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. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

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><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>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 ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

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9.7K 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

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 ...

연구

의학

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

마우스 신장 이식 : 동종 이식 거부의 모델

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

쥐의 전체 두께 피부 이식

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

면역학 및 감염병학

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

마우스의 소설 미세 수술 이소성 모델, 욕실 공산권 가슴 벽, 흉선, 및 심장 이식

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

마우스에서 소성을 뒷다리 이식

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

쥐에 있는 이종 심혼 및 심장 근육 세포 이식을 위한 면역학 모형

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

무균 기술, 마취 및 진통의 현대 표준과 일치하는 수정된 쥐 이종 심장 이식 프로토콜

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

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 ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.