우리는 T.를 제공하는 존 켈리 마틴 테일러 (위생 및 열대 의학의 런던 학교) 감사합니다 비. brucei GVR35-VSL-2 및 생체 내 이미징에 대한 조언 박사 안드레아 Zelmer (LSHTM). 이 작품은 빌과 멜린다 게이츠 재단 글로벌 건강 프로그램 (허가 번호 OPPGH5337)에 의해 지원되었다.

윤리학 모든 작업은 영국 홈 오피스 동물의 승인 (과학적인 절차) 법 1986 위생 및 열대 의학 동물 복지와 윤리 심의위원회의 런던 학교에서 실시 하였다. 가이드 라인에 도착하는이 보고서에 따른다. 발광 생물 트리파노소마 (Trypanosoma) brucei brucei의 생체 항로 1. <ol><li> T.의 냉동 보관 재고 (라고 stabilate)를 제거 비. brucei 변형 액체 질소에서 9 (MTA를 통해 위생 및 열대 의학의 런던 학교 교수 존 켈리에서 구입 가능) GVR35-VSL-2 및 실온에 평형 수 있습니다. 멸균 인산 완충 포도당 식염수 (PBS-G)의 pH 8.0 (1 L PBS + 15g 포도당) (10)에 2 × 10 4 트리 파노 솜 / ㎖로 stabilate을 희석. 참고 : T.를 비. brucei GVR35-VSL-2는 인간에게 비 감염성이며 SAPO 실험을 수행 할 시설 허가가 필요합니다. </li><li> INJ요법 25 G 바늘로 복강 내 경로 (IP)를 통해 trypanosomes에 희석 0.2 ml의와 20-25g 여성 CD1 마우스입니다. 이것은 &quot;기증자 마우스&quot;입니다. 특정 병원체 무료 (SPF) -cage 및 사료 광고 임의로에 감염된 마우스를 놓습니다. 참고 : CD1 마우스는 이계 변형 및 T. 있습니다 비. brucei GVR35 감염이 변형에 잘 특징이다. 그러나이 프로토콜은 근친 또는 유전자 변형 계통에 적용 가능하지만, 촬상 -6,9-의 민감도에 영향을 미칠 수 털 색깔 중요하다. </li><li> 혈액 샘플링에 의해 주변 기생충을 모니터링합니다. 플라스틱 제한 수단에 마우스를 놓고 21 G 바늘이나 사혈와 venepuncture에 의해 꼬리에서 혈액의 5 μl를 타고 붉은 혈액 세포를 용균 쉽게 계산 할 수 있도록 0.85 % 멸균 염화 암모늄 1/10 섞는다. </li><li> 노이 바 우어 혈구에서 희석 된 혈액을 계산합니다. <ol><li> 두 챔버로 희석 된 혈액을로드합니다. 전체 중앙 GR 카운트하나의 챔버의 ID는 n 개의 트리 파노 솜을 얻었다. 혈액 = N × 104 트리 파노 솜 / ㎖에서 트리 파노 솜의 농도는 모든 희석 계수를 조정하고 두 챔버들의 평균을 계산한다. 주 : 후 약 5~7일 후 감염, 기생충 주연 2 × 104 트리 파노 솜 / ml의 농도로 감염을 수행하기에 충분히 상승한다. </li></ol></li><li> 트리파노소마 수준은 1.6 단계로 이동 필요한 생쥐의 수만큼 높은 경우. 하나의 공여 마우스 6 × 104 트리파노소마 / ml의 기생충 레벨 10 마우스 악성 충분하다. 참고 : 통계적으로 유의 한 그룹 크기를 들어, N = 6 마우스의 각 치료 그룹 8 필요하다. 감염의 다른 모델의 경우, 파워 계산은 해당 그룹의 크기를 결정하기 위해인가 될 필요가있을 것이다. </li><li> (약 5 ~ 10 분 동안 28 ~ 30 ℃에서) 열 상자에 장소 마우스는 꼬리 정맥이 팽창 할 수 있도록합니다.터미널 마취 유도 20 밀리그램 / 25 G 바늘 kg 펜토 바르 비탈 정맥을 관리합니다. 확인 마우스 부드럽게 움츠림 반사가 없는지 확인하는 발바닥을 눌러 마취. </li><li> 비쥬 튜브에 5000 USP 단위 / ㎖의 헤파린을 5 ㎖ 분취하고 및 1 mL의 주사기에 부착 된 바늘로부터 회 흡인하여 21 G 바늘을 Heparinize. </li><li> 헤파린 21 G 바늘과 1 ML의 주사기를 가지고 가고, 흉곽 아래 중앙에 바늘을 삽입하여 마취 통로 마우스의 심장 천자를 수행 (혈액의 비드 바늘의베이스에 풀 것) 부드럽게 플런저를 철수 그래서 같은 마음의 실을 축소 할 수 없습니다. 혈액의 0.7 ML의 최소를 수집합니다. </li><li> 위의 단계 1.1로 PBS-G의 pH 8.0에서 2 × 10 4 트리 파노 솜 / ㎖의 접종을 생성하기 위해 감염된 혈액을 희석. 주 :이 시점에서 남아있는 혈액을 8 % 글리세롤, 20 % 열 - 불 활성화 된 태아 (CA)와 혼합하여 stabilate 생산​​ 동결 보존 할 수있다LF 혈청 (하이 - FCS)와 72 % 감염된 혈액. 천천히 액체 질소로 전송하기 전에, C / 분 ° -1 냉각 속도를 달성하기 위해 컨테이너를 동결 느린 동결을 사용하여 -80 ℃에서 동결. </li></ol> 실험 쥐의 2. 감염 <ol><li> 부드럽게 따뜻한 정맥을 팽창하기 위해 가열 상자에 CD1에게 여성 마우스 (~ 21-25g)을 놓습니다. 플라스틱 구속으로 가온 마우스를 넣고, 마우스 당 4,000 트리파노소마 동등한 꼬리 정맥 내로 25 G 바늘 정맥 0.2ml의 접종 물을 희석하여 주입. 출혈을 막기 위해 후 주사 부위에 압력을 넣습니다. 복강 내 감염은 감염의 유효한 경로이며, 이전의 연구 (10)에 사용되어왔다, 그러나 우리는 정맥 경로보다이 덜 재현 발견 :합니다. </li><li> SPF-배출 케이지 3 그룹에 감염된 마우스와 케이지를 무작위로. 대조군으로서, 야생형 비 생물 발광 기생충, T.와 CD1 암컷 마우스를 주입 비. brucei GVR35 (N =삼). </li><li> 혈액 영화 현미경을 통해 감염을 모니터링 <ol><li> 스팟 5 μL의 유리 슬라이드 상 venepuncture 또는 사혈에서 혈액 (부드럽게 박막을 생성하기 위해 상기 슬라이드를 통해 혈액의 방울을 밀어 제 유리 슬라이드를 이용) 혈액 도말 표본을 수행한다. 공기 건조에 슬라이드를 허용합니다. </li><li> DH 2 O로 세척하기 전에 10 분 동안 김사 100 % 메탄올 및 얼룩과 혈액 도말 표본을 수정하고 공기 건조를 할 수 있습니다. 100X 목적에서보기 (FOV)의 10 분야에서 트리 파노 솜의 수를 계산합니다. 주 : 혈액 촬영이 일찍 일일 감염 후 같은 전체 동물의 비 침습적 영상과 함께 실시된다. 동물들을 처리 할 때 샘플을 실온에서 저장하고 나중에 계산 될 수 있으므로, 트리파노소마 정량이 방법은 혈구 계수보다 선호된다. </li></ol></li></ol> 3. 생물 발광 이미징 감염을 추적하는 참고 : 감염을 모니터링하려면 전체 ANIM알 비파괴 이미징을 이용할 수있다. <ol><li> 30 ㎎ / ㎖에서 둘 베코 인산염 완충 생리 식염수 (DPBS)에서 D-루시페린, 반딧불 루시 페라 기판의 재고 솔루션을 준비합니다. 필터 소독 및 -20 ° C에서 분량 씩 냉동. 참고 : D-루시페린 민감한 빛 때문에 호일에 분량 씩 포장하여 빛으로부터 보호. 이 활동 11에 영향을 미칠 수있는 반복 루시페린 - 해동 냉동하지 마십시오. </li><li> 실온 D-루시페린의 나누어지는을 따뜻하게. (nonbioluminescent 야생 형 기생충 균주에 감염 제어 마우스 포함) 케이지에서 각 마우스를 제거하고 복강 25 G 바늘과 150 밀리그램 / (DPBS에 희석) 가온 D-루시페린의 kg을 주입. / 분 2.5 L에서 2 % 이소 플루오 란 / O 2 믹스 마취를 유도하기 위해 약 5 분 동안 이소 플루오 란 챔버에 마우스를 놓습니다. 참고 : 그들이 마취 할 때 마우스가 움직이지된다. 여기에 사용되는, 이미 저를 들어, 마우스 3 번에 처리 될 수있다. 마우스는 anesthet 경우이상 30 분 동안화된 안과 인공 눈물 연고 마르지 마우스의 눈을 방지하기 위해 적용될 수있다. </li><li> 여기에 설명 된 이미 저를 들어, 소프트웨어를 열고 제조업체의 지침에 따라 소프트웨어를 사용하여 시스템을 초기화합니다. 카메라 가열 플레이트가 온도에 도달 한 후에는 이미지를 획득. 참고 :이 장비는 보통 하룻밤 배경 교정을 수행하는 시스템을 가능하게 꺼져되지 않습니다. 그것은 다시 시작해야하는 경우, 먼저 교정 검사를 실행해야합니다. </li><li> (3.5 참조) 이미 저에 마취 마우스를 놓습니다. 어떤 밝은 생물 발광 신호를 통해 출혈을 최소화하기 위해 마우스 사이에 큰 검은 분리기를 사용합니다. 노출 시간의 세트를 이용하여, 화상을 마우스; 뷰 E 매체 비닝 1 F / 정지 개방 필터 및 필드 1, 3, 10, 30, 60 및 180 초 (12.5 X 12.5 cm 2). 참고 : 야생형 nonbioluminescent GVR35 기생충에 감염 제어 마우스가, 배경을 제공운드, 생물 발광 값입니다. 이 모델과 함께 루시페린의 반응 속도 실험 (그림 6)에서, 우리는 10 분에서 생물 발광 신호 피크 관리를 게시하고 영상은 약 3 마우스 5 분 걸리는 것으로 나타났습니다. 마취와 쥐를 이미징 할 때 고려 사항으로이 척도를 가져 가라. </li><li> 쥐의 철저한 영상을 보장하기 위해, 복부, 지느러미와 측면보기를 얻기 위해 회전합니다. 순차적 동물 사이의 이미징을위한 방향의 순서로 일관성을 유지한다. </li><li> 촬영 후, 마우스는 SPF-케이지에 반환하기 전에 관찰하에 마취에서 회복 할 수 있습니다. </li><li> 21 일 후 감염 될 때까지 매 7 일마다 마우스 이미징 계속합니다. 전체 동물을 통해 생물 발광 신호의 꾸준한 증가가 있어야합니다. </li><li> D21, 이미지와 혈액 필름 / kg diminazene aceturate 복강 40 mg을 가진 쥐를 상기하고, 선량으로 쥐에서. 이 약은 주변 기생충을 취소되지만 혈액 꼰 로프 코를 교차하지 않습니다N 장벽, 따라서 CNS 감염 나은 시각화를 가능하게한다. 참고 Diminazene aceturate의 초기 단계 동물 트리파노소마 증을 치료하기 위해 사용하는 잘 확립 된 약물이다. </li><li> D28과 D35에서 이미징 및 모니터링을 계속합니다. </li></ol> 4. CNS 감염을 확인 <ol><li> D35에서, 위에서 설명한 바와 같이 이미지 마우스 (3.1-3.4 단계). </li><li> 촬영 후, 추가 분석을 위해 혈액을 수집, 단계 1.6-1.8에 설명 된대로 마우스는 마취에게서 피를 뽑다 마우스를 복구 할 수 있습니다. </li><li> 심장 천자 후, 20 ml의 PBS와 마우스를 관류 (옵션 : 3 밀리그램 / 관류 액에 루시페린 ml의 추가) 기관에서 혈액을 취소하고 시간 간격 뇌 영역에서 삭제했을 수 있습니다 루시페​​린 레이블을 재 도입하는 마음을 통해 단계 4.1. </li><li> 부드럽게 뇌 있도록, 만곡 가위를 이용하여 두개골의 원주 둘레베이스로부터 절단하고, 두개골 상부를 올려 뇌를 제거부드럽게 곡선 집게 함께 해제된다. </li><li> 적절한 루시페린이 절제 뇌에 추가 된 보장하기 위해, 검은 색 플라스틱 피펫 이전 이미지로 기관을 통해 15 ㎎ / ㎖ 루시페린의 50 μL의 한 부분에 절제된 뇌를 놓습니다. 위와 같이 설정을 사용하여 이미지. 이것은 뇌 반구에 감염의 지역화를 시연 할 예정이다. 주 : 앞서 8 바와 같이 절제 뇌 기생충의 하류 정량화 qPCR에 의해 수행 될 수있다. 다른 대안 다운 스트림 응용 프로그램은 고정 다음 뇌 조직에 기생충을 식별하기 위해 조직 학적 또는 면역 조직 화학을 포함 할 수 있습니다. </li></ol> 생물 발광 이미징 5. 정량 주 : 생물 발광 이미징 소프트웨어이자 (ROI)의 영역을 사용하여 정량화 배경 생물 발광 대해 보정 될 수있다. <ol><li> 투자 수익 (ROI) 도구를 사용하면, 전체 동물의 정량 A의 직사각형 ROI를 생성차 마우스의 꼬리의베이스에 코 끝을 충당하기 위해 마우스를 이미지 위에 올려 놓으. 각 동물마다 포인트에 동일한 크기의 창을 사용. 뇌에 대한 ROI를 볼 때, 이미지에 마우스 두부를 위에 위치시키는, 동일한 방법으로 원형 ROI를 사용한다. </li><li> 원하는 경우, 감염의 시각적 표현에 대해 동일한 스펙트럼에 나타나는 이미지를 조정합니다. 참고 : 여기에 사용되는 소프트웨어가 자동으로 최소 및 최대 광자 계수 조정 각 이미지의 색상 맵을 생성한다. 일련의 이미지 사이의 시각적 비교를 사용하려면, 컬러 스케일은 모든 이미지에 대해 동일한 값으로 설정해야합니다. <ol><li> 가장 높은 생물 발광 및 도구 팔레트에서 &#39;이미지 조정&#39;탭을 갖는 이미지를 사용하여, 로그 스케일과 적절한 컬러 규모의 최소 및 최대 (경험적으로 결정되는)을 선택합니다. 실험을 통해 모든 이미지에이 비율을 적용합니다. </li></ol></li></ol>

<ol>
	<li>Brun, R., Blum, J., Chappuis, F., Burri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis.">Human African trypanosomiasis.</a> Lancet. 375 (9709), 148-159 (2010).</li><li>Rodgers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosomiasis+and+the+brain.">Trypanosomiasis and the brain.</a> Parasitology. 137 (14), 1995-2006 (2010).</li><li>Barrett, M. P., Boykin, D. W., Brun, R., Tidwell, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+pharmacological+re-engagement+with+a+neglected+disease.">Human African trypanosomiasis: pharmacological re-engagement with a neglected disease.</a> Br. J. Pharmacol. 152 (8), 1155-1171 (2007).</li><li>Barrett, M. P., Vincent, I. M., Burchmore, R. J. S., Kazibwe, A. J. N., Matovu, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+resistance+in+human+African+trypanosomiasis.">Drug resistance in human African trypanosomiasis.</a> Future Microbiol. 6 (9), 1037-1047 (2011).</li><li>Jennings, F. W., Whitelaw, D. D., Urquhart, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+duration+of+infection+with+Trypanosoma+brucei+in+mice+and+the+efficacy+of+chemotherapy.">The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy.</a> Parasitology. 75 (2), 143-153 (1977).</li><li>Andreu, N., Zelmer, A., Wiles, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+biophotonic+imaging+for+studies+of+infectious+disease.">Noninvasive biophotonic imaging for studies of infectious disease.</a> FEMS Microbiol. Rev. 35 (2), 360-394 (2011).</li><li>Sadikot, R. T., Blackwell, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging.">Bioluminescence imaging.</a> Proc. Am. Thorac. Soc. 2 (6), 537-540 (2005).</li><li>Burrell-Saward, H., Rodgers, J., Bradley, B., Croft, S. L., Ward, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+reproducible+in+vivo+imaging+mouse+model+for+evaluation+of+drugs+against+late-stage+human+African+trypanosomiasis.">A sensitive and reproducible in vivo imaging mouse model for evaluation of drugs against late-stage human African trypanosomiasis.</a> J. Antimicrob. Chemother. 70 (2), 510-517 (2015).</li><li>McLatchie, A. P., 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=Highly+sensitive+in+vivo+imaging+of+Trypanosoma+brucei+expressing+'red-shifted'+luciferase.">Highly sensitive in vivo imaging of Trypanosoma brucei expressing 'red-shifted' luciferase.</a> PLoS Negl. Trop. Dis. 7 (11), e2571 (2013).</li><li>Jennings, F. 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=Human+African+trypanosomiasis:+potential+therapeutic+benefits+of+an+alternative+suramin+and+melarsoprol+regimen.">Human African trypanosomiasis: potential therapeutic benefits of an alternative suramin and melarsoprol regimen.</a> Parasitol. Int. 51 (4), 381-388 (2002).</li><li>Wiles, S., Robertson, B. D., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+monitoring+of+in+vivo+colonization+and+clearance+dynamics+by+light-emitting+bacteria.">Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.</a> Methods Mol. Biol. 574, 137-153 (2009).</li><li>Rodgers, J., Bradley, B., Kennedy, P. G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+chemotherapy+with+a+substance+P+receptor+antagonist+(aprepitant)+and+melarsoprol+in+a+mouse+model+of+human+African+trypanosomiasis.">Combination chemotherapy with a substance P receptor antagonist (aprepitant) and melarsoprol in a mouse model of human African trypanosomiasis.</a> Parasitol. Int. 56 (4), 321-324 (2007).</li><li>Kamerkar, S., Davis, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+on+the+brain:+understanding+host-pathogen+interactions+in+chronic+CNS+infection.">Toxoplasma on the brain: understanding host-pathogen interactions in chronic CNS infection.</a> J. Parasitol. Res. 2012, 589295 (2012).</li><li>Franke-Fayard, 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=Murine+malaria+parasite+sequestration:+CD36+is+the+major+receptor,+but+cerebral+pathology+is+unlinked+to+sequestration.">Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration.</a> Proc. Natl. Acad. Sci. U.S.A. 102 (32), 11468-11473 (2005).</li><li>Hitziger, N., Dellacasa, I., Albiger, B., Barragan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+of+Toxoplasma+gondii+to+immunoprivileged+organs+and+role+of+Toll/interleukin-1+receptor+signalling+for+host+resistance+assessed+by+in+vivo+bioluminescence+imaging.">Dissemination of Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-1 receptor signalling for host resistance assessed by in vivo bioluminescence imaging.</a> Cell. Microbiol. 7 (6), 837-848 (2005).</li><li>Lewis, M. 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=Bioluminescence+imaging+of+chronic+Trypanosoma+cruzi+infections+reveals+tissue-specific+parasite+dynamics+and+heart+disease+in+the+absence+of+locally+persistent+infection.">Bioluminescence imaging of chronic Trypanosoma cruzi infections reveals tissue-specific parasite dynamics and heart disease in the absence of locally persistent infection.</a> Cell. Microbiol. 16 (9), 1285-1300 (2014).</li></ol>

The authors have nothing to disclose.

생물 발광의 T. 개발 비. brucei GVR35 변형 후반 단계에 이른에서 트리파노소마 감염의 시각화 할 수 있습니다. 이전 감염 모델 기생충은 뇌에있는 경우 혈액 막 현미경에서 실시간으로, 말기를 검출 할 수 없었다 및 기생 부하 (12)를 결정하기 위해 감염된 마우스에서 뇌의 컬링 및 제거가 필요했다. 하나의 마우스 감염의 전체 및 모든 단계에서 관찰 할 수 감염의 빠른 질적 평가...

Human African trypanosomiasis (HAT), or sleeping sickness, is caused by the vector-borne protozoan parasites of the Trypanosoma brucei spp1. Estimated numbers of current cases is fewer than 7 thousand every year with almost 70 million people exposed to the risk of the parasite infection within the African continent. The disease, which is most often lethal if left untreated, comprises an early hemolymphatic stage where parasites are present in the blood, progressing to the late stage when parasites invade the central nervous system (CNS) and are no longer susceptible to treatment by early stage trypanosomal drugs2. The current drug therapies for late-stage HAT have both complex, prolonged, treatment regimens and severe adverse effects as well as reported resistance, therefore research into new drug therapies is imperative3,4. 
The study of late-stage human African trypanosomiasis (HAT) within traditional mouse models is lengthy and complex, with the removal of brain tissue being required to monitor parasitic burden5. The animal infective strain T. b. brucei is used as the study model of trypanosomiasis with the late stage appearing 21 days post infection (dpi). To monitor the wild type nonbioluminescent parasite infection in the mouse model, peripheral blood films or quantitative PCR are the only methods to determine parasite burden. For parasite burden in the brain, the mouse needs to be culled, brain excised and qPCR carried out on tissues, making it impossible to track parasites through multiple time points in the late stage infection. This results in the inability to follow real-time infections within the central nervous system (CNS). 
In vivo bioluminescence imaging (BLI) can provide highly sensitive, non-invasive detection of parasite dissemination and disease progression in a mouse model that can be followed in a single animal for the entirety of the experiment6. BLI is based on the emission of light in the visible spectrum produced by a luciferase-catalyzed reaction. The emitted photons are then detected by a charge coupled device (CCD) camera7. For this purpose, the pathogen is genetically modified to express a luciferase protein and the substrate, luciferin, is introduced at time points of interest by injection. The main advantage of this method is the ability to carry out longitudinal studies, in which the same animal can be imaged several times with minimal adverse effects. The acquired bioluminescence signal can be quantified, thus indicating the pathogen burden.
The optimization and validation of a red-shifted bioluminescent T. b. brucei has enabled the investigation of the late stage infection through non-invasive procedures, detecting parasites earlier than blood film microscopy and greatly reducing the time, cost and numbers of animals needed to study CNS infection and drug screening in late-stage trypanosomiasis8,9. In this protocol we demonstrate infection of mice with bioluminescent trypanosomes and how to then visualize the parasites in vivo for quantification of disease progression and CNS penetration.

<table><tbody><tr><td>PBS</td><td>Sigma, UK</td><td>P4417</td><td>tablets pH 7.4</td></tr><tr><td>Glucose</td><td>Sigma, UK</td><td>G8270</td><td>99.5% (molecular) grade</td></tr><tr><td>Ammonium chloride</td><td>Sigma, UK</td><td>A9434</td><td>99.5% (molecular) grade</td></tr><tr><td>Heparin (lithium salt)</td><td>Sigma, UK</td><td>H0878</td><td></td></tr><tr><td>Hi-FCS</td><td>Gibco, Life Technologies, UK</td><td>10500-064</td><td>500 ml</td></tr><tr><td>DPBS</td><td>Sigma, UK</td><td>D4031</td><td>Sterile filtered</td></tr><tr><td>Mr. Frosty</td><td>Nalgene, UK</td><td></td><td></td></tr><tr><td>Giemsa</td><td>Sigma, UK</td><td>G5637</td><td></td></tr><tr><td>D-Luciferin</td><td>Perkin Elmer, UK</td><td></td><td></td></tr><tr><td>Sigma, UK</td><td>115144-35-9</td><td></td><td></td></tr><tr><td>Diminazene aceturate</td><td>Sigma, UK</td><td>D7770</td><td>Analytical grade</td></tr><tr><td>IVIS Lumina II</td><td>Perkin Elmer, UK</td><td></td><td>other bioimagers available &lt;em&gt;e.g.&lt;/em&gt; from Bruker, Kodak</td></tr><tr><td>Living Image v. 4.2</td><td>Perkin Elmer, UK</td><td></td><td>proprietary software for Perkin Elmer IVIS instruments; other instruments may have their own</td></tr><tr><td>1 ml Syringe</td><td>Fisher Scientific, UK</td><td>10142104</td><td></td></tr><tr><td>20 ml Syringe</td><td>Fisher Scientific, UK</td><td>10743785</td><td></td></tr><tr><td>25 G Needles</td><td>Greiner Bio-one</td><td>N2516</td><td></td></tr><tr><td>21 G Needles</td><td>Greiner Bio-one</td><td>N2138</td><td></td></tr><tr><td>Twin-frosted microscope slide</td><td>VWR, UK</td><td>631-0117</td><td></td></tr><tr><td>1.5 ml Microcentrifuge tube</td><td>StarLab, UK</td><td>I1415-1000</td><td></td></tr><tr><td>7 ml Bijou tube</td><td>StarLab, UK</td><td>E1412-0710</td><td></td></tr><tr><td>Mouse restrainer</td><td>Sigma, UK</td><td>Z756903</td><td>our restrainer was made in-house, this is a similar model</td></tr></tbody></table>

bioluminescence imaging to detect late stage infection of african trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

이 프로토콜은 T.와 마우스의 감염 다음 질병의 진행을 수행하는 방법을 보여줍니다 비. brucei는 인간의 아프리카 트리파노소마 증의 모델이. 그림 1 치료 및 이미징 단계의 시간표를 보여주는 실험 프로토콜의 타임 라인을 보여줍니다.이 주변 기생충을 정량하는 데 사용되는 고정 김사 묻은 혈액 도말 검사에서보기의 전형적인 필드?...

Watch this Scientific Journal Video about Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis at JoVE.com

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

생물 발광 영상은 아프리카 트리파노소마 증의 늦은 단계의 감염을 감지하는

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

bioluminescence-imaging-to-detect-late-stage-infection-african

Research

JoVE Journal

Immunology and Infection

7.7K 조회수.  London School of Hygiene & Tropical Diseases. This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

비디오: Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

라이브 호스트에 트렌스 제닉 Leishmania의 기생충의 생체내 이미징에서

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

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Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

HIV / AIDS의 종합 및 비용 효과적인 실험실 감시 : 아프리카 역할 모델

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

다른 동물 모델에서 NADPH 산화 효소 활동의 Bioluminescence 영상

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

신경 침윤성 Encephalitic 바이러스의 생체 이미징 시스템 (IVIS) 검색에서

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

높은 처리량 유전자 태그에<em&gt; 트리파노소마 (Trypanosoma) brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

트리파노소마증 검출 및 분류를 위한 AI 프로그램

Superior Auto-Identification of Trypanosome Parasites by Using a Hybrid Deep-Learning Model

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of hotspot areas under active surveillance is a fundamental procedure for controlling disease transmission. Microscopic examination is a commonly used diagnostic method. It is, nevertheless, primarily reliant on skilled and experienced personnel. To address this issue, an artificial intelligence (AI) program was introduced that makes use of a hybrid deep learning technique of object identification and object classification neural network backbones on the in-house low-code AI platform (CiRA CORE). The program can identify and classify the protozoan trypanosome species, namely Trypanosoma cruzi, T. brucei, and T. evansi, from oil-immersion microscopic images. The AI program utilizes pattern recognition to observe and analyze multiple protozoa within a single blood sample and highlights the nucleus and kinetoplast of each parasite as specific characteristic features using an attention map.
To assess the AI program&#39;s performance, two unique modules are created that provide a variety of statistical measures such as accuracy, recall, specificity, precision, F1 score, misclassification rate, receiver operating characteristics (ROC) curves, and precision versus recall (PR) curves. The assessment findings show that the AI algorithm is effective at identifying and categorizing parasites. By delivering a speedy, automated, and accurate screening tool, this technology has the potential to transform disease surveillance and control. It could also assist local officials in making more informed decisions on disease transmission-blocking strategies.

저자 스포트라이트: 현미경 이미지에서 AI 기반 트리파노솜 종 검출 

Worldwide medical blood parasites were automatically screened using simple steps on a low-code AI platform. The prospective diagnosis of blood films was improved by using an object detection and classification method in a hybrid deep learning model. The collaboration of active monitoring and well-trained models helps to identify hotspots of trypanosome transmission.

하이브리드 딥러닝 모델을 사용한 트리파노솜 기생충의 우수한 자동 식별

Trypanosomiasis is a significant public health problem in several regions across the world, including South Asia and Southeast Asia. The identification of ...

생체공학

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

종양 진행 및 혈관 신생의 이중 생물 발광 이미징

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

생물 발광 영상은 아프리카 트리파노소마 증의 늦은 단계의 감염을 감지하는

요약

더 많은 비디오 탐색

이 비디오의 챕터

라이브 호스트에 트렌스 제닉 Leishmania의 기생충의 생체내 이미징에서

HIV / AIDS의 종합 및 비용 효과적인 실험실 감시 : 아프리카 역할 모델

다른 동물 모델에서 NADPH 산화 효소 활동의 Bioluminescence 영상

신경 침윤성 Encephalitic 바이러스의 생체 이미징 시스템 (IVIS) 검색에서

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

높은 처리량 유전자 태그에

하이브리드 딥러닝 모델을 사용한 트리파노솜 기생충의 우수한 자동 식별

하이브리드 딥러닝 모델을 사용한 트리파노솜 기생충의 우수한 자동 식별

하이브리드 딥러닝 모델을 사용한 트리파노솜 기생충의 우수한 자동 식별

종양 진행 및 혈관 신생의 이중 생물 발광 이미징