We thank the imaging platform of the Institut de la Vision. We acknowledge the French Muscular Dystrophy Association (AFM) for a PhD fellowship to O.V. and Allergan INC. This work performed in the frame of the LABEX LIFESENSES [reference ANR-10-LABX-65] was supported by French state funds managed by the ANR. We thank Peggy Barbe, and M&#233;lissa Desrosiers for technical assistance with AAV preparations. We are grateful to St&#233;phane Fouquet for excellent technical assistance in confocal microscopy and his expert input with the interpretation of the results.

본 연구에 사용 된 모든 동물은 안과 및 비전 연구에서 동물의 사용을위한 ARVO 방침에 따라 마음에 든다고 및 처리되었다. HEK-293 세포를 17, 18의 일시 발현에 의해 재조합 AAV (rAAV) 1. 생산 참고 : 맥 클루 C, (2011) 19 조브를 참조하십시오. <ol><li> AAV 벡터 플라스미드의 대규모 플라스미드 제조 (적어도 1 ㎎ / ㎖)을 정제. 3 플라스미드를 사용합니다. AAV 역전 된 말단없이 복제 및 캡시드 유전자를 운반하는 AAV 헬퍼 플라스미드 LTR에, AAV ITR이 전사 플라스미드로 지칭 운반 유전자 발현 카세트, 및 아데노 바이러스 헬퍼 유전자 E2, E4 및 VA RNA 유전자를 공급하는 플라스미드를 지칭 됨 반복 pHELPER으로합니다. </li><li> 이 3 개의 플라스미드 폴리에틸렌 이민 (또는 다른 형질 전환 시약)을 사용하여 293 세포를 형질 감염. 형질 전환 효율이 80 % 이상인 양호한 VI에 필요RAL 수익률. </li><li> iodixanol 기울기에 293 세포 용 해물에서 재조합 AAV를 정화. 15 %, 25 %, 40 % 및 60 % iodixanol 그라데이션을 획득하기 위해 사용된다. , AAV 입자를 함유하는 40 % iodixanol 분획 수집 0.001 % 플루로 PBS에 대해 1 시간 및 완충액 교환 (인산 완충 생리 식염수)에 대해 500,000 XG에서 초 원심 분리 한 후 분획을 농축시킨다. </li><li> 바이러스 게놈 역가 (20)를 확인하려면, QPCR 다음에 DNase의 ProteinaseK 다이제스트를 수행합니다. 정방향 및 역방향 프라이머는 AAV2 ITR 위치한 62 BP 영역을 증폭. 표준 플라스미드, 프로브는 FAM으로 표지하고 (BHQ) 소광 블랙홀 갖는다. </li><li> ITR 프라이머, 100 nM의 순방향 ITR 프라이머, 100 nM의 AAV2 ITR 프로브, 0.4 mM의 dNTPs를, 2 mM의 MgCl2를, 1X 백금의 Taq 완충액, 1U 백금 역방향가 340nm를 사용하여 25 μL의 최종 부피의 qPCR (정량적 폴리머 라제 연쇄 반응)을 수행 DNA 형성 촉매 5 μL 템플릿 (표준, 샘플 및 템플릿없이 제어). </li><li> 용표준은, 연속 희석을 배 10 7 X에 형질위한 플라스미드를 사용하여 (5 μL 10월 10일에서 10월 4일까지 게놈 / ㎖의 최종 농도에서 얻어진 2 × 109 3 10 2 × 게놈 / ㎖ 각각). </li><li> 변성의 40주기 다음에 15 분 동안 95 ° C에서 초기 변성 단계에 의해 프로그램을 시작 1 분, 어닐링 / 신장 95 ℃에서 1 분, 60 ° C에서. 단기 기억이나 장기 저장 기간 동안 -20 ℃에서 4 ℃에서 보관. </li></ol> AAV 2. 유리체 강내 주입 <ol><li> 케타민 (50 ㎎ / ㎏)과 자일 라진 (10 ㎎ / ㎏)와 C57BL6J 마우스를 마취의 반사, 적절한 마취를 나타내는 철수 반사와 꼬리 핀치 응답을 바로 잡고의 손실을 확인합니다. neosynephrine 5 %의 각막 응용 프로그램에 의해 동공을 확장하고 안약 0.5 % mydriaticum. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> 초 미세 30 G의 disposab 전달제작 : 적도를 통해 바늘과 유리체에, 윤부 옆에 (애기 K, 주피터 (2007) 21 참조). 유리체의 중심에 바늘의 직접 관찰과 ShH10-GFP 또는 AAV5-GFP의 1 10 × 4 (11) 부사장을 포함하는 1 μL 재고 (그림 1)을 주입한다. ILM 실험에 대한 제어로 한쪽 눈을 사용하십시오. BRB 실험에 두 눈을 주입한다. 주입 눈에 항 염증 항균 국소 치료를 적용합니다. </li><li> neosynephrine와 동공을 확장하고 mydriaticum 눈 이미징 떨어진다. GFP (녹색 형광 단백질) 식 일주, 이주 1 월, 유리체 강내 주입되어 2 개월 후 (그림 1)을 따라 안저 카메라로 안저 검사를 수행합니다. CO 2 흡입 1-2개월 후 주사로 쥐를 희생. </li></ol> 3. 면역 조직 화학 <ol><li> 망막 저온부 (그림 1)의 경우, L을 제거하는 탈핵 눈을 해부1 시간 동안 4 % 파라 포름 알데히드에 ENS와 각막 및 침수 수정. <ol><li> 실온에서 1 시간, 실온에서 또 다른 시간 동안 20 % 수 크로즈 10 % 수크로오스에 미리 고정 눈 Cryoprotect. 밤새 4 ° C에서 30 % 자당 용액 Cryoprotect. 동결 등을 알아내는 매트릭스로 수지를 포함에 eyecups을 포함합니다. 10 μm의 저온부를 확인하고 특수 냉동 조직 절편 치료를 슬라이드에 마운트하고 그 염색시 조직의 준수를 향상시킬 수 있습니다. </li><li> 5 분 동안 PBS pH를 7.4에서 0.1 % 트리톤 X100와 섹션을 Permeabilize 하시려면. PBS에서 세척 배 및 블록 1 % BSA와 함께 PBS에서 실온에서 1 시간, 0.1 % 트윈 20 대. </li></ol></li><li> 망막 flatmounts (도 1)의 경우, 박리를 용이하게하기 위해 15 분 동안 4 % 파라 포름 알데히드에서 탈핵 쳐다. <ol><li> 15-30 분에서, 각막 및 렌즈를 제거하는 눈을 해부. ORA 세라 주위 절단함으로써 망막 색소 상피 (RPE) 및 공막으로부터 망막을 분리하고,시신경. 망막 및 형질 망막 세포에 형광 단백질 (고정이 24 시간을 초과 할 수 없습니다) 해결하기 위해 또 다른 30 ​​분 동안 4 % 파라 포름 알데히드에 담가. 멸균 PBS, pH가 7.4에서 5 분을 씻으십시오. </li><li> (1 % BSA, 2 % 정상 염소 또는 당나귀 혈청 PBS, 0.5 % 트리톤 X-100) 버퍼를 차단하는 PBS를 변경하고 실온에서 4 시간 또는 4 박 ° C 중 하나에 대한 버퍼를 차단에 품어. </li></ol></li><li> 실온에서 또는 4 ° C에서 하룻밤 중 4 시간 동안 차단 버퍼에 차 항체와 조직을 품어. PBS로 세척 배. 참고 : 다음과 같이 사용 된 항체는 다음과 같습니다 항 라미닌 (/ 1,000 1) 라벨 ILM, 안티 로돕신 클론 4D2 (1/500) 표시 막대, 안티 - 글루타민 합성 효소 클론 GS-6 (1 / 1,500) 라벨 뮐러 세포 , PNA 렉틴 (1/40) 라벨 콘. </li><li> 방 템피에서 1 시간 (저온부) 또는 2 시간 (flatmounts)에 대한 버퍼를 차단에 알렉사 형석 복합 이차 항체의 500 희석 : 1 조직을 품어rature와 빛으로부터 보호. PBS로 세척 배. </li><li> 망막 완화 컷을 확인하고는 광 수용체 또는 망막 신경절 세포 (RGC) 어느 한쪽이 위쪽으로 향하게하여 유리 슬라이드에 flatmount. 4 ° C에서 coverslip에 저장을 적용, 수성 mountingmedium를 추가합니다. </li><li> 레이저 스캐닝 공 초점 현미경에 공 초점 현미경을 수행합니다. 여기 및 발광 크로스 토크를 줄이기 위해, 이미지 라인으로 순차적으로, 라인을 획득. 표본화 정리에 따라 스텝 크기를 정의합니다. 최종 이미지에 과포화 픽셀을 최소화 노출 설정을 사용합니다. Z-프로젝트 기능에서 최대 강도를 사용하여 마지막 8 비트 RGB 색상 모드로 변환 한면에 피지와 프로세스 12 비트 이미지, 프로젝트 Z 섹션. </li></ol> 마우스의 혈액 샘플 4. PCR 분석 <ol><li> 마취 C57BL6J 마우스의 음경 정맥 AAV-GFP의 1 내지 10 행 × 4 11 입자를 함유하는 100 μL 스톡 (5 % 이소 플루 란을 주입초 미세 6mm 바늘 인슐린 주사기를 사용하여 가까운 챔버 유도 및 수술 중에 코 콘을 통해 이소 플루 란의 2.5 %)에 대한. 이 동물은 AAV 입자 순환의 양성 대조군이 될 것입니다. </li><li> 주입하고 3 시간, 24 시간 2 일, 3 일 ShH10 유리체 강내 주입 후 또는 intrapenile 주입 (그림 1) 전후 마우스 꼬리에서 샘플 혈액. 약 10 ~ 20 μL 헤파린 튜브에 수집됩니다. 절차의 완료 후 CO 2 흡입 쥐를 희생. </li><li> 당신의 선택의 추출 키트를 사용하여 혈액 샘플에서 게놈 DNA를 추출합니다. </li><li> 게놈 DNA의 PCR 증폭을 수행합니다. 이 프로토콜은 다음 프라이머 쌍을 사용 GFP를, 5&#39;-CGACACAATCTGCCCTTTCG-3 &#39;, 안티센스 5&#39;-CATGGACGAGCTGTACAAGGGA-3&#39;을 감지. 다음 PCR 프로그램을 수행 하였다 : 95 ° C에서 2 분 (1 사이클); 95 ° C 45 초, 55 ℃ 1 분, 72 ℃에서 45 초 (30 회) 72 ° C 5 분 (1주기)4 ° C 유지. </li></ol> 5. 선택 에반스 블루 방법 주 : 에반스 블루 제 10 이용한 망막 혈관에서 알부민의 누설을 측정함으로써 혈관 투과성 정량화. <ol><li> 5 ㎛의 필터를 통해 5 분간 초음파 세정기에서, 여과 및 생리 식염수 (6 ㎎ / ㎖), 초음파 처리에 의해 용해 에반스 블루 염료를 준비한다. </li><li> (단계 4.1, 이소 플루 란 흡입) C57BL6J 마우스를 마취 반사, 철수 반사와 적절한 마취를 나타내는 꼬리 핀치 응답을 바로 잡고의 손실을 확인하고 음경 정맥 (그림 1)을 통해 에반스 블루 (45 ㎎ / ㎏)을 주입. 쥐의 발, 총구, 귀이, 에반스 블루 주입에 따라 염료의 흡수와 분포를 확인 명확 블루가 있는지 확인합니다. </li><li> 케타민 (50 ㎎ / kg)과 자일 라진 (10 ㎎ / kg)와 염료의 주사 후 C57BL6J 마우스에게 3 시간 및 마취 rightin의 손실을 검증g는 적절한 마취를 나타내는 리플렉스. 샘플 intracardially 500 헤파린 튜브에 혈액 μL 및 시트르산 완충액 좌심실을 통해 2 분 동안 마우스를 관류 (0.05 M, pH가 3.5, 시트르산 7.8 g, 증류수 1 L 시트르산 나트륨 3.8 g을 용해) 혈관 수축을 방지하기 위해 37 ° C로 미리 예열. 마우스는 관류를 통해 희생된다. </li><li> 신중하게 관류, 명백히하다 및 후 망막을 수집하는 운영 현미경으로 두 눈을 해부하다 (3.2 절 참조). 원심 증발기에서 건조 망막 하룻밤, 무게, 그리고 (100 XG)를 진탕 65 ° C에서 18 시간 동안 포름 아미드 100 ㎕와 망막을 배양하여 에반스 블루 염료를 추출합니다. 4 ° C에서 2 시간 동안 20,798.5 XG에 30K 오메가 필터 튜브 원심 분리기 망막 샘플 및 원심 분리기 혈액 샘플을 4 ℃에서 15 분 동안 20,798.5 XG에. </li><li> 흡광도를 측정하기 위해 두 상층 액을 사용합니다. 최대의 흡광도 FO를 감산하여 각 시료의 흡광도를 결정740 nm에서 흡광도 최소값이 620 nm에서 에반스 블루 R. 포름 아미드 에반스 블루의 표준 곡선에서 혈장과 망막 에반스 블루 농도를 계산한다. 시간당 건조 망막 (플라즈마 / g 망막 건조 중량 / 인사 μL)의 그램 당 에반스 블루의 마이크로 리터의 BRB 투과성을 표현한다. </li></ol> 6. 선택적 형광 안저 주 : 마우스에서 망막 혈관으로부터 누설 나트륨 플루오 레세 인의 복강 내 주사에 의해 시각화 될 수있다. <ol><li> C57BL6J 마우스 마취 (이소 플루 란 흡입을, 단계 4.1 참조)과 눈 (neosynephrine 및 mydriaticum)를 드랍 동공을 확장. 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> 형광 안저 0.1 ml의 멸균 식염수에 10 %의 나트륨 형광의 복강 0.01-0.02 ml를 주입한다. </li><li> 안저 카메라를 사용하여 주사 후 양쪽 눈의 영상을 수집한다. 프로 후 공동으로 2 흡입 쥐를 희생시저. 참고 : 망막 혈관 조영술에 표시 될 20 초 필요하다. 형광 이미지는 주사 후 3-10 분 사이에 최대 포착되어야한다. 누설 명소 (있는 경우) 2.5 분 후에 관찰 할 수 있습니다. 나중 시점에서 화질 인해 나트륨 플루오 레세 곧 분석에 사용 된 후에 얻어지는 유리체, 따라서로 이미지 만이 확산 손실된다. </li></ol>

<ol>
	<li>Halfter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+retinal+basal+lamina+during+early+embryonic+development+leads+to+a+retraction+of+vitreal+end+feet,+an+increased+number+of+ganglion+cells,+and+aberrant+axonal+outgrowth.">Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth.</a> J Comp Neurol. 397 (1), 89-104 (1998).</li><li>Halfter, W., Dong, S., Balasubramani, M., Bier, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary+disruption+of+the+retinal+basal+lamina+and+its+effect+on+retinal+histogenesis.">Temporary disruption of the retinal basal lamina and its effect on retinal histogenesis.</a> Dev Biol. 238 (1), 79-96 (2001).</li><li>Halfter, W., Willem, M., Mayer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane-dependent+survival+of+retinal+ganglion+cells.">Basement membrane-dependent survival of retinal ganglion cells.</a> Invest Ophthalmol Vis Sci. 46 (3), 1000-1009 (2005).</li><li>Abdelkader, E., Lois, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+limiting+membrane+peeling+in+vitreo-retinal+surgery.">Internal limiting membrane peeling in vitreo-retinal surgery.</a> Surv Ophthalmol. 53 (4), 368-396 (2008).</li><li>Dalkara, D., 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=Inner+limiting+membrane+barriers+to+AAV-mediated+retinal+transduction+from+the+vitreous.">Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous.</a> Mol Ther. 17 (12), 2096-2102 (2009).</li><li>Bringmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muller+cells+in+the+healthy+and+diseased+retina.">Muller cells in the healthy and diseased retina.</a> Prog Retin Eye Res. 25 (4), 397-424 (2006).</li><li>Eichler, W., Kuhrt, H., Hoffmann, S., Wiedemann, P., Reichenbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+release+by+retinal+glia+depends+on+both+oxygen+and+glucose+supply.">VEGF release by retinal glia depends on both oxygen and glucose supply.</a> Neuroreport. 11 (16), 3533-3537 (2000).</li><li>Kaur, C., Foulds, W. S., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-retinal+barrier+in+hypoxic+ischaemic+conditions:+basic+concepts,+clinical+features+and+management.">Blood-retinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management.</a> Prog Retin Eye Res. 27 (6), 622-647 (2008).</li><li>Kaur, C., Sivakumar, V., Foulds, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+response+of+neurons+and+glial+cells+to+hypoxia+in+the+retina.">Early response of neurons and glial cells to hypoxia in the retina.</a> Invest Ophthalmol Vis Sci. 47 (3), 1126-1141 (2006).</li><li>Xu, Q., Qaum, T., Adamis, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+blood-retinal+barrier+breakdown+quantitation+using+Evans+blue.">Sensitive blood-retinal barrier breakdown quantitation using Evans blue.</a> Invest Ophthalmol Vis Sci. 42 (3), 789-794 (2001).</li><li>Amato, R., Wesolowski, E., Smith, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+visualization+of+the+retina+by+angiography+with+high-molecular-weight+fluorescein-labeled+dextrans+in+the+mouse.">Microscopic visualization of the retina by angiography with high-molecular-weight fluorescein-labeled dextrans in the mouse.</a> Microvasc Res. 46 (2), 135-142 (1993).</li><li>Yang, G. 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=Virus-mediated+transduction+of+murine+retina+with+adeno-associated+virus:+effects+of+viral+capsid+and+genome+size.">Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size.</a> J Virol. 76 (15), 7651-7660 (2002).</li><li>Li, W., 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=Gene+therapy+following+subretinal+AAV5+vector+delivery+is+not+affected+by+a+previous+intravitreal+AAV5+vector+administration+in+the+partner+eye.">Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye.</a> Mol Vis. 15, 267-275 (2009).</li><li>Koerber, J. T., 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=Molecular+evolution+of+adeno-associated+virus+for+enhanced+glial+gene+delivery.">Molecular evolution of adeno-associated virus for enhanced glial gene delivery.</a> Mol Ther. 17 (12), 2088-2095 (2009).</li><li>Klimczak, R. R., Koerber, J. T., Dalkara, D., Flannery, J. G., Schaffer, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+adeno-associated+viral+variant+for+efficient+and+selective+intravitreal+transduction+of+rat+Muller+cells.">A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Muller cells.</a> PLoS One. 4 (10), e7467 (2009).</li><li>Vacca, O., 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=AAV-mediated+gene+delivery+in+Dp71-null+mouse+model+with+compromised+barriers.">AAV-mediated gene delivery in Dp71-null mouse model with compromised barriers.</a> Glia. , (2013).</li><li>Choi, V. W., Asokan, A., Haberman, R. A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+viral+vectors.">Production of recombinant adeno-associated viral vectors.</a> Curr Protoc Hum Genet. 12 (Unit 12 19), (2007).</li><li>Choi, V. W., Asokan, A., Haberman, R. A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+recombinant+adeno-associated+viral+vectors+for+in+vitro+and+in+vivo+use.">Production of recombinant adeno-associated viral vectors for in vitro and in vivo use.</a> Curr Protoc Mol Biol. 16 (Unit 16 25), (2007).</li><li>McClure, C., Cole, K. L., Wulff, P., Klugmann, M., Murray, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+titering+of+recombinant+adeno-associated+viral+vectors.">Production and titering of recombinant adeno-associated viral vectors.</a> J Vis Exp. (57), e3348 (2011).</li><li>Aurnhammer, C., 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=Universal+real-time+PCR+for+the+detection+and+quantification+of+adeno-associated+virus+serotype+2-derived+inverted+terminal+repeat+sequences.">Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences.</a> Hum Gene Ther Methods. 23 (1), 18-28 (2012).</li><li>Chiu, K., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreous+injection+for+establishing+ocular+diseases+model.">Intravitreous injection for establishing ocular diseases model.</a> J Vis Exp. (8), 313 (2007).</li><li>Kolstad, K. D., 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=Changes+in+adeno-associated+virus-mediated+gene+delivery+in+retinal+degeneration.">Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.</a> Hum Gene Ther. 21 (5), 571-578 (2010).</li><li>Sene, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+implication+of+Dp71+in+osmoregulation+and+vascular+permeability+of+the+retina.">Functional implication of Dp71 in osmoregulation and vascular permeability of the retina.</a> PLoS One. 4 (10), e7329 (2009).</li><li>Benard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Quantifiable+Blood+Retinal+Barrier+Breakdown+Model+In+Mice.">A New Quantifiable Blood Retinal Barrier Breakdown Model In Mice.</a> ARVO Annual Meeting. , (2011).</li></ol>

The authors have nothing to disclose.

BRB는 혈액과 망막 사이 분자의 교환을 제어한다. 그 내역은 당뇨병 성 망막증 또는 연령 - 관련 황반 변성 (AMD)과 같은 다양한 질병과 연관된다. 우리는 최근에 투과 BRB를 표시하는 디스트로핀 넉 아웃 (knock-out) 마우스에, 망막은 아데노 관련 바이러스 성 벡터 (AAV)에 의해 매개 유전자 전달에 더 관대하게 보여 주었다. 그러나, BRB 투자율 AAV 입자에도 불구하고 안구 내이 모델의 안구 구획에 국한 유...

뮐러 세포는 망막의 폐해 주요 성분이다. 형태 학적으로, 이들은, 유리체와 접촉하는 반경 방향 및 망막 endfeet을 스팬 ILM 후자의 비밀 컴포넌트를 직면한다. ILM는 약 10 가지 세포 외 기질 단백질 (라미닌, 아 그린, perlecan, nidogen 콜라겐 여러 헤파린 설페이트 프로테오글리칸)로 이루어지는 기저막이다. 개발하는 동안, 그 존재는 신경절 세포 1-3의 망막 histogenesis, 시신경 축삭의 탐색 및 생존을위한 필수적이다. 그러나, ILM 성인 망막 본질적인이고 수술 사 망막 손상시키지 않고 특정 병리에서 제거 될 수있다. 유전자 치료에있어서, 상기 멤브레인 주입술 5 AAVs를 이용한 망막의 효율적인 전달을위한 물리적 장벽이된다. 그들의 프로세스의 광범위한 arborization을 통해 뮐러 세포 영양 및 규제 suppo을 제공RT 망막 신경 세포와 혈관 세포 모두에. 뮐러 세포도 BRB (6)의 형성과 유지에, 망막 항상성의 조절에 관여한다. 꽉 접합 망막 모세 혈관 내피 세포 사이에, 뮐러 세포, 성상 세포와 혈관 주위 세포는 BRB를 형성한다. BRB는 당뇨 망막 병증, 망막 정맥 폐쇄 및 호흡기 질환 등 retina.In 많은 질병에 들어가는 특정 물질을 방지, 망막의 저산소증은 BRB 7-9을 통해 누출이 발생합니다. 이 파열은 vasogenic 부종, 망막 박리와 망막 손상으로 이어지는 혈관 투과성의 증가와 연관되어있다. 뮐러 세포와 밀접 모두 BRB ILM 무결성에 중요한 역할을하고, 혈관 기저막과 연관된다. 따라서, 뮐러 아교 세포의 레이블을 다음 망막 장벽의 물리적 상태의 연구에 특히 관련이있다. 고전적인아군, BRB 투과성 혈장 알부민 비공유 결합 에반스 청색 염료의 전신 주입 이루어진 에반스 블루 분석법을 이용하여 측정된다. 상기 분석은 망막 혈관 (10) (프로토콜 섹션 5 참조)에서 알부민 누설 (중간 크기의 단백질, ~ 66 kDa의)을 측정한다. 또한, 혈관 누출 형광의 누설 증명 형광 망막 혈관 조영술에 의해 가시화 될 수있다 (작은 분자 ~ 359 다; 프로토콜 제 6 장 참조) 11. 그럼에도 불구하고, 두 방법은 작은 분자와 단백질에 BRB 투과성의 평가를 할 수 있지만 ILM의 무결성에 대한 정보를 제공하지 않습니다. 따라서, BRB 투과성 공부, 우리는 큰 분자 (예를 들어, AAV 입자, 25 nm의 직경)에 BRB 투과성에 대한 정보를 제공 AAV 기반 방법을 사용 하였다. 사실, 우리의 방법은 그 제안하는 혈액에 AAV 전이 유전자의 존재를 감지 할 수 있습니다 ~ 25 nm의 직경 입자 것혈류로 침투 할 수 있습니다. 이 방법은 또한, 병리 적 상태의 ILM 및 세포 외 기질 단백질의 구조에 대한 정보를 제공한다. 두 AAV 변종은 연구에 유용 : AAV5 및 ShH10. Subretinally 주입, AAV5은 광 수용체와 망막 색소 상피 (12)에 대한 자연적인 굴곡 운동이 있지만 그대로 ILM 5,13 야생 형 망막의 유리체에 투여시는 외부 망막에 걸쳐 얻을 수 없습니다. ShH10 특별히 신경 세포 (14, 15)을 통해 아교 세포를 대상으로 설계되었습니다 AAV의 변종이다. ShH10 선택적으로 손상된 장벽 (16)와 망막의 효율성 향상과 건강하고 병에 걸린 망막 모두에서 뮐러 세포 레이블. immuhistochemistry 및 혈액 DNA 분석과 결합 된 이러한 바이러스 도구 망막 장벽의 상태 및 질환에서의 관여 (도 1)에 대한 정보를 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="643">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Name of Material/ Equipment</td>
			<td style="width:107px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:193px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">C57BL6J mice strain</td>
			<td style="width:107px;">JANVIER LABS</td>
			<td style="width:119px;"></td>
			<td>mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Ketamine 500</td>
			<td style="width:107px;">Virbac France</td>
			<td style="width:119px;"></td>
			<td>anesthetic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Xylazine Rompun 2%</td>
			<td style="width:107px;">Bayer Healthcare</td>
			<td style="width:119px;"></td>
			<td>anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Neosynephrine 5% Faure</td>
			<td style="width:107px;">Europhta</td>
			<td style="width:119px;"></td>
			<td>dilatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Mydriaticum 0,5%</td>
			<td style="width:107px;">Thea</td>
			<td style="width:119px;"></td>
			<td>dilatant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sterdex</td>
			<td style="width:107px;">Novartis</td>
			<td style="width:119px;"></td>
			<td>anti-inflammatory</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Cryomatrix embedding resin</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Superfrost Plus Adhesion Slides</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:119px;">10143352</td>
			<td>slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">anti-laminin&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">L9393</td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">anti-rhodopsin clone 4D2&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">MABN15</td>
			<td>antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">anti-glutamine synthetase clone GS-6&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;">MAB302</td>
			<td>antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Anti-Glial Fibrillary Acidic Protein</td>
			<td style="width:107px;">Dako</td>
			<td style="width:119px;">334</td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">PNA Lectin&#160;</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;">L32459</td>
			<td>probe</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Alexa fluor conjugated secondary antibodies&#160;</td>
			<td style="width:107px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td>antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Fluorsave reagent</td>
			<td style="width:107px;">Calbiochem</td>
			<td style="width:119px;">345789</td>
			<td>mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">QIAmp DNA Micro Kit&#160;</td>
			<td style="width:107px;">QIAGEN</td>
			<td style="width:119px;">56304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">GoTaq DNA polymerase</td>
			<td style="width:107px;">Promega</td>
			<td style="width:119px;">M3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Evans Blue dye&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">E2129&#160;</td>
			<td>dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">5 &#181;m filter&#160;</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Sodium Citrate&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">S1804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Citric acid&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">C1909-2.5KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Formamide spectrophotometric&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">295876-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Fluorescein</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:119px;">F2456&#160;</td>
			<td>dye</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:225px;">Micron III</td>
			<td style="width:107px;">Phoenix Research Labs</td>
			<td style="width:119px;"></td>
			<td style="width:193px;">Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Insulin Syringes</td>
			<td style="width:107px;">Terumo</td>
			<td style="width:119px;">SS30M3109&#160;</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Syringe 10 &#181;l Hamilton</td>
			<td style="width:107px;">Dutscher</td>
			<td style="width:119px;">74487</td>
			<td>Seringue 1701</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Needle RN G33, 25 mm, PST 2&#160;</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:119px;">11530332</td>
			<td>Intravitreal Injection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">UltraMicroPump UMP3</td>
			<td rowspan="2" style="width:107px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3</td>
			<td style="width:193px;">Versatile injector uses microsyringes to deliver picoliter volumes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller</td>
			<td style="width:119px;">UMP3-1</td>
			<td>Digital controller</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:225px;">Binocular magnifier SZ76</td>
			<td style="width:107px;">ADVILAB</td>
			<td style="width:119px;">ADV-76B2</td>
			<td style="width:193px;">Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Spring scissors straight - 8,5cm</td>
			<td rowspan="3" style="width:107px;">Bionic France S.a.r.l</td>
			<td style="width:119px;">15003-08</td>
			<td rowspan="3" style="width:193px;">Retinas dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Micro-ciseaux de Vannas courbe</td>
			<td style="width:119px;">15004-08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Pince Dumont 5</td>
			<td style="width:119px;">11254-20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Veriti 96-Well Thermal Cycler</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:119px;">4375786</td>
			<td>Thermocycler</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Ultrasonic cleaner&#160;</td>
			<td style="width:107px;">Laboratory Supplies</td>
			<td style="width:119px;">G1125P1T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Nanosep 30k omega tubes&#160;</td>
			<td style="width:107px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:225px;">Speedvac</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:119px;">SC 110 A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:225px;">Spectrofluorometer&#160;</td>
			<td style="width:107px;">TECAN&#160;</td>
			<td style="width:119px;">infinite M1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

using adeno-associated virus as a tool to study retinal barriers in disease

뮐러 세포는 망막의 주요 신경교 세​​포이다. 최종 - 피트는 외측 및 내측 제한 멤브레인 (ILM)에서 망막의 한계를 형성하고, 성상 세포, 외막 세포 및 내피 세포와 함께 이들은 혈액 망막 장벽 (BRB)을 확립한다. ILM은 망막과 유리체 사이에 조직 학적 경계를 정의하는 기저막 역할을하면서 BRB는 혈류와 망막 사이의 물질 이동을 제한합니다. 뮐러 세포 레이블링이 세포는 BRB와 ILM의 중요한 부분으로, 망막 장벽의 물리적 상태를 연구하는 것이 특히 관련이있다. BRB와 ILM 모두 자주 망막 질환에서 변경 및 질병 증상에 대한 책임이 있습니다. 이러한 에반스 블루 분석 또는 형광 안저 촬영 등의 BRB의 무결성을 연구하는 몇 가지 잘 확립 된 방법이있다. 그러나 이러한 방법들은 BRB 투과도 (T)의 범위에 대한 정보를 제공하지 않는다나노 미터 범위의 더 큰 분자, O. 또한, 그들은 같은 ILM 다른 망막 장벽의 상태에 대한 정보는 제공하지 않는다. 망막 ILM과 함께 BRB 투과성을 연구하기 위해, 우리는 질병 상태의 ILM과 세포 외 기질 단백질의 상태를 나타내는 동안 큰 분자 BRB의 투과성에 대한 정보를 제공 AAV 기반의 방법을 사용했다. 두 AAV 변종은 연구에 유용 : AAV5 및 ShH10. AAV5은 광 수용체에 대한 자연적인 굴곡 운동이 있지만 ILM이 손상되지 않은 경우 (야생 형 망막에 즉,) 유리체에 투여시는 외부 망막에 걸쳐 얻을 수 없습니다. ShH10는 신경 교세포 대한 강한 향성을 가지고 있으며, 선택적으로 건강하고 병에 걸린 망막 모두에서 뮐러 아교 세포 레이블을 것입니다. ShH10는 ILM이 손상되어 망막에보다 효율적인 유전자 전달을 제공합니다. 면역 조직 화학 및 혈액 DNA 분석과 함께 이러한 바이러스 도구는 질병 망막 장벽의 상태에 빛을 흘렸다.

동물 모델은 ILM (- B도 2a)의 구조를 도시 섭동 있다면 우리는를 사용 ShH10 뮐러 글 리아 세포의 형질 도입을 증가 망막 예상된다. 예를 들어, 우리가 Dp71의 부재에 그를 보여, ShH10 목표를 구체적으로하지만, 더 효율적으로 뮐러 야생형 마우스 (16) (그림 2C - F)에 비해이 마우스 라인에서 ILM의 투과성 증가를 나타내는 유리체 강내 주입하여 신경 교세포. </...

Watch this Scientific Journal Video about Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease at JoVE.com

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

도구로 아데노 관련 바이러스를 사용하여 질병 망막 장벽을 연구하는

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

using-adeno-associated-virus-as-tool-to-study-retinal-barriers

Research

JoVE Journal

Medicine

14.9K 조회수.  Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968. To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

비디오: Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

재조합 Adeno - 관련 바이러스 혈청형 3 벡터에 의해 간암 세포의 고효율 전달

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

연구

의학

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Polycystic 신장 질환과 함께 인간과 마우스의 염색체 이상을 공부하는 스펙트럼 Karyotyping

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

편리한 도구로 안저 사진은 역학 연구에서 심혈관 질환의 위험 요인에 대한 미세 혈관 응답을 연구하기 위해

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

인간 Videofluoroscopic 삼키기 연구 방법을 적응하는 것은 검색 및 설치류 질병 모델에서 연하의 특성을

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

재조합 아데노 - 관련 바이러스 성 벡터의 정위 주사를 통해 파킨슨 병에 대한 알파 - 시누 클레인 기반 쥐 모델 개발

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

망막 화상 진 찰을 사용 하 여 치 매 공부 하기

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

Adeno 관련 바이러스 중재 배달 심장 유전자 쥐에서 편집에 대 한 CRISPR의

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

The Dyspepsia Educational Tool As a Novel Aid in Dyspepsia Management

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from this development, as it enables patient engagement in self-management. Dyspepsia is a condition thought to arise from gastric and duodenal perturbations, brain-gut axis disturbances, and dietary factors. Behavioral interventions are a major part of dyspepsia treatment, hence patient engagement and motivation through education is essential. Protocols that describe the development process of such educational tools are scarce. We provide a methodology describing development of a dyspepsia educational tool. Assessment of users' needs is the first step, followed by a literature search. The content is developed based on the main themes and entered into a content management system, to build the program. Final adjustments are made after a pilot test of the tool. The presented protocol can be used as a guide for development of a digital dyspepsia educational tool or as a tool for similar situations.

This protocol describes the development process of a digital dyspepsia educational tool. Assessment of unmet needs and literature, content development, and building of the tool are presented. The methodology can be used as a guide for future development of digital educational tools.

소화 불량 장애 관리에 새로운 원조로 소화 불량 교육 도구

Digital educational tools have a well-established role in current healthcare. In particular, disorders that are managed non-pharmacologically benefit from ...

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

작은 동물 모델에서 아데노 관련 바이러스 벡터의 결막하 투여

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...