This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

참고 : 쥐의 사용을 포함하는 모든 실험 절차는 듀크 대학의 동물 관리 및 사용위원회에 의해 승인 된 프로토콜에 따라 수행되었다. 기관 흡인 삽관 정맥 1. 준비 <ol><li> 펜치와 2 인치 14 G의 angiocathether 바늘의 바늘 끝을 잘라. </li><li> 바늘의 루멘 바늘 코 펜치 폐쇄 핀치. </li><li> 절단 집게 오픈 루멘의 가장자리 부까지 조여진 부분을 절단하고, 나머지 30 단부 양쪽 가장자리를 컷 - 니들 에지에 45 ° 각도. </li><li> 바로 슬립 조인트 플라이어의 개방 영역으로 캐 뉼러의 4mm와 약간 위쪽으로 곡선 끝에서 형성 될 때까지 위쪽으로 당겨 - 아래 3 놓습니다. 참고 :이 기관 (그림 1)에 카테터를 연출 촉진 할 것이다. </li><li> 캐 뉼러의 끝 가장자리를 부드럽게 미세 사포를 사용한다. </li><li&gt; angiocatheter 튜브에 무딘 악기를 돌려줍니다. 튜브가 무딘 바늘보다 약간 짧은 있는지 확인하십시오. </li></ol> 2. 사전 외과 적 치료 <ol><li> 조작 영역 위에 10X 배율로 설정 운영 현미경을 배치하여 절차 사이트를 설정. </li><li> 운영 지역에 온난화 패드를 놓고 깨끗한 흡수 패드 커버. </li><li> 수술 부위 근처의 심장 박동과 혈액 산소 모니터를 설정합니다. </li><li> 무게 기반 약물 투여 (예를 들면, 수술 후 진통제, 항생제, 또는 림프구 고갈 항체)에 대한 쥐의 무게를. 참고 : - 450 g의 일반적인 성인 쥐 (350) 사이의 무게. </li><li> 유도 챔버를 사용하여 기화 3 % 이소 플루 란 O-2 (3 L / 분)와, 진정 쥐 활성탄을 함유하는 폐 마취제 가스 소기 배기 시스템. 마취 유도 5 분 기관 내 삽관에 필요한 깊은 마취를 제공 할 수 있습니다. </li><li> 피하 쥐 이전에 삽관 및 수술을 진행하기로 카프로 펜 또는 Meloxicane (4.4 ㎎ / ㎏)을 관리합니다. </li><li> 삽관 전에 마취 쥐의 목과 가슴을 면도 전기 면도기를 사용합니다. </li></ol> 3. 삽관 <ol><li> 삽관 정맥의 끝 부분에 윤활 소량 (예를 들어, KY 젤)을 마찰하여 삽관 설치를 준비합니다. 커넥터 튜브를 통해 이소 플루 란 가스의 흐름을 시작하기 위해 인공 호흡기를 켭니다. </li><li> 삽관 장치에 쥐를 전송하고 그 상부 앞니 (그림 2A)에 의해 금속 막대에 쥐를 일시 중지합니다. 주 : 특정 삽관 장치가 온라인으로 구입하실 수 있습니다. 여기에 표시된 모델은 부탄 토치를 사용하여 2 × 4 인치 허리 부분의 섹션 주위에 성형 아크릴 시트를 사용하여 만든되며, 보강을 클로로포름으로 연결됩니다. 바 (또는 금속 와이어) (S) 상에 배치 된 마스킹 테이프 위에 천공 홀에 의해 부착되고아크릴의 IDE는 분열 또는 갈라짐을 방지합니다. </li><li> 목 (그림 2B)의 복부 표면에서 2cm - 유연한 고휘도 광원 1을 배치하여 쥐의 목을 횡단는-조명. </li><li> 부드럽게 위쪽과 아래쪽 치아의 측면에 혀를 당겨 학생 표준 패턴 집게 한 쌍을 사용합니다. 파지 한 손의 엄지 손가락과 집게 손가락의 혀의 하단부에 대해 포셉 일측의 평평한 내측 표면 사이에 배치하는 동안 혀. 눌러 복부 후두 (그림 2C)의 후두개와 조리개를 노출한다. </li><li> 성대를 시각화하고 angiocatheter의 허브가 앞니에 닿을 때까지기도로 열린 성문을 통해 전방 삽관 정맥의 위로 향하게 한, 무딘 끝을 안내합니다. </li><li> 금속 탐침을 제거하고, 이소 플루 란과 환기를 시작 angiocatheter 개방에 마취 튜브를 연결합니다. 참고 : 일반적인 산소 플로인공 호흡기 w 비율은 3 % 이소 플루 란 3 L / 분이다. <ol><li> 14 mmHg로 - ~ (12)의 압력을 달성 볼륨 조절 환기 모드 60 호흡 / 분의 속도로 인공 호흡기를 설정한다. 3cm의 H 2 O의 기말 양압 (PEEP)을 사용하여 </li></ol></li><li> 기관 내 튜브의 적절한 배치를 보장하기 위해 인공 호흡기와 동기화 양측 흉벽 확장을 관찰합니다. </li><li> 마취 동안 건조를 방지하기 위해 쥐의 눈에 수의사 권장 눈 연고를 적용합니다. </li><li> 마취 동안 건조를 방지하기 위해 쥐의 눈에 수의사 연고를 적용합니다. </li><li> 천 테이프의 스트립 쥐의 머리에 기관 내 튜브를 고정합니다. 기관 내 튜브와 마취 유입 튜브 사이의 확고한 연결을 확인합니다. </li><li> 쥐의 발에 혈액 산소와 심장 박동 모니터를 연결하고 모니터링을 시작합니다. 아무 반응에에 존재하지 확인하여 적절한 마취를 확인전자 핀치. </li></ol> 4. 개흉술과 흉선 <ol><li> 프로 시저의 전체 표준 무균 기술을 따르십시오. <ol><li> 작업 영역과 운영 테이블을 청소하고 70 % 에탄올 용액으로 소독. </li><li> 절차를 수행하는 동안 멸균 수술 장갑을 사용하고, 절차를 수행하는 동안 사용되는 모든 장비와 자료를 압력솥. </li><li> 전체 가슴 부분에 프로 비돈 요오드를 적용하고 건조 할 수 있습니다. 그 후 거즈에 70 % 에탄올로 피부의 표면을 세정. 멸균 운영 필드를 표시하도록 구멍을 절단 투명한 플라스틱 포장으로 쥐를 커버. </li></ol></li><li> 상부 흉부 지역에서 흉골 노치를 확인합니다. 흉골 노치 위 3mm와 무딘 팁 시어 가위를 사용하여 흉골을 따라 말단 중간 선 연장 - 2를 시작하는 피부를 통해 2cm의 중간 선 절개를합니다. </li><li> 다시 무딘 팁 시어 가위를 사용하여 흉골 노치에서 1.5 cm 중간 흉골 절개를 수행합니다. 케이그냥 천천히 흉골 미리 아래에있는 가위의 아래쪽 가장자리를 EEP​​. </li><li> 사전 기관 스트랩 근육 (sternohyoid 및 sternothyroid 근육)을 나타 내기 위해 단지 분리 된 흉골 및 오픈 아래에 작은 셨기 견인기를 삽입합니다. 무딘 Graefe 씨의 집게를 사용하여 이전 기관 스트랩 근육을 분리합니다. 주 :이 시점에서, 기관지를 볼 수 있고, 삽관 튜브는 기관 내부 가시화되어야한다. </li><li> 분리 된 스트랩 근육과 흉골 아래에있는 작은 셨기 트랙터의 접지 단자를 배치합니다. 흉선의 우수한 측면을 노출 트랙터를 엽니 다. </li><li> 낮은 흉선 로브를 흉선 조직의 측면 가장자리를 해제하고 노출 미세 뒤몽 집게를 사용합니다. </li><li> 상대 정맥, 쇄골과 경동맥 혈관과 접촉을 피하도록주의하면서 오픈 절개 부위에 부드럽게 우량 흉선을 당기고 흉선과 폐 사이의 섬세한 흉막 안감의 중단을 최소화 할 수 있습니다. </li><li> t으로그는 혈관 해부 및 공개됩니다 흉선을 분할하는 마이크로 가위를 사용합니다. 필요한 경우 지혈을 위해 혈관에 압력을 유지하기 위해 면봉을 사용합니다. </li><li> 절개로 낮은 흉선 로브를 제공하고 급격히 후방 첨부 파일을 용해. 그대로 흉선을 제거하고 조심스럽게 누락 부분에 대한 제거 흉선을 검사합니다. 참고 : 작은 흉부 림프절은 흉선을 둘러싼 시각 종종 흉선 조직에서 구별하기 어려운 할 수있다. 이들은 흉선 조직과 유사한 외관 이산 라운드 노드되지만 흉선과 연속성되지 않습니다. </li><li> 셨기 견인기를 제거하고 두 5-0 맥슨 중단 봉합과 sternohyoid과 sternothyoid 근육을 닫습니다. </li><li> 양압 환기에서 종격동를 밀봉 봉합에 걸쳐 수술 학년 시아 노 아크릴 레이트 조직 접착제 2 방울을 적용합니다. 참고 :이 기흉과 혈종의 발생을 감소합니다. </li><li> 성을 닫습니다두와 ernum는 절단 바늘에 4-0 실크 봉합사를 중단. 기본 근육층을 피하기 위해 바로 흉골 아래 바늘을 안내하는주의하면서 갈비뼈 사이의 틈을 통해 바늘을 삽입합니다. </li><li> 실행 4-0 나일론 봉합사로 피부 층을 닫습니다. 수술 마취 회복 기간을 단축하는 것이이 시점에서 이소 플루 란을 중지. </li><li> 생리 식염수에 적신 거즈로 절개 부위와 주변의 피부를 청소합니다. </li><li> 절개를 밀봉 절개를 통해 시아 노 아크릴 레이트 접착제 2 방울 - 1 다음 국소 마취에 대한 Bupivicaine (0.25 %)의 몇 방울을 적용합니다. </li><li> 쥐가 독립적 인 호흡 노력의 흔적을 보여줍니다과 사지를 움직이기 시작 때까지 환기를 계속합니다. 쥐를 튜브를 빼내하고 온난화 패드에 배치 새장에 가까운 관찰에서 복구 할 수 있습니다. </li><li> 프레 놀핀를 관리 (0.01-0.05 ㎎ / ㎏) 피하 쥐에 즉시 수술 후 48 시간 포마다 8 ~ 12 시간을 반복세인트 작동 가능 (일반적으로 5 총 용량). 24 및 48 시간에서 수술 후, 카프로 펜 또는 Meloxicane (4.4 ㎎ / ㎏) 피하 (수술 전 투여 량을 포함, 3 곳의 총 투여 량)을 관리 할 수​​ 있습니다. </li><li> 쥐가 새장 주위를 이동 될 때까지 지속적으로 모니터링. 호흡 곤란의 흔적에서, 재 삽관과 가슴의 재 탐사를 수행합니다. <ol><li> 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 방치하지 마십시오. </li><li> 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. </li></ol></li><li> 스킨 층 봉합 일주 수술 후 제거합니다. </li></ol>

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Transplant.</a> Proc. 37 (4), 1947-1948 (2005).</li><li>Groen, H., Klatter, F., Pater, J., Nieuwenhuis, P., Rozing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporary,+but+Essential+Requirement+of+CD8++T+Cells+Early+in+the+Pathogenesis+of+Diabetes+in+BB+Rats+as+Revealed+by+Thymectomy+and+CD8+Depletion.">Temporary, but Essential Requirement of CD8+ T Cells Early in the Pathogenesis of Diabetes in BB Rats as Revealed by Thymectomy and CD8 Depletion.</a> Clin. Dev. Immunol. 10 (2-4), 141-151 (2003).</li></ol>

The authors have nothing to disclose.

전체 흉선에 대한 본 프로토콜은 합병증을 최소화 할 수 있도록 설계 수술 접착제 응용 프로그램과 3 층 절개 폐쇄와 최소 침습 접근 방식을 제공합니다. 흉선의 완전한 제거는 나이브 T 세포의 손실에 의해 및 종격동 아세포의 림프계 조직 IHC 염색으로 증명되었다. 20 % 인해 늑막, 심장과 함께 흉선의 하엽의 친밀한 관계에 기인 대부분의 사망시 합병증에 - 성인 쥐의 흉선의 ...

1960 년대 초 이후, 흉선은 중앙 면역 관용의 개발에서의 중요한 역할을 인정하고있다. 쥐의 흉선은 동종 이식 및 종양 전이의 설정에서 림프구의 분화, 자기 관용, 그리고 immunotolerance에서 흉선의 역할을 정의하는 필수적인 절차로 입증되었습니다. 래트 흉선 제거는 T 세포 고갈 또는 네이티브 나이브 T 세포의 재 출현없이 정의 T 세포 개체군의 양자를 포함하는 연구 전송을 허용한다. 신생아 설치류 Thymectomies 안정적인 결과 1 흡인 기술을 사용하여 달성 될 수있다. 성인 쥐에서,이 기술은 대략 20 %의 사망율과 연관되며, 종종 불완전 흉선이 초래한다. 지속적으로 성인 쥐의 완전한 흉선을 달성하기 위해, 중간 흉골 절개를 통해 종격동의 오픈 노출이 필요합니다. 그러나,이6 % 2 - 4 - 절차는 1.5에 이르기까지 전반적인 사망률을 선도하는 기관 손상, 출혈과 기흉 등의 합병증과 관련이 있습니다. 지난 20 년 동안 흉선 기술의 개선은 수술 후 합병증을 감소하고 생존율을 향상시켰다. 양압 환기를 허용 기관 내 삽관은 기흉 총 5 적어있다. 삽관하는 방법은 이전에 직접 성대 시각화를 사용하여 덜 침습적 방법으로 기관에 공개 노출 범위를 설명했다. 삽관 절차와 관련된 합병증은 심장에 구멍 상대 정맥의 열상으로 인한 기관 손상, 성대 파열, 의도하지 않은 식도 삽관, 출혈 등이 있습니다. 또한, 흉막 안감에 낮은 흉선 로브의 근접은 기흉이 발생할 수 있습니다. 여기에 우리의 기술을 설명무딘 엔드 angiocatheter 및 경피 기관 조명을 사용하여 기관 내 삽관의 간단한 방법에 따라 최소 침습 2cm의 피부 절개를 통해 흉선. 흉선은 1.5 cm의 흉골과 종격동을 봉인하고 출혈 및 호흡기 합병증의 발생을 최소화하기 위해 수술 접착제 응용 프로그램과 삼층 폐쇄를 포함한다. 흉선 및 IHC 염색에 흉선 조직의 유무 다음 CD4 +와 CD8 + 나이브 T 세포의 소실에 의해 입증되는 바와 같이이 방법은 확실하게 완료 흉선 초래한다. 수술 시간 및 근교 절차 사망률은 최소로 유지된다.

<table border="0" cellpadding="0" cellspacing="0" width="718">
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			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
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			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
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			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

신생아 쥐의 흉선은 면역 학적 연구를위한 설립하고 신뢰할 수있는 절차입니다. 그러나, 성인 쥐, 흉막 중단에서 출혈과 기흉의 합병증은 상당한 사망률이 발생할 수 있습니다. 이 프로토콜은 미니 흉골 절개술과 기관 내 삽관을 이용하여 쥐의 흉선의 간단한 방법이다. 삽관은 비 침습적 쉽게 재현 방법으로 달성 및 기흉을 방지하기 위해 양압 환기 및 흉막 혼란을 최소화하기 위해주의 흉선 절제술을위한 충분한 시간을 허용하는 제어기도를 허용한다. 여전히 흉선의 전체 시각화를 제공하면서 1.5 cm 흉골 절개, 종격동 혈관 및 흉막과의 접촉을 감소시킨다. 종격동의 노출 후, 흉선 배율 하에서 박리 (blunt dissection)에 의해 제거된다. 흉막은 수술 후 접착제의인가 다음에 미리 기관 근육 봉합사에 의해 밀봉 폐쇄된다.흉부 후 피부의 봉합 폐쇄 다음에 흉골 봉합 폐쇄에 의해 폐쇄된다. 유세포 나이브 T 세포의 면역 조직 화학 (IHC) 종격동 조직 염색 및 부재에 의해 입증되는 바와 같이 모든 thymectomies 완료했고, 절차는 96 %의 생존율을 가졌다. 최소한의 합병증 완료 흉선은 흉선 성인 쥐에서 더 면역 학적 연구를 위해 요구되는 경우이 방법은 적합하다.

이 절차는 성인 루이스 래트 (N = 26)에서 수행 하였다. 평균 수술 시간은 15 ± 3 분이었다. 어떤 수술 사망률은 없었다. 평균 삽관 시간 - 환기의 설립에 삽관 장치에 쥐의 배치에서이 - 45 ± 5 초였다. 24 쥐 수술 후 일 (POD) POD 4 14. 한 쥐 개발 호흡 곤란을 통해 호흡 곤란 또는 출혈의 증거와 함께 작업에서 완전히 회복했고, 종격동을 탐구 재수술을 시행 하였다. 래트 폐 붕괴 로브를 갖는 것으로 밝혀?...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

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 ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K 조회수.  Duke University Medical Center. 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. 

비디오: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

백혈구 호밍 및 마이 그 레이션의 비침습 영상 생체내에</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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면역학 및 감염병학

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Zebrafish 애벌레의 분해해 칸디다 증의 비침습 이미징

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

마우스의 림프 Vasculature의 비 침습적 광학 이미징

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

신생아와 성인 중추 신경계에서 배양 소교 세포

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Neuropathogenic의 비 침습 모델<em&gt; 대장균</em&gt; 신생아 쥐에 감염

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

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

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

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

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

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

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

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

결막에서 세포를 분리하고 표현하기위한 비 침습적 방법

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...