:119px;">K 2-7510</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Tricaine Hydrochloride 0.5%</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">NDC 0065-0741-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Healon</td>
			<td style="width:71px;">Abbott</td>
			<td style="width:119px;">Healon OVD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">7-0 Sutures</td>
			<td style="width:71px;">Alcon</td>
			<td align="right" style="width:119px;">8065</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">2.5% Phenylephrine HCl</td>
			<td style="width:71px;">Alcon</td>
			<td style="width:119px;">NDC 61314-342-02</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">1% Tropicamide</td>
			<td style="width:71px;">Bausch &#38; Lomb</td>
			<td style="width:119px;">NDC-24208-585-59</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hamilton Syringe</td>
			<td style="width:71px;">Hamilton</td>
			<td style="width:119px;">7654-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">33 gauge needle</td>
			<td style="width:71px;">Hamilton</td>
			<td align="right" style="width:119px;">90033</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cell Strainer (100 &#956;m nylon)</td>
			<td style="width:71px;">BD Falcon</td>
			<td align="right" style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hemocytometer</td>
			<td style="width:71px;">Cardinal Health</td>
			<td style="width:119px;">B3175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine corneal transplantation: a model to study the most common form of solid organ transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

각막 이식의 쥐 모델을 성공적으로 각막 이식 거부 19-23 각막 이식 수용 13, 15, 16, 18, ​​24 ~ 27 두의 메커니즘을 특성화하기 위해 20 년 이상 사용되어왔다. 이 모델은 FasL의 결여 즉 동물에서 각막 이식에서 수용 FasL의 발현의 중요성을 설정하는 데 사용 된 각막 동종 이식 (15)을 받아 들일 수 없었다. 이는 또한 혈관 내피 성장 인자 수용체 1 모르 처리 크게 각막 이식 생존 <...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

쥐의 각막 이식 : 고체 장기 이식의 가장 일반적인 형태를 연구하기위한 모델

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

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9.6K 조회수.  Saint Louis University. Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

비디오: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Orthotopic 대동맥 이식 : 만성 Vasculopathy의 개발 연구에 생쥐 모델

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

연구

의학

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Murine 아일릿 절연 및 Subcapsular 신장 이식을위한 방법

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Orthotopic 폐 이식의 Murine 모델에 Obliterative Bronchiolitis의 개발

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

수정 커프 기술을 사용하여 쥐의 자궁 심장 이식 모델

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Bowditch 효과 조사 : 압력 볼륨 관계를 측정하기 위해 마우스의 심장 도관

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

분할 각막 단추를 사용하여 돼지 각막 내피 내피 기관 문화 모형

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

각막 내피 세포 손실의 비 침습적, 레이저 보조 실험 모델의 개발

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

직교 간 이식의 쥐 모형에 있는 동맥 재연결 후에 감소된 합병증

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

24시간 장기 보존 및 지속적인 원격 분석을 사용하여 돼지 모델에서 직소 신장 자동 이식

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 모델에서 심근 경색 후 MSC 로딩 주사용 하이드로겔의 자궁 내 이식

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

Methods Article

쥐의 각막 이식 : 고체 장기 이식의 가장 일반적인 형태를 연구하기위한 모델