The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services (DHHS) or of the institutions and companies affiliated with the authors. This work was funded in part through Battelle Memorial Institute's prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272200700016I. M.R.H., K.J., D.P., L.B., and J.W. performed this work as employees of Battelle Memorial Institute. Subcontractors to Battelle Memorial Institute who performed this work are: R.B., an employee of Charles River Laboratories - Insourcing Solutions; L.K. and M.R.L., employees of MEDRelief Staffing Inc.; M.G.L. as an employee of Lovelace Respiratory Research Institute, Inc.; and J.H.K. as an employee of Tunnell Government Services, Inc.

이 프로토콜은 다음과 동물 보호 지침을 준수합니다. 동물 평가 및 실험 동물 관리 국제 인증 협회의 인증을 받았으며 시설에 수용되었다. 모든 실험 절차는 알레르기 및 전염병 국립 연구소, 임상 연구 부문, 동물 관리 및 사용위원회의 승인 및 동물 복지법 규정, 공공 보건 서비스 정책을 준수이었고, 관리 및 사용에 대한 가이드되었다 실험 동물 추천. 1. ( &quot;콜드 사이드&quot;에) CT 스캐너를 준비 <ol><li> X 선관을 컨디셔닝 <ol><li> 어떤 직원이 스캔 방에없고 주제 테이블이 비어 있는지 확인합니다. </li><li> 스캐너 제조업 자의 사양에 따라 튜브 컨디셔닝 절차를 실행. </li></ol></li><li> 에어 교정을 수행 <ol><li> 어떤 물체가 검색 필드에 없는지 확인합니다. </li&gt; <li> 스캐너 제조업 자의 사양에 따라 공기 교정 절차를 수행한다. 많은 플랫폼에서이 교정은 자동 또는 반자동이다. </li></ol></li><li> ( &quot;핫 사이드&quot;에) 품질 관리 검사를 수행 <ol><li> 추가 단계를 추가하여 (참고 5에서 자세히 설명)을 ABSL-4 정장 실험실 항목의 절차를 완료합니다. 절차는 방사성 물질에 대한 노출을 포함하는 경우, 보호 복하에 수술에 선량계 클립. 방사선 노출의 계산을 위해 매 2 개월 선량계에 돌립니다. </li><li> 스캔 테이블에 적절한 홀더, 공기, 물, 테플론으로 만들어진 된 CT의 머리와 몸 팬텀을 놓습니다. </li></ol></li><li> ( &quot;콜드 사이드&quot;에) 품질 관리 검사를 수행 <ol><li> 추위 측면에서, 보어 (6)의 중앙에 팬텀을 진행합니다. </li><li> ENS에 실험실 표준화 품질 관리 (QC) 파라미터를 이용하여 스캔을 취득URE 스캐너 제조업체의 사양에 있습니다. </li><li> 제조 업체의 프로토콜에 따라 각각의 재료 종류 (공기, 물 또는 테프론) 위에 중심이자 지역 내에서 신호 강도의 평균과 표준 편차를 계산하는 이미지의 QC 컴퓨터 분석을 수행합니다. </li><li> 중부 표준시 스캐너를 사용할 준비가되었음을 CM 직원에게 통지합니다. </li><li> 형상 관리 직원과 동물 정보 (식별 번호, 중량, 생년월일)를 확인합니다. </li></ol></li><li> 환자 데이터베이스에 주제 정보를 등록합니다. <ol><li> (이하 &quot;콜드 측&quot;에서) CT 스캐너 콘솔에서 스캐너의 방사선 정보 시스템 (RIS)를 액세스 할 수 있습니다. 를 RIS는 스캔 일정의 세부 사항으로 채워되어 있는지 확인합니다. </li><li> 현재의 제목과 같은 식별 번호, 나이, 생년월일, 및 시험 날짜와 인구 통계를 선택합니다; 다음, 시험 유형이 자동으로 채울한다. </li><li> 수동으로 대상 무게를 입력합니다. </li><li> 올바른셔서 선택(예를 들어, 첫번째 머리, 첫 발, 경향, 부정사) CT 갠트리에 bject 방향입니다. </li></ol></li></ol> 2. ABSL-4 소송 실험실에서 작업 영역을 준비 <ol><li> 동물 절차 룸에서 클래스 II 바이오 안전성 캐비닛 (BSC) 또는 하향 통풍 표를 준비합니다. BSC (또는 하향 통풍 표)를 켜고 최소 10 사용하는 분은 이전. <ol><li> 깨끗하고 승인 살균제와 같은 5 % 이중 급 암모늄하여 BSC 또는 하강 기류 테이블의 상부면의 내부 표면 소독 (알킬 N-N-알킬 디메틸 벤질 암모늄 클로라이드, 디메틸 에틸 벤질 암모늄 클로라이드) 소독액. BSC에 또는 하강 기류 테이블의 상부면의 내부 표면 스프레이 및 접촉 시간의 10 분 후 건조 표면을 닦으십시오. 스프레이 잔류 소독제를 제거하기 위해 70 %의 에탄올로 표면을 닦아주십시오. </li><li> 장소 장비 및 마취 유도 상자와 동물 취급 장갑을 포함하여 BSC 또는 하강 기류 표에 필요한 공급. </li></<html> 올&gt; </li><li> 중부 표준시 스캐너 침대를 준비합니다. <ol><li> 설정 동물 유지 장치를 CT 침대에. </li><li> 셋업 생체 징후 심박수, 산소 포화도를 검출하기 위해 모니터링한다. 스캐닝 세션 이상 10 분, 온도 모니터링 경우. </li></ol></li><li> 마취 기계를 준비합니다. <ol><li> 기화기의 이소 플루 란 볼륨을 확인하고 필요한 경우보다 이소 플루 란을 추가합니다. </li><li> , 산소와 마취 호흡 회로를 가압 탈출 가스 검사, 시각적으로 누출 1 마취 기계를 검사하여 누설 테스트를 수행합니다. <ol><li> 누출이 검출되면, 소스를 결정하는 문제를 해결하고, 적절하게 구현 된 보정을 검증하는 후속 누설 테스트를 수행한다. </li></ol></li><li> 폐기물 마취 가스를 캡처 일회용 청소 용기의 무게를. 캐니스터는 캐니스터의 초기 무게 이상 ≥50 g의 경우, 용기 (7)를 교체합니다. </li></ol></li></ol>&quot;(핫 측&quot;제목에) CT 스캐너 룸에서 동물 절차 룸과 마취 유도의 제조에서&gt; 3. 동물 운송 &quot; <ol><li> 클래스 II BSC에 이미징 스위트 룸에 인접한 설치류를 들고 방 중 하나에서 케이지 주택 기니 돼지를 전송합니다. <ol><li> 동물의 ID를 확인합니다. </li><li> 케이지 카드를 제거하고 동물로 유지. </li></ol></li><li> 케이지에 뚜껑을 열고 양압 정장 장갑을 통해 동물 보호 가죽 장갑을 돈. 부드럽게, 기니피그를 선택 마취 유도 상자에 동물을 배치하고 뚜껑 상자를 커버한다. </li><li> 클래스 II BSC에서 동물을 포함하는 마취 유도 상자를 제거하고 수송 카트에 놓습니다. </li><li> 카트를 사용하여 CT 스캐너 방 동물을. </li><li> 중부 표준시 스캐너 룸에서 즉시 유도 상자에 청소 용기와 마취 기계를 연결합니다. 유도 상자에 산소 가스를 켜고 납품에 기화기를 설정마취 8의 초기 유도 어 4 % 이소 플루 란. </li><li> 마취의 적절한 깊이 마취 유도시 동물을 모니터링 (예를 들어, 외부 자극, 근육 톤, 안정된 호흡과 심장 박동에 응답하지) 8. </li><li> 기니 돼지가 완전히 마취시 유도 상자에 마취를 끕니다. </li></ol> 4. ( &quot;핫 측&quot;에서) CT 스캐너의 이미징 침대에 주제를 정렬 <ol><li> 인덕션 박스에서 마취 기니 돼지를 제거하고 이미징 침대에 놓습니다. <ol><li> 경향이 위치에 유지 쿠션에 기니 돼지를 놓습니다. </li><li> 건조에서 각막 상피를 보호하기 위해 두 눈 안과 연고를 적용합니다. </li></ol></li><li> 유지 보수 아이소 플루 란 마취를 관리 할 수 ​​있습니다. <ol><li> 기니 돼지 코 콘을 놓고 코 콘 8 일 L / 산소 분을 제공하기 위해 가스를 켜십시오. </li><li> 2 ~ 3 %에 전달하기 위해 기화기를 설정노즈 콘 통해 기니피그에 이소 플루 란. </li><li> 피사체가 마취의 원하는 평면 (아무 운동, ~ 분당 60 숨을)에 도달하면, 마취의 유지 보수를 위해 이소 플루 란의 1.0-1.5 %를 제공하기 위해 설정 기화기을 줄일 수 있습니다. </li><li> 체온, 심박수 및 호흡 수를 포함한 생체 신호를 모니터링한다. 호흡은 각각 9, 이소 플루 란의 비율을 가속 또는 감속, 증가 또는 감소하기 시작합니다. </li></ol></li><li> C가, 필요한 경우, 데이터 수집 및 마취 깊이의 예상되는 길이에 따라이 8,9 유지 ° 37 내지 39 기니피그 체온을 유지하기 위해 별도의 열원을 제공한다. </li><li> 유지 쿠션의 맨 위에 플라스틱 덮개를 고정합니다. </li><li> 수용 튜브에 영상 침대를 진행합니다. </li></ol> 5.보기의 이미지 필드 설정 <ol><li> 중부 표준시 (SC)의 &quot;콜드 측&quot;에 레이저 시스템 (이미징 직원) 활성화anner 객실은 CT 스캔의 기니피그를 배치합니다. 레이저 십자선에서 관심의 해부학을 커버하고 &quot;아웃&quot;버튼 &quot;에서&quot;테이블을 사용합니다. </li><li> 시야를 설정하기 위해, 관심의 해부학 위에 시작점을 설정하는 레이저 버튼을 사용한다. </li><li> &quot;뜨거운 쪽&quot;에 연락 CM 직원은 자신과 CT 스캐너 사이의 리드 방패를 배치합니다. </li></ol> 6. 획득 이미지 <ol><li> 제조 업체의 프로토콜에 따라 CT 연구 이미지 슬라이스 배치를위한 설문 조사 스캔을 획득. </li><li> 설문 조사 이미지에 CT 연구 이미지 조각의 위치를 ​​규정. </li><li> 대조적 주입 사용될 경우 &quot;고온 측&quot;직원과 업무. </li><li> 제조 업체의 프로토콜에 따라 연구 스캔을 획득. </li><li> 제조사의 프로토콜에 따라 스캐너 콘솔 CT 영상을 재구성. </li><li> 사진 보관 및 통신 시스템에 이미지를 전송합니다. 더 질적 ...로서를 수행ntitative 제조 업체의 프로토콜에 따라이 보관 시스템에서 분석합니다. </li></ol> 7. 스캔 후 복구 <ol><li> 음식과 간식 깨끗한 microisolator 케이지에 스캐너 침대에서 기니피그를 전송합니다. </li><li> 카트를 사용하여, 다시 주택 지역에 동물 새장을 운반 할 것. </li><li> 완전히 마취에서 회복 될 때까지 동물을 모니터링합니다. </li><li> 완전히 회수되면, 고효율 입자상 공기 (HEPA)에 케이지 복귀 통풍 선반 -filtered. </li></ol> 스캐너 베이 및 장비 8. 소독 <ol><li> 스캐너 Bay 지역 소독 (예를 들어, 동물, 스캐너 베드 스캐너 실내 바닥, 도어 핸들과 직접 접촉했다 표면)에서 10 분의 접촉 시간 동안 5 %의 이중 급 암모늄 용액. </li><li> 린스 이중 급 암모늄 용액에 10 분 노출 후 70 % 에탄올 용액으로 표면. </li><li> 직접 분사 할 수없는 항목을 닦아에탄올 포화 된 천을이어서 이중 급 암모늄 포화 천 이중 급 암모늄 용액 (민감한 전자 장비)과. </li></ol>

<ol>
	<li>Jahrling, P. B., 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=The+NIAID+Integrated+Research+Facility+at+Frederick,+Maryland:+a+unique+international+resource+to+facilitate+medical+countermeasure+development+for+BSL-4+pathogens.">The NIAID Integrated Research Facility at Frederick, Maryland: a unique international resource to facilitate medical countermeasure development for BSL-4 pathogens.</a> Pathog Dis. , (2014).</li><li>de Kok-Mercado, F., Kutlak, F. M., Jahrling, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NIAID+Integrated+Research+Facility+at+Fort+Detrick.">The NIAID Integrated Research Facility at Fort Detrick.</a> Appl Biosafety. 16, 58-66 (2011).</li><li>Keith, L., Moyer, B. R., Cheruvu, N. P. S., Hu, 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=.">.</a> Preclinical imaging in BSL-3 and BSL-4 envrionments: imaging pathophysiology of highly pathogenic infectious diseases. , (2014).</li><li>Chosewood, L. C., Wilson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in microbiological and biomedical laboratories. , (2009).</li><li>Janosko, K., 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=Safety+precautions+and+operating+procedures+in+an+(A)BSL-4+laboratory:+1.+Laboratory+suite+entry+and+exit+procedures.">Safety precautions and operating procedures in an (A)BSL-4 laboratory: 1. Laboratory suite entry and exit procedures.</a> J Vis Exp. , (2016).</li><li>Diagnostic Radiology Committee Task Force on CT Scanner Phantoms. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Phantoms for evaluation and quality assurance of CT scanners. , (1977).</li><li>Collins, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACUP+712.01+Waste+anesthetic+gas+scavenging+systems.">ACUP 712.01 Waste anesthetic gas scavenging systems.</a> Institutional Animal Care and Use Committee. , (2013).</li><li>Gargiulo, 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=Mice+anesthesia,+analgesia,+and+care,+Part+I:+anesthetic+considerations+in+preclinical+research.">Mice anesthesia, analgesia, and care, Part I: anesthetic considerations in preclinical research.</a> ILAR J. 53, E55-E69 (2012).</li><li>Office of the Press Secretary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Executive order 13546. Optimizing the security of Biological Select Agents and Toxins in the United States. The White House. , (2010).</li><li>Escaffre, O., Borisevich, V., Rockx, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Hendra+and+Nipah+virus+infection+in+humans.">Pathogenesis of Hendra and Nipah virus infection in humans.</a> J Infect Dev Ctries. 7, 308-311 (2013).</li><li>Sueblinvong, V., 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=Critical+care+for+multiple+organ+failure+secondary+to+Ebola+virus+disease+in+the+United+States.">Critical care for multiple organ failure secondary to Ebola virus disease in the United States.</a> Crit Care Med. , (2015).</li><li>Mazur, 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=Safety+precautions+and+operating+procedures+in+an+(A)BSL-4+laboratory:+2.+General+practices.">Safety precautions and operating procedures in an (A)BSL-4 laboratory: 2. General practices.</a> J Vis Exp. , (2016).</li><li>Bohannon, J. K., 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=Safety+precautions+and+operating+procedures+in+an+(A)BSL-4+laboratory:+3.+Aerobiology.">Safety precautions and operating procedures in an (A)BSL-4 laboratory: 3. Aerobiology.</a> J Vis Exp. , (2016).</li></ol>

The authors have nothing to disclose.

이 시리즈의 이전 기사 세부 사항, 안전 절차 및 최대 봉쇄 실험실 12, 13에서 안전하게 작업하는 데 필요한 추가 공학적 관리에 대한 광범위한 교육,주의를 강조했다. 안전하게 작업을 수행하면이 실험실에서 가장 높은 우선 순위입니다. 감염된 동물에 물린 또는 상처를 입힐 또는 에어로졸 (7)을 생성하는이 철학 때문에 이러한 가능성 등의 추가 위험에 더욱 살아있는 동물과 함께...

국립 알레르기 전염병 연구소 (NIAID) 프레드릭 MD (IRF-프레드릭)에서 포트 데 트릭에서 통합 연구 시설의 임무는의 정도와 상관 관계 임상 질병 과정을 이해하는 전염병 연구 신흥 수행하는 미생물에 의한 질병. IRF-프레드릭은 ABSL-4 실험 1에서 높은 결과 병원균의 동물 모델에서 의료 영상을 수행 할 수있는 독특한 기능이 있습니다. 연구자가 사용할 수있는 이미징을 포함 : 컴퓨터 단층 촬영 (CT), 자기 공명 영상 (MRI), 양전자 방출 단층 촬영 (PET), 단일 광자 단층 촬영 (SPECT), 초음파, X 선 및 형광 투시. 연구진은 종 연구에서, 이러한 약물 치료 및 예방 접종 등의 개입의 효과를 질병의 진행을 모니터링하고 평가하는 데 사용할 수 이미징 기능을 사용합니다. IRF-프레드릭의 영상 방식은 구체적이었다높은 억제 2,3- 외부 장비의 핵심 구성 요소를 유지하도록 설계 및 유지 보수를위한 접근. 이 디자인은 &quot;핫&quot;(포함 병원체)과에 촬영 슈트 분리 &quot;차가운면을.&quot; 이러한 분리를 달성하기 위해 특별히 설계된 튜브는 각각의 영상 기법 (도 1)의 구멍으로 높은 수납 공간을 확장하도록 구성 하였다. 생물학적 봉쇄를 제공 할뿐만 아니라,이 튜브는 높은 봉쇄 실험실의 오염을 제거하는 데 사용되는 가스 및 화학 물질 촬영 장비를 보호한다. 비교 의학 (CM) 직원 손잡이가 &quot;뜨거운 쪽&quot;에 동물을 모니터링하면서 이미징 과학자와 기술자는 &quot;콜드 측&quot;에서 스캐너를 운영하고 있습니다. 형상 관리 직원이 실험을 조정 이미징 과학자들과 긴밀하게 협력해야하기 때문에, 이러한 분리는 통신 문제가 발생할 수 있습니다. 옵션을 사용할 수 평가 한 후, CM 직원은 밖으로 있었다외부 봉쇄 촬상 직원 전화를 사용하는 휴대폰을 단파장 초고 주파수의 전파를 송신 블루투스 이어 피스 장착. 때문에 설비의 설계, 무선 액세스 포인트는 &quot;핫&quot;사이 시멘트 강 층과 &quot;저온 측&quot;에 의한 신호 간섭을 극복하기 위해 각 객실에 설치되어야했다. 따라서, 높은 봉쇄 외부 소음 양압 정장 및 이미징 직원을 입고 CM 직원 사이의 통신은 이제 신뢰할 수있다. 카메라는 또한 &quot;뜨거운 쪽&quot;의 활동을보고 직원 이미징 이미징 객실의 뜨거운 측면에 설치되어 있습니다. 카메라로, 영상 직원은 동물의 위치 또는 이미징 프로토콜에 대한 마지막 순간의 변화와 CM 기술자를 안내 할 수 있습니다. IRF-프레드릭 ABSL-4 정장 실험실에서 모든 작업 정장 (4)를 캡슐화 긍정적 인 압력을 착용하는 직원을 필요로한다. 이 정장을 입고 이동성을 감소시키고, 무거운 말X 장갑 소송에 부착 플러스 장갑의 최대 세 개의 추가 레이어 손재주를 타협. 결과는 절차를 완료하는 데 시간이 더 걸릴 미세 운동 기술이 필요한 작업이 훨씬 더 어려운 것입니다. 바이오 안전성 레벨이 증가, 동물 취급 및 조작으로 더 도전적이고 시간이 특히 작은 동물로,이 소요된다. ABSL-4 실험실에서 절차는 ABSL-2 실험실보다 2 ~ 3 배의 시간이 걸릴 수 있습니다. 시각적으로 예로서 기니 돼지 CT 스캔 절차를 사용하여 ABSL-4 환경에서 이미징 동물 모델과 관련된 문제를 설명하기 위해이 문서의 목적이다.

<table border="0" cellpadding="0" cellspacing="0" width="916">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micro-Chem Plus</td>
			<td style="width:237px;">National Chemical Laboratories</td>
			<td style="width:139px;">255</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CT scanner</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CT phantom</td>
			<td>Philips Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isovue-300 (CT contrast reagent)</td>
			<td>Bracco Diagnostics</td>
			<td>NDC 0270-1315-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ventilated rack</td>
			<td>Lab Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-isolator cage</td>
			<td>Lab Products</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety cabinet</td>
			<td>Nuaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia machine</td>
			<td>SurgiVet</td>
			<td>WWV9000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia induction box</td>
			<td>VetEquip</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anesthesia mask</td>
			<td>Henry Schein</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waste gas scavenging canister</td>
			<td>Fisher</td>
			<td>F/AIR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Holding cushion</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic ointment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vital signs monitor</td>
			<td>Bionet</td>
			<td>BM3Vet</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mobile phone</td>
			<td>Spectralink</td>
			<td>8440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue Tooth ear piece</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wireless access points</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sperian positive-pressure suit</td>
			<td>Honeywell Safety Products</td>
			<td>BSL 4-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Outer suit gloves (latex, Ansell Canners and Handlers)</td>
			<td>Fisher</td>
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</table>

safety precautions and operating procedures in an (a)bsl-4 laboratory: 4. medical imaging procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

동물 취급에 대한 모든 안전 절차 및 표준 운영 절차에 대한 엄격한 준수는 ABSL-4 실험실에서 안전하게 작업을 위해 필수적이다. 촬상 스위트 동물 절차 방에서 유도 박스 내에서 감염된 동물을 전송하는 공통 통로의 오염의 위험을 최소화한다. 절차는 필요에 따라, 더 실험실 획득 감염이나 동물 과목의 교차 오염은 IRF-프레드릭에 ABSL-4 연구를 수행하는 동안 기록되지 않?...

Watch this Scientific Journal Video about Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures at JoVE.com

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 4. 의료 영상 절차

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

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Research

JoVE Journal

Immunology and Infection

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

10.8K 조회수.  National Institutes of Health (NIH). Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

비디오: Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

조류 종의 소화 기관을 통과 한 후 감염성 프리온을 확인하기위한 절차

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

연구

면역학 및 감염병학

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 : 1. 바이오 안전성 레벨 4 정장 연구소 스위트 출입국 절차

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

이미징 면역 시냅스 역학에 대한 내피 평면 셀 모델

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 2. 일반 사례

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 3. 공중 생물학

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축<em&gt; 녹농균</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

비 침습적<em&gt; In Vivo</em&gt; 활성화 가능한 형광 이미징 에이전트를 사용한 염증성 MMP 활성의 형광 광학 이미징

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

뮤린 소장에서 상피 내 림프구의 활력 영상

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

심질 마우스에 있는 인간 흉막 중피종의 정형외 모형의 PET/CT에 의한 이식 및 모니터링

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 4. 의료 영상 절차

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