Name: sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice
Uploaded: 2021-06-28T13:00:00
Description: Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

저자는 상파울루 대학의 의학 클리닉에서 포스트 졸업 프로그램 덕분에; 코르데나카오 데 아페르페이소아멘토 데 페소알 드 니벨 슈페리어(CAPES) 및 상파울루 의과대학(FMUSP).

이 프로토콜은 USP 의과 대학의 동물 사용에 대한 윤리위원회에 의해 승인되었다, Num. 프로젝트: 1343/2019-CEUA: FMUSP. 이 프로토콜을 위해, C57BL/6 마우스는 6주, 20± 2g의 무게가 사용되었다(n = 9/그룹).
1. 라파로토미
<ol><li>자일라진(10 mg/kg)과 케타민 용액(80 mg/kg)으로 동물을 마취시키고, 1mL 주사기와 13x0.45mm 바늘 26G 1/2를 사용하여 피하(0.1 mL/10g의 체중)를 피하한다. 발가락을 꼬집어...

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타우로콜레이트 나트륨 주입에 의한 급성 췌장염을 유도하는 방법은 이미 랫트22,23,24에서 나타났다. 2008년, 2010년, 2015년에 출판된 세 개의 유사한 작품은 프로토콜3,14,15에 대한 참조역할을 했습니다. 이 작업에서는 C57BL/6 마우스에서 이 방법을 재현하기 위...

인간에서, 담석의 존재는 담석의 말단 부분의 방해로 인한 췌장염의 가장 흔한 원인이며, 췌장 분비물의 흐름을 방해하고 췌장에 있는 강렬한 염증 과정을 일으키는 원인이 되며, 혈청 및 염증 성 중재자에서 소화 효소의 농도가 증가한다1,2.
급성 췌장염의 발달을 설명하기 위해 두 가지 다른 이론이 제안되었습니다 (AP). "일반적인 채널" 이론은 담낭에 존재하는 돌이 담즙 분비가 췌장 덕트로 역행하는 것을 허용하는 말단 일반적인 담관 시스템을 방해한다는 것을 건의합니다. 두 번째 이론 ("덕트 방해" 이론)은 과잉 담석에 의한 췌장 덕트의 방해가 십이지장 분비의 흐름에 막힘을 일으켜 덕덕 고혈압을 유발한다는 것을 시사합니다. 급성 담즙 췌장염으로 이끌어 내는 기계장치는 완전히 이해되지 않더라도, 결과는 강렬한 선동적인 프로세스입니다. 소화효소 분화와 췌장자가 소화는 조직병리학적 변화, 염증성 사이토카인(IL-1β, IL-6, TNF-α)의 증가, 급성상 단백질4,5,6의 증가로 이어집니다.
가혹한 심각한 심각한 췌장염은 다중 기관의 관여 및 높은 사망 리스크 때문에 임상주의를 받을 자격이 있는 상태입니다. 급성 췌장염의 번식을 위한 동물 모델은 질병의 병리학적 메커니즘을 설명하고 질병의 초기 단계에서 시작하여 염증 성 사건의 진화를 모니터링하는 데 도움이되기 때문에 중요합니다. 이것은 일반적으로 진료소에서 불가능2,7. 또한, 췌장 조직에 대한 접근은 임상 전 연구에서 쉽게, 동위원소 종과 함께 작업할 가능성과 함께 임상 조건에 연결된 변화의 용해성을 선호8, 바람직하지 않은 변수를 제거하고, 인간 조건에서 관찰 된 결과와 임상 유사성을 미러링9.
쥐와 쥐 종에 있는 급성 췌장염의 유도를 위한 담즙 및 비 담즙 모형은 과학문헌에서 빈번하게 공부되었습니다. 유도의 비 담즙 방법은 cholecystokinin 분비학 또는 그 아날로그 cerulein10의 수막 자극 복용량의 관리를 포함한다; L-아르기닌의 거의 치명적인 복용량의 관리; 또는 ethionine11로 보충 콜린 결핍 다이어트의 관리. 이러한 방법은 재현하기 쉽고 췌장 염증을 초래하지만 이론적으로 AP (즉, 췌장 덕트로 담즙 분비물의 역류)를 트리거하는 메커니즘을 복제하지 않습니다. 담즙 모형을 해결하는 기술은 췌장 덕트에 담즙산의 역행 주입을 기반으로 하고 이 프로토콜을 실행하기 위하여 잘 훈련된 연구원을 요구합니다. 여러 연구는 쥐에서이 방법을 사용 하 여 발표 되었습니다 (이러한 실험 외과 수술을 포함 하기 때문에 분명히 기술적 인 이유로)12,13. 그러나, 마우스에 있는 접근은 염증3,14,15의 연구 결과에 있는 더 흥미로운 결과를 제안할 수 있습니다. 이 연구에서는, 우리는 C57BL/6 마취 마우스에 있는 타우로콜린나트륨의 주입에 의하여 가혹한 급성 췌장염의 재생산을 위해 따라야 할 단계의 체크리스트를 보여줄 것입니다.
항체를 이용한 실험의 필요성과 유전자 및 단백질 발현의 분석을 수반하는 작업의 경우, 마우스의 사용은 이러한 동물에 대한 물질의 무기고가 크고 동종 및 녹아웃 종과 함께 작업할 가능성 때문에 바람직하며, 그 중에서도 연구와 관련이 있는 것으로 나타났다16. 마우스 C57BL/6은 원래 항종양 활성 및 면역학 연구를 위해 개발된 마우스의 근친 변형이다. 이 긴장은 점점 동종인 것에 대 한 연구원에 의해 선호 되 고, 결과의 더 큰 재현성을 허용, 실험에 동물의 작은 수의 사용 및 동일한 그룹 사이의 결과의 적은 가변성을 의미할 수 있습니다17,18.
Perides 외. (2010)14 나트륨 taurocholate 주입에 의해 마우스에서 AP 유도에 대 한 프로토콜을 발표 했다. 여기서 우리는 C57BL/6 마우스에서 더 높은 나트륨 타우로콜린 농도(2.5%)를 사용하여 이 모델을 업데이트하며, 주입의 부피 및 속도(도 1). 심각도의 최대 수준은 마우스에 있는 유도의 12 시간 안에 도달합니다. 혈청 과 복막 구멍에서 IL-6의 농도의 상승은 AP의 진행과 상관된다. 연습을 통해 마취 유도에서 주입 완료까지의 총 예상 시간은 동물 당 25 분입니다. 숙련된 연구원이 이 실험을 수행하는 것이 필수적입니다. 용액이 일반적인 담관에 제대로 주입되도록 타우로콜레이트 나트륨 대신 메틸렌 블루를 사용하여 여러 파일럿 트레이닝 세션을 수행합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm needle</td>
			<td>INTRAG MEDICAL TECH</td>
			<td>90183210</td>
			<td>30G</td>
		</tr>
		<tr>
			<td>&#160;0.54 mm polyethylene tube</td>
			<td>Tygon</td>
			<td>730010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Styrofoam block</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>masking tape for mounting the mouse</td>
			<td>Missner</td>
			<td>1236</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Infusion pump scheduled to 10&#181;L / min.</td>
			<td>Havard aparatus-Peristaltic Pump Series</td>
			<td>MA1 55-7766</td>
			<td>&#160;Model 66 Small Peristaltic</td>
		</tr>
		<tr>
			<td>Scissors and forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic providine iodine</td>
			<td>Pfizer</td>
			<td>12086OR</td>
			<td>antisepsis</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SIGMA</td>
			<td>459836</td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Lord</td>
			<td>bdk9a1ghk6</td>
			<td>For trichotomy</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td>86339- 1G</td>
			<td>CAS NUMBER- 345909-26-4</td>
		</tr>
		<tr>
			<td>microvessel clip</td>
			<td>Medicon Surgical</td>
			<td>56.87.35</td>
			<td>Approximator, opening 4.0 mm, closing pressure 30 -&#160;40 g</td>
		</tr>
		<tr>
			<td>6-0 prolene</td>
			<td>Bioline</td>
			<td>5162</td>
			<td>Suture line</td>
		</tr>
		<tr>
			<td>Ketamin NP (cloridrato de dextrocetamina) 50mg/mL</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xilazine 2%</td>
			<td>Syntec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline solution (0.9% (wt/vol) saline)</td>
			<td>Farmace</td>
			<td>105851</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Blue</td>
			<td>Sigma-Aldrich Chemicals</td>
			<td>M5528</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX&#160;MAP&#160;Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay</td>
			<td>MERCK</td>
			<td>MCYTOMAG-70K</td>
			<td>Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.</td>
		</tr>
		<tr>
			<td>Amylase Assay</td>
			<td>Labtest</td>
			<td>11</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmarres retractor 13-mm 
			width</td>
			<td>ROBOZ</td>
			<td>RS-6672</td>
			<td></td>
		</tr>
	</tbody>
</table>

sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice

타우로콜린성 주입에 의한 담즙 급성 췌장염 유도는 임상 담즙 췌장염의 발병에 대응하는 염증 성 사건의 인간 임상 상태 및 재생의 표현으로 인해 과학 계에 의해 널리 사용되고 있다. 췌장 손상의 심각도는 주입된 담즙산의 농도, 속도 및 부피를 측정하여 평가될 수 있다. 이 연구는 프로토콜 재생에 사용되는 재료 및 방법의 업데이트 된 체크리스트를 제공하고이 급성 췌장염 (AP) 모델의 주요 결과를 보여줍니다. 이전 출판물의 대부분은 쥐에서이 모델을 재현하는 자신을 제한했다. 우리는 연구 결과와 관련있을 수 있는 추가이 (즉, 마우스의 유전자 변형 된 균주와 함께 작동의 가능성과 함께 이 동물을 위한 시약 및 항체의 무기고의 가용성)를 제공하는 마우스에 이 방법을 적용했습니다. 마우스의 급성 췌장염 유도의 경우, C57BL/6 마우스에서 3분 동안 주입 속도 10 μL/min에서 2.5%의 나트륨 타우로콜레이트의 정의된 투여량을 가진 체계적인 프로토콜을 제시하며, 이는 유도의 12시간 이내에 최대 정도의 심각도에 도달하고 그 결과를 강조하는 방법을 검증하는 결과를 강조한다. 연습과 기술로 마취 유도부터 주입 완료까지 총 예상 시간은 동물 당 25 분입니다.

췌장염 중증도는 부재에 해당하는 슈미트의 scale21 에 따라 0-3 사이에서 득점되었으며, 1은 경미한 존재(<25%)에 해당하며, 2는 중등도(25~50%)에 해당하며 3은 강렬한 존재감(> 50%)에 해당한다. 수행된 측정은 혈장 아밀라제 활성, 췌장 부종, 아시나르 세포, 부상/괴사, 췌장 염증(H&E 염색 섹션의 히스토로지 분석에 의한) 및 혈청 및 PerC 유체의 IL-6 ?...

Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

가혹한 급성 췌장염을 위한 동물 모형은 염증성 사건의 진화의 관측을 용이하게 하는 초기 단계에서 병리학적 변화의 연구를 가능하게 합니다. 여기서 우리는 마취 C57BL/6 마우스의 췌장 덕트에 타우로콜린산 나트륨의 역그레이드 주입에 의하여 가혹한 급성 담즙 췌장염의 유도를 위한 프로토콜을 제공합니다.

C57BL/6 마우스에서 타우로콜레이트 나트륨 유도 심한 급성 췌장염

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

이 방법은 급성 담즙 췌장염에서 발생하는 염증 성 사건을 재현하는 데 사용할 수 있습니다. 그것은 그것의 초기 단계에서 췌장 조직에 있는 변경을 공부하는 것이 가능합니다, 이는 일반적으로 진료소에서 가능하지 않습니다. 이 기술의 주요 장점은 훈련 된 연구원을 위해 빠르고 쉽게 재현할 수 있으며 인간의 급성 췌장염과 유사한 결과를 제공한다는 것입니다.이 방법은 췌장 및 인접 한 장기에서 염증 성 중재 세포의 연구를 허용하기 때문에 약물 분자 및 기타 치료 적 개입의 약리적 면역 반응을 연구하는 데 사용할 수 있습니다. 이 프로토콜을 시도할 때 장기를 찾고 캐니언 영역을 설정하는 것이 중요합니다. 장기의 위치를 암기하고 메틸렌 블루와 같은 색의 용액을 사용하여 먼저 주입 응용 프로그램을 행사합니다.발가락 핀치로 마취의 깊이를 확인한 후 마취마우스의 복부 부위를 5%의 포비도네 요오드 용액으로 청소하십시오. 트리머를 사용하여 가슴과 하복부 사이의 머리카락을 제거하고 약 2 평방 센티미터의 수술 부위를 70 % 알코올로 청소하십시오. 수술 판에 동물을 고정하고 복부의 상부에 수평으로 피부의 5 밀리미터를 절단하고 xiphoid 과정 아래 1 센티미터를 절단 가위를 사용합니다.복막에 컷을 반복하여 캐비티를 최소화하여 복강경 절제술을 수행합니다. 리트랙터를 사용하여, 창자에서 약 1 센티미터에서 머리쪽으로 간을 당기고 췌장과 십이지장 의 지역을 찾습니다. 집게를 사용하여 간을 동물의 머리쪽으로 들어 올립니다.소장의 부분을 부드럽게 당기면, 일반적인 담관의 탈반 부분을 더 잘 볼 수 있는 6-0 폴리프로필렌 봉합사로 두 측면 끝을 고정하십시오. 마이크로 용기 클립을 사용하여 근위 공통 담관을 일시적으로 폐색합니다. 복강에서 장기를 노출시다.그런 다음 8-0으로 탈모 한 일반적인 담관의 임시 폐색을 합니다. 봉합 사. 일반적인 담관에 접근하려면 소장 벽의 희끄무레한 부분으로 나타나는 복리어 지역을 뚫습니다. 0.54mm 폴리에틸렌 튜브에 0.4 밀리미터 바늘을 연결한 후, 3분간 10그램당 10마이크로리터의 일정한 속도로 2.5%의 타우로콜린산나트륨 용액 주입을 위한 주입 펌프를 시작합니다.주입 후, 마이크로 용기 클립을 제거, 임시 8-0 봉합사, 췌장 담관에서 주사 바늘. 6-0 비흡수성 모노필라멘트 폴리프로필렌 봉합사로 복부를 봉합합니다. 복강경과 최종 봉합사 사이의 시간은 30 분 이상이어야합니다.수술 후, 나무 면도와 물과 음식 광고 리비툼 줄 지어 폴리에틸렌 상자에 동물을 집. 피부를 뒤로 당긴 동물을 부드럽게 잡고 안구의 약간의 돌출을 촉진하고, 눈이 위쪽으로 향하여 동물을 배치합니다. 동물의 눈에 국소 마취제를 함유한 눈 연고 한 방울을 주입합니다.모세관 튜브의 끝을 눈의 내측 모서리에 놓고 약 30~45도 각도로 안구 아래에 부드럽게 삽입합니다. 혈류가 시작될 때까지 모세관 튜브를 회전시십시오. 컬렉션이 끝나면 눈꺼풀을 가벼운 압축으로 닫아 두어 항상성을 보장하십시오.나중에, 27 게이지 바늘을 사용하여, 마우스의 복막 구멍에 얼음 차가운 PBS의 4 밀리리터를 주입. 주사 후 복막을 10초 동안 부드럽게 마사지하여 부착된 세포를 제거합니다. 가위와 핀셋을 사용하여 내피부와 근육에 0.5cm의 작은 컷을 만들어 복강을 노출시한다.회막에 전구 파이펫을 삽입하고 지방 조직을 흡인하지 않도록주의하면서 가능한 한 많은 액체를 수집합니다. 추가 처리를 위해 헤드 리전에서 췌장의 일부를 수집합니다. 수집된 유체를 얼음 위에 보관하는 튜브에 보관하십시오.그런 다음 복막 유체를 5분 동안 250회 G로 원심분리하고 인터류빈-6 투약용 상퍼를 비축합니다. 혈청을 얻으려면 수집된 혈액 샘플을 15분 동안 700배 G로 원심분리하고 아밀라제 및 인터류신-6 투약용 상퍼를 비축한다. 급성 췌장염을 유도한 후 12시간 동안 췌장 조직의 분석은 가짜 그룹에서 조직 샘플과 비교했을 때 영향을받는 장기에서 강렬한 염증을 보였다.유사한 결과는 샴과 APTA에서 조직 견본의 혈소실린 eosin 염색 후에 조직학적 검사에 의해 표현되었습니다. 혈청의 분석은 샴그룹에 비해 AP 군에서 혈청 아밀라제 및 인터류킨-6 시토카인 농도의 현저한 증가를 제시했다. 동시에, 인터류신-6 농도는 AP 군에서 복막 구멍 유체에서 상당히 높았다.이 모델에서 췌장염의 중증도는 주입의 농도, 부피 및 압력 및 급성 췌장염 유도 시에 비례적으로 의존합니다. 이 기술의 도움으로, 심한 급성 췌장염에 지속적인 간원 암비의 효과 연구 할 수있다.

sodium-taurocholate-induced-severe-acute-pancreatitis-in-c57bl6-mice

Research

JoVE Journal

Medicine

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

만료 CO 2</sub응급실에서 Intubated 또는 자연스럽게 호흡 환자> 측정

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

연구

의학

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

마우스의 급성 폐 상해를 유발하는 압력 제어 환기

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

마우스의 급성 신장 손상 및 사후 상해 섬유증의 허혈 - 재관류 모델

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

세로 두 광자 이미징 다음 마우스의 급성 뇌 외상

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6 마​​우스의 전신 홍 반성 루푸스의 BM12 유도 성 모델 (SLE)

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Murine 원심 인공, C57BL/6 마우스에 전환 Colitis의 새로운 모델

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

쥐의 급성 심근 경색의 실시간 압력-볼륨 분석

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

직접 경포당증 주입에 의해 마우스에 있는 심각한 폐 상해를 유도하는

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

성인 얼룩말 피시에 있는 Cisplatin에 의해 유도된 급성 신장 상해 모형

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...