Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

저자는 실험 중에 비디오 데이터를 수집하는 데 도움을 준 Liang Zhang에게 감사를 표합니다. 이 연구는 중국 국립 자연 과학 재단 (보조금 번호 : 82070479)과 중앙 대학 기초 연구 기금 (보조금 번호 : 3332022128)의 지원을 받았습니다.

이 프로토콜은 기관 검토를 거쳐 중국 의학원 후와이 병원 기관 동물 관리 및 사용 위원회(FW-2021-0005)의 승인을 받았습니다. 모든 실험 절차는 국립 보건원에서 발행 한 실험실 동물의 관리 및 사용 가이드에 따라 수행되었습니다.
참고 : 수컷 Sprague-Dawley 쥐 (체중 : 500-600g, 연령 : 12-14 주)는 음식과 물에 자유롭게 접근 할 수있는 표준 실험실 조건에서 보관되었습니다. 래트를 무...

<ol>
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(133), e56986 (2018).</li><li>Ha, J. Y., Kim, J. S., Kim, S. E., Son, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+activation+of+mitophagy+and+autophagy+by+staurosporine+protects+against+dopaminergic+neuronal+cell+death.">Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.</a> Neuroscience Letters. 561, 101-106 (2014).</li><li>Yamamoto, A., Yue, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+its+normal+and+pathogenic+states+in+the+brain.">Autophagy and its normal and pathogenic states in the brain.</a> Annual Review of Neuroscience. 37, 55-78 (2014).</li><li>You, X. M., 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=Rat+cardiopulmonary+bypass+model:+Application+of+a+miniature+extracorporeal+circuit+composed+of+asanguinous+prime.">Rat cardiopulmonary bypass model: Application of a miniature extracorporeal circuit composed of asanguinous prime.</a> Journal of Extra-Corporeal Technology. 37 (1), 60-65 (2005).</li><li>Chen, Q., Lei, Y. Q., Liu, J. F., Wang, Z. C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+chlorogenic+acid+treatment+on+neuroinflammation+after+deep+hypothermic+circulatory+arrest+may+be+mediated+through+CYLD/NF-&#954;B+signaling.">Beneficial effects of chlorogenic acid treatment on neuroinflammation after deep hypothermic circulatory arrest may be mediated through CYLD/NF-&#954;B signaling.</a> Brain Research. 1767, 147572 (2021).</li><li>Li, Y. 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=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, 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=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, R., 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+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, 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=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

캐뉼러는 쥐에서 DHCA를 확립하는 가장 기본적인 절차입니다. 캐뉼레이션 전에 동맥을 2 % 리도카인 0.5mL로 담그면 캐뉼러가 더 쉬워집니다. 캐뉼레이션 후, 외부 경정맥을 통해 500 IU / kg 헤파린으로 헤파린 화가 미세 혈전 형성을 피하기 위해 필요합니다17. 우리는이 용량의 헤파린이 활성화 된 응고 시간 (ACT) >480 초의 목표를 달성 할 수 있음을 반복적으로 발견했습니다. 재...

심부 저체온 순환 정지 (DHCA)는 1953 년부터 심장 수술에 사용되었습니다1. DHCA는 신체의 혈류를 전 세계적으로 차단하기 전에 환자의 심부 온도를 심히 저체온 수준(15-22°C)으로 낮추는 것을포함합니다2. 순환 정지는 상대적으로 무혈 수술장을 제공 할 수 있습니다. 깊은 저체온증은 특히 뇌와 심근에서 신진 대사를 감소 시키며, 이는 허혈에 대한 효과적인 보호 방법입니다3. DHCA는 일반적으로 복잡한 선천성 심장 질환, 대동맥 궁 질환, 심지어 대정맥혈전 4,5가 있는 신장 또는 부신 종양에 대한 수술 중에 적용됩니다. 따라서 DHCA 동물 모델을 확립하는 것은 임상 환경에서 절차의 개선과 합병증 예방에 중요한 참고 자료를 제공합니다.
개6, 토끼7 및 기타 동물로 모델을 설정할 수 있지만 조작성과 저렴한 비용 때문에 쥐를 사용하는 것이 바람직합니다. DHCA 래트 모델은 2006년 Jungwirth et al.8에 의해 처음으로 기술되었다. 순환 정지 기간이 신경 학적 결과에 영향을 미치는 것으로 나타났습니다. 그 이후로 DHCA 쥐 모델이 광범위하게 조사되었습니다. DHCA가 전신 염증 반응 증후군 (SIRS)을 유발할 수 있음이 밝혀졌습니다 9. 후속 연구에서 약리학자들은 SIRS에 의해 유도된 DHCA 관련 신경염증이 레스베라트롤10 및 트립톨라이드11에 의해 감쇠될 수 있음을 발견했습니다. 우리 팀은 또한 DHCA 관련 신경 염증이 감기 유도 성 RNA 결합 단백질12를 억제함으로써 약화 될 수 있음을 발견했습니다. 심혈관계에서 슈퍼옥사이드 디스뮤타제는 DHCA13 동안 허혈/재관류(I/R) 손상에 심장 보호 효과가 있습니다. 이러한 결과는 DHCA 관련 병태생리학적 과정에 대한 이해를 넓히고 DHCA의 결과를 개선하기 위한 새로운 방향을 제시했습니다. 그러나 DHCA 후 내독소혈증, 산화 스트레스 및 자가포식에 관한 결과는 결정적이지 않습니다. DHCA는 심폐 우회술(CPB)14과 동일한 운영 기술을 사용하지만 관리 전략이 다르며 DHCA를 생성하는 단계는 다양한 팀마다 다릅니다 8,9,10,11. 본 연구는 래트에서 DHCA 절차를 확립하는 방법을 제공하는 것을 목적으로 한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
			<td>Micro-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Changzhou Prefluid Technology Co.,Ltd</td>
			<td>BL100</td>
			<td></td>
		</tr>
		<tr>
			<td>SD Rat</td>
			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>SPACECABS</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of deep hypothermic circulatory arrest in rats

심부 저체온 순환 정지 (DHCA)는 복잡한 선천성 심장 질환 및 대동맥 궁 질환의 수술 중에 일상적으로 적용됩니다. 본 연구는 래트에서 DHCA를 확립하는 방법을 제공하는 것을 목표로 한다. 활력 징후에 대한 DHCA 과정의 영향을 평가하기 위해 순환 정지가 없는 정상 온도 심폐 우회(CPB) 쥐 모델을 대조군으로 사용했습니다. 예상대로 DHCA는 체온과 평균 동맥 혈압을 크게 감소시켰습니다. 혈액 가스 분석 결과 DHCA는 젖산 수치를 증가 시켰지만 혈액 pH와 헤모글로빈, 헤마토크릿, Na+, Cl-, K + 및 포도당 농도에는 영향을 미치지 않았습니다. 또한, 상온 CPB 래트와 비교하여 투과 전자 현미경의 결과는 DHCA 래트에서 해마 자가포식소체의 경미한 증가를 보여주었습니다.

대조군으로서, 순환기 정지가 없는 정상 온도 CPB(NtCPB) 래트는 전체 시술 동안 안정적인 평균 동맥 혈압(MAP) 및 체온을 보인 반면, DHCA 래트의 MAP는 심정지 동안 감소하였다(p < 0.01, 도 3A). DHCA 랫트의 온도는 냉각 단계 동안 빠르게 떨어졌고 재가온 단계 동안 점차적으로 회복되었습니다. 쥐를 DHCA 회로에서 떼어냈을 때, DHCA 쥐의 온도는 정상으로 돌아왔다(<strong class="xfig...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

이 프로토콜은 전신 염증 반응 증후군, 허혈 / 재관류 손상, 산화 스트레스, 신경 염증 등을 조사하는 데 적용될 수있는 쥐에서 심부 저체온 순환 정지의 확립을 제시합니다.

쥐의 심부 저체온 순환 정지 확립

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

쥐에서 깊은 저체온 순환 정지의 확립. 심부 저체온 순환 정지, DHCA는 복잡한 선천성 심장병 및 대동맥 궁 질환의 수술 중에 일상적으로 적용됩니다. 본 연구는 래트에서 DHCA를 확립하는 방법을 제공하는 것을 목표로 한다.이 프로토콜은 기관 검토를 받았으며 중국 의학원 Fuwai 병원의 기관 동물 관리 및 사용위원회의 승인을 받았습니다. 준비 작업. CPB 회로에 모프 스포이드, 롤러 펌프, 열교환기, 멤브레인 산소 공급기, 연결 튜브 및 회로를 연결하는 물 탱크로 수정된 저장소가 포함되어 있는지 확인하고 롤러 펌프가 부드럽게 회전하는 상태에서 40밀리리터 프라이밍 용액으로 12밀리리터의 하이드록시 전분과 1밀리리터의 식염수로 회로를 프라이밍합니다.둘째, 마취와 캐뉼러. 튜브를 인공 호흡기 및 측면 매개 변수와 연결하십시오. 전기 난방 담요와 오른쪽을 놓고 테이프로 오른쪽을 향하십시오.건조를 방지하기 위해 눈에 안과 용 연고를 바르십시오. 면도기로 오른쪽 사타구니 부위, 오른쪽 자궁 경부 및 꼬리의 머리카락을 면도하십시오. 그런 다음 알데히드와 알코올로 피부를 소독하십시오.피부와 왼쪽 사타구니 부위를 자르고 근육과 조직 연골을 해부하여 왼쪽 대퇴 정맥과 동맥을 노출시킵니다. 동맥을 조심스럽게 분리하십시오. 22 게이지 정맥 카테터를 왼쪽 대퇴 동맥으로 캐뉼러합니다.결찰 된 동맥과 실크 카테터. 응고를 피하기 위해 캐뉼라를 플러시하기 위해 헤파린이 함유 된 식염수를 사용하십시오. 카테터를 프레스 센서와 연결하여 혈압을 모니터링합니다.꼬리의 피부를 자른 다음 메스를 사용하여 꼬리 동맥의 표면 종을 절단하여 수술 분야의 중간에있는 꼬리 동맥을 노출시킵니다. 22 게이지 정맥 카테터로 꼬리 동맥을 캐뉼러하십시오. 실크로 동맥과 카테터를 결찰하십시오.응고를 피하기 위해 헤파린이 함유 된 식염수를 사용하여 카테터를 세척하십시오. 정맥의 오른쪽 경정맥의 피부를 자른 다음 근육과 조직을 분리하여 정맥을 노출시킵니다. 16 게이지 수제 다연령 정맥 카테터를 정맥의 오른쪽 외부 경정맥에 삽입하고 오른쪽 하대 정맥 또는 우심방에 조심스럽게 넣습니다.오염을 피하기 위해 오른쪽 외부 정맥이 각 캐뉼러 부위를 촉촉한 공으로 덮는 곳에 헤파린 나트륨을 투여하십시오. 셋째, 꼬리 동맥의 카테터와 회로를 연결하고 펌프를 분당 1-2 밀리리터의 낮은 속도로 유지하십시오. 그런 다음 저장소를 오른쪽 외부 경정맥의 카테터와 연결하십시오.저수지에 항상 약 1 센티미터의 혈중 농도가 있는지 확인하십시오. 물 탱크를 켜고 먼저 수온을 섭씨 37도로 설정하십시오. 혈압이 안정되면 분당 80-100 밀리리터까지 부드럽게 펌프를 증가시킵니다.넷, 냉각. 실내 온도를 섭씨 20도 정도로 설정하십시오. 이 일회용 장갑에 얼음 조각을 넣은 다음 오른쪽 머리와 옆에 놓습니다.탱크의 온도는 쌀의 직장 온도에 따라 실시간으로 제안되었습니다. 혈액 가스 분석 결과에 따라 인공 호흡기의 관련 매개 변수를 적절하게 변경하십시오. 다섯, 깊은 저체온증 순환 정지.그런 다음 직장 온도가 섭씨 15도에서 20도까지 떨어집니다. 순환 정지 중에 깊은 저체온증을 유지하기 위해 얼음 팩을 교체하고 장갑을 폐기하십시오. 롤러 펌프를 중지하십시오.저장소가 환경과 접촉하도록 유지하고 외부 경정맥에서 저장소로 혈액을 천천히 배출하십시오. 혈압 개혁에주의하십시오. 혈압과 심장 박동이 없으면 배수를 멈추고 저수지를 닫아 두십시오.인공 호흡기를 끕니다. 여섯, 워밍업 및 재관류. 일회용 장갑을 모두 제거하고 실내 온도를 섭씨 25도까지 올립니다.정맥 배액관 클리핑을 유지하면서 메모리 산소 간 환기를 복원하십시오. 롤러 펌프를 켜서 저장소의 혈액이 천천히 올바른 몸으로 돌아가는지 확인합니다. 저수지의 혈중 농도가 1 센티미터로 유지 된 후 인공 호흡기를 켜십시오.배수관을 사용하여 우심방에서 저수지로 혈액을 천천히 배출하십시오. 가열 램프, 가열 패드 및 물 탱크를 켭니다. 물 탱크의 온도를 섭씨 25도로 설정합니다.그런 다음 적시에 왼쪽 온도로 조정하십시오. 직장 온도가 섭씨 34도까지 돌아온 후 가열 램프를 제거하십시오. 일곱, CPB를 끊습니다.천천히 그리고 점차적으로 펌프 유량의 롤러를 줄이고 유속으로 대정맥 속 속도를 조정하여 분당 1 밀리리터로 줄입니다. 저수지를 환경과 접촉하도록 유지하십시오. 회로에 남아있는 혈액을 분당 1 밀리리터의 유속으로 주입하십시오.메모리 산소 공급과 롤러 펌프를 중지하십시오. 기계 환기와 깊은 마취 후 안락사. 대표 결과.대조군으로서는 상온 CPB, NtCPB, 랫트 등이 있다. 순환기 정지 없이 전체 시술 동안 안정적인 평균 동맥 혈압 MAP 및 체온을 보였다. 반면 DHCA 래트의 MAP는 심정지 동안 감소하였다.DHCA 쥐의 온도는 냉각 단계에서 빠르게 떨어졌고 회전 단계에서 점차적으로 회복되었습니다. DHCA 회로의 이유식은 DHCA 랫트의 온도를 정상으로 되돌렸다. 위의 데이터는 우리의 DHCA 절차가 클리닉에서 환자의 실제 온도 및 MAP 변화를 모방했음을 나타냅니다.DHCA 프로세서의 상승 및 효과는 혈액 가스 분석에 의해 조사되었다. 전혈을 1 차 용액과 접촉 한 후, 헤모글로빈의 농도는 두 그룹 모두에서 데실 리터 당 6 그램보다 높았다. DHCA 표면에서 이유식을, CPB 회로에 남아있는 혈액을 우측으로 재주입하기 때문에 데실리터당 9그램으로 증가하였다.헤마토크릿 인 HCT는 HB와 유사한 경향을 보였다. DHCA 재관류 후 젖산은 빠르게 증가하여 DHCA 그룹에서 더 두드러졌습니다. DHCA 절차 후 pH가 감소했는데, 이는 젖산 축적의 결과일 가능성이 큽니다. 전체 실험 동안 나트륨, 염화물, 칼륨 이온 및 포도당의 농도는 각 10 점에서 큰 차이를 나타내지 않았습니다.이러한 결과는 DHCA가 락테이트산으로의 증가만을 유도하였을 뿐 혈중 pH 및 헤모글로빈, 헤마토크릿, 나트륨 이온, 클로라이드 이온, 칼륨 이온 및 글루코스의 농도에는 영향을 미치지 않았음을 시사한다. DHCA가 신경 자가포식에 미치는 영향을 평가하기 위해 투과 전기 현미경을 사용했으며 놀랍게도 DHCA 쥐의 해마에서 자가포식소체의 수를 발견하고 증가시켰습니다. 위의 데이터는 우리의 DHCA 모델이 클리닉에서 환자의 실제 온도 및 MAP 변화를 모방했음을 나타냅니다.이 모델은 전신 염증 반응 증후군, 신경 염증 등을 조사하는 데 적용될 수 있습니다.

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

쥐에서 Orthotopic 힌드 - 림브 이식

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

연구

면역학 및 감염병학

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

연구실 고양이와 마우스에 외부의 뜻과 Endoparasites의 진단

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

엡스타인 - 바 바이러스 성장 변화 Lymphoblastoid 전지 라인 구축

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

설립<em&gt; 체외</em&gt; 시스템의 세포 내 행동을 연구하는<em&gt; 칸디다 글라 브라</em&gt; 인간 THP-1 식세포에

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

비 침습적 기관 내 삽관 성인 쥐에서 전체 흉선

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

히트의 예방에 의한 흰쥐의 반작용을 스트레스<em&gt; 고초균</em&gt; 스트레인

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

열 등 한 베 나 정 맥의 협 착 증: 깊은 정 맥 혈전 증의 Murine 모델

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

스테이 시스 고주파 초음파에 의해 모니터링 하는 쥐에 의해 유도 된 깊은 정 맥 혈전 증

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

깊은 피부 주입으로는 모델 조직학 분석 Candida albicans 피부 감염의

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

바이러스 성 감염 및 초파리 Melanogaster 상호 작용 호스트-바이러스의 분석의 설립

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...