Funding from the Australian Research Council (ARC) and National Health and Medical Research Council (NHMRC) is gratefully acknowledged.

The authors have nothing to disclose.

쥐 장간막 림프절 삽관 모델은 다양한 물질로 도전하는 농도와 다양한 세포와​​ 소장에서 림프에 응답하여 발생하는 이들에 대한 변경 (예 : 지질 및 약물 등) 분자의 전송 속도를 직접 정량화 할 수 있습니다 (다이어트 항원 약물, 제형 등) 10,27 질병 (암, 바이러스, 대장염, 인슐린 저항성 등) 5-7. 림프 수집 성분은 또한 상기 추가의 실험에 이용 될 수있다. 예를 들?...

림프는 각 소장 융모 1에 포함 된 단일 lacteals에서 유래 단방향 프로세스를 통해 소장에서 흐른다. Lacteals 따라서 유체, 거대 분자 및 세포 림프 형성에 상대적으로 투과성 lacteals에 이러한 요소의 항목으로 시작한다. lacteals의 초기 림프 이후 림프 미세 혈관의 네트워크, 수집 (구 심성) 림프관, 장간막 림프절 궁극적으로 사후 노드 (원심성) 림프 혈관의 시리즈를 통해 소장에서 흐른다. 노드 내 림프 교환 혈액에서 노드를 입력 노드에 상주 면역 세포뿐만 아니라 물질로 발생하는 수질 부비동의 시리즈를 통해 전달합니다. 소장에서 흐르는 모든 림프 결국 원심성 장간막 림프절 덕트 및 이후 치스 테르 나디 라티 나에 chyli에 수렴한다. 치스 테르 나디 라티 나에 chyli 또한 림프 꼬리 주변 조직을 배출, intest를 수집원고 판은, 간 및 요추 지역과는 종격동과 몸의 뇌 부분에서 림프와 함께 흉부 림프 덕트를 결합한다. 흉부 림프 덕트는 왼쪽 경정맥과 쇄골 하 정맥의 교차점에 정맥 시스템에 직접 림프를 비 웁니다. 장간막 림프절에서 직접 림프 덕트의 수집을 가능하게 기재되어있는 프로토콜, 따라서 장 림프계를 통해 전신 (일반)에 순환 부위에서 직접 천이 다양한 인자의 분석을 용이하게한다. 장내 림프계에 할당 중요한 생리 학적 기능은 유체 밸런스를 유지하기 위해 지질 친 유성 분자의 흡수를 용이하게하기 위해, 적절한 한 면역 반응을 활성화한다. 종양 세포와 바이러스는 2-4 및 주요 변경 사항은 여러 가지 염증 및 대사 병리 5-7 림프관 내에서 발생하는 장 림프관을 통해 전파. 수장간막 다양한 세포 및 분자의 농도 및 전송 속도의 장 림프관을 통한 벌크 유체 흐름의 분석뿐만 아니라 정량을 가능 장간막 림프절 내에 덕트 nulation은 림프를 수집한다. 다양한 문제 (예를 들면, 다이어트, 항원, 약물)과 질병 모델에 대한 응답으로 농도 또는 이러한 요소의 대중 교통에 대한 변경 사항은 (예를 들어, 대장염, HIV, 당뇨병)도 평가 될 수있다. 이 광범위하게 분석하고 여기에 비교 될 수있다 각 림프 구성 요소를 설명하는 것은 불가능 동안, 장간막 림프절은 단순하게 수성, 지질 및 세포 단계로 구성된다. 성상에 대한 관심의 구성 요소는 항원 또는 tolerogens 8, 사이토 카인 및 비만 세포 매개 (9), 및 인크 레틴 10 대사 매개 면역 메신저로 펩타이드 및 단백질을 포함한다. 포스트 마디 장간막 림프절의 세포 분획 lymphocyt의 (99 %), 거의 전적으로 구성되어ES (11). 다양한 면역 세포 (수지상 세포, 비만 세포, 등)은 사전 마디 장간막 임파선를 입력하지만 노드 (12) 내에 남아있다. 심성 림프 세포 내의 관심있는 경우, 장간막 림프절의 제거를 통해 이러한 세포를 수집 할 수있다 장간막 림프절 덕트 (12)의 삽관 전에 몇 일 노드. 이러한 방식으로, 구 심성, 원심성 및 림프 도관에 직접 연결되고 심성 림프에서 임파구는 장간막 림프절 도관에 직접 전달한다. 장 림프관 통과 다양한 면역 세포의 표현형 및 운송 따라서 검사 할 수있다. 아마도 현재까지 장간막 림프절을 수집하기위한 인용 가장 일반적인 이유는, 그러나, 처리 장내 흡수식이 지질 친 유성 분자 (10)의 수송을 연구하는 것이다. 섭취 후,식이 지질은, 예를 들면, 트리글리 세라이드의 지방산과 모노 글리세리드, phosp로 (소화지방산 및 리소 인지질, 지방산 및 콜레스테롤 등) 콜레스테롤 에스테르로 holipid 담즙에서 양친의 첨가를 통해 작은 미셀 포성 구조로 장내 루멘 내에 분산 (인지질, 콜레스테롤 및 담즙 염)과의 작용 췌장 효소 (10, 13). 여기에서 그들은 장 세포로 흡수된다. 흡수 성분의 비율은, 흡수성 세포 (장 세포) 내 트리글리 세라이드, 인지질 및 콜레스테롤 에스테르를 형성하기 위해 재 에스테르 화. 이러한 재 - 에스테르 화 된 지질은 담즙 분비, 점막 지질 풀 또는 장내 혈액 (13)로부터 공급 외생 섭취 지질 성분 및 내인성 지질 성분의 조합으로부터 조립된다. 여기에서, 에스테르 화 지질 어느 장 세포 내에 저장되어 있거나 함께 다양 아포 단백질과 다른 분자와의 친 유성 장내 지단백질 (칠로 마이크론, 초 저밀도 지단백질 (VLDL)) (조립 <em&gt; 예를 들어, 비타민) (10, 13). 장 lacteals이 장 모세 혈관보다 자신의 항목 침투성이 더으로 장 세포를 종료 한 후, 지질 단백질은 특히 장간막 림프 시스템을 통해 전신 순환 장에서 수송된다. 흡수 된 지질 성분의 비율은 또한, 관련되지 않은 단일 지단백질 모세 혈관과 간문맥 통해 체순환으로 소장에서 반송 14 분자이다. 그러나, 일반적으로, 간문맥 수송로 짧고 중간 사슬 길이 지질의 흡수에만 상당한 플레이어이다. 장간막 림프절의 컬렉션은 따라서 소장에서 지질 단백질 및 관련 구성 요소 (지질, 친 유성 분자, 아포 단백질)의 전송에 대한 평가를 할 수 있습니다. 지단백질 정량화 및 장간막 임파선 지단백질, 일반적 nascen에 유리하게 특성화 될 수있다t 상태 그들은 광범위하게 이러한 지단백 리파제 (15) 전신 효소에 의해 수정되지 않은 때문이다. 장간막 림프절 삽관 된 래트 모델은 아마도 역사적으로 가장 광범위 소장에서 지질 / 지단백 교통 분석에 기술 된 반면, 관심을 확대 영역은 지용성 약물, 전구 약물 및 다른 생체이 13,16의 수송 림프관의 역할은 여기에서 설명하는 모델의 초점이다. (일반적으로 긴 사슬 중성 지방&gt; 50 ㎎ / g에서 로그 P&gt; 5 용해도의 예외가 분명 있지만) 친 유성 약 17, 18, ​​19 및 기타 생체이 13, 16 중 하나를 수동적으로 또는하여 장 림프관에 액세스 할 수 있습니다 드럭 적극적으로 장 지단백 전송 경로 (19)에 통합. 쥐 장간막 림프절 삽관 기술은 이렇게 많은 응용 프로그램이 있습니다. 볼만 등 알. 먼저 techniq 설명변형 모델의 다음 번호가 설명 되었기 때문에 단말. 1948 20 래트에서 장간막 림프절 덕트 cannulate한다. 쥐 다양한 마취제 (21, 22)로 마취 할 때 예를 들어, 수집이 발생할 수 있습니다, 또는 의식 상태에있는 동안은 (23, 24)를 이동 15 자유롭게을 억제. (- 5 ㎖ / hr로 대개 0) 25 래트 재수 다른 솔루션과 같은 위장, 소장 또는 비경 다른 속도로 지질 및 약물 제제와 같은 다른 물질을 투여 할 수있다. 몇몇 연구에서는 흉부 림프 덕트보다는 장간막 림프절 덕트 흉부 림프 덕트는 서로 림프를 받으면이 관심 요인에 따라, 소장에서 운송 과대 수도 있지만, 림프관을 통해 소장에서 전송을 추정 삽관 지역 22, 26. 림프 삽관 모델은 쥐 15,27, 미니 PI 등 여러 가지 다른 종에서 기술 된GS 12, 양 28, 29, 돼지 30 개 (31). 그러나, 래트 모델은 가장 널리 일관 인용. 의식이 25 림프의 수집 뒤에 또는 22 쥐 15,27을 마취 장간막 림프절 덕트의 삽관에 대한 자세한 프로토콜은 이전에 출판되었으며, 관심있는 독자는 이러한 프로토콜에 관한 것이다. 이 프로토콜은 시각화 형식으로 기술을 보여 처음이다. 림프 삽관 된 래트 모델은, 비용면에서 수술 윤리적 고려 용이성 큰 동물 모델에 비해 이점을 갖는다. 마우스 모델에 비해 마우스 모델은 트랜스 제닉 동물 (27)에서 더 상세한 연구를 가능하게하더라도, 장간막 림프절 삽관 수술은 또한 쥐 쉽다. 그럼에도 불구하고, 쥐 모델, 생리의 차이와 관련된 특히, 그 한계 extrapolat의 몇 가지 제한 사항이 있습니다다른 전임상 및 임상 상황에 이온. 예를 들어, 쥐 담즙 흐름에 상수 및 높은 종 식품 반면 식품 섭취 또는 지질 독립적 담즙 흐름 (32)를 자극한다. 이것은 큰 종과 인간에서 볼 반영 쥐의 대표 사전 및 식후 환경을 얻기에 어려움을 만듭니다. 현실적인 인간의 투여 량의 투여 25을 형성 한 후 림프 전송을 평가할 때 약물 전달 연구의 경우, 큰 종은 또한 바람직 할 수있다. 최근 연구에서, 장간막 림프절에서 지질 전송 속도는 종 (27)에 걸쳐 지질 전송 데이터를 외삽에 자신감을 제공하는 등가 질량과 지질의 종류를 투여 한 후 종 (마우스, 쥐, 개)에 걸쳐 유사한 것으로 밝혀졌다. 그러나, 동물의 크기 (즉, 개&gt; 쥐&gt; 마우스)의 순서로 순위 모델 지용성 약물, 할로 판 트린의 전송. 스케일링 인자 따라서 EX해야 할 수도다른 종 쥐에서 임파 약물 전송 데이터인지 추정. 림프 삽관 모델의 제한은, 일반적으로, 림프관은 용기 (33)를 캐 뉼러를 한번 변경되는 압력 구배에 반대 보낸 림프 흐름 및 전송을 수정할 수 림프관부터 직접 해당 수동 림프 컬렉션이다. 림프 삽관 모델은 기술에 익숙하지 않은 실험실에서 파악하는 작업이 쉽지 않을 수도 있습니다. 대체 모델은 따라서 설명되었다. 예를 들면, 지질 단백질 및 친 유성 분자로서 장내 림프계를 통해 요인 통과 간접적 혈액의 컬렉션을 통해 연구되어왔다. 그러한 모델 오랄 존재 관리 및 억제제의 부재 다음과 같은 지질 및 / 또는 약물의 혈중 농도를 비교 포함한다 (예를 들면, 콜히친, 플루 L81, 사이클로 헥시) 림프 전송 블록 34 장 지질 단백질 생산. 장점혈액 샘플 수집을 통해 간접적으로 림프 전송을 정량화 모델의 침습 수술이 35 필요하지 않습니다으로는 인간의 림프 수송의 어떤 평가를 할 수 있다는 것입니다. 그러나, 림프 교통 억제제는 특정하지 않은 및 림프관을 통해 수송된다 요인이 희석과 같은 평가를 복잡하게 전신 순환에 수정됩니다. 체외 대안도 설명 하였다. 예를 들어, 카코 -2 세포 또는 격리 장 세포 배양 물보다 상세히 림프관 36-38 입력 분자의 장내 분비를 연구하기 위해 사용되어왔다. 인간의 장내 미세보다 잘 나타내는 고급 체외 모델은 최근 39 설명했다. 이 모델에서 림프관 내피 세포층 림프관으로 소장에서 물질 전달의보다 상세한 분석이 가능 카코 -2 세포와 공 배양된다. 그러나, vitr에서오 전지 시스템은 교환 흐름을 부족 및 장 루멘과 기본 혈액과 림프 혈관 공급 즉, 상호 연결을 전송합니다. 대안적인 접근법에서, Kassis는 외. 혈관 수축, 림프 흐름 및 장간막 림프관 33 형광 지질 농도 사이의 양적 비교를 가능 시츄 촬상 시스템의 듀얼 채널 (고속 명 시야 화상과 형광)을 설치했다. 시험 관내 시스템에서의 상기 이상이 모델의 이점은 림프관을 통한 면역 세포의 통로의 정확한 추적을 가능하게한다는 것이다. 대량 지질 (또는 약물) 전송의 절대 측정은, 그러나, 아직 이미징 방법을 사용하여 설립. 시험관과 실리에 구체적으로도 40 ~ 42을 게시 한 장 림프관을 통해 친 유성 약물 수송의 정도를 예측하기 위해 접근되지 않습니다. (C)의 여러 예를 들어, 생체 친 화성플라즈마 카일로 마이크론에 대한 ompounds 생체 (41)에서의 림프 전송으로 합리적으로 상관 관계가 밝혀졌다. 이어서, 동일 그룹들은 다수의 물리 화학적 특성에 기초하여 40 킬로 미크론 치료약 친 화성을 ​​예측하기 실리 모델을 확립. 홀름 등. 또한 명백한 분자량 디스크립터 (42)의 기초 림프 친 유성 화합물의 수송을 예측 모델에 실리 비교적 복잡한 설립. 이러한 모델은 알려지지 약물 수송 림프관의 정도를 예측하기위한 유용한 방법을 제공 할 수있다. 약물 및 다른 실험실에 걸쳐 광범위한 모델 검증 그러나, 정확성 및 재현성을 확인하기 위해 필요할 것이다. 장간막 림프절 덕트 삽관 따라서 직접 소장 및 인자 (세포 단백질의 복소 배열의 운송 속도를 배수 림프절의 내용을 검사 할 수있는 유일한 수단 남아생체 내 상황에서 림프 펩타이드, 지질, 약물). 여기에서 우리는 장간막 림프절 및 마취 쥐 전신 혈액의 수집을 가능하게하는 장간막 림프절 덕트 및 경동맥 동맥 삽관을위한 프로토콜을 설명합니다. 대표 데이터는 모델 장간막 림프계를 통해 소장에서 지질 및 약물 수송을 검사 할 수있는 방법을 보여준다. 이 모델과 문제 해결 가이드를 확립 발생할 수있는 문제에 대한 논의가옵니다. 설립되면 모델은 장 림프 전송을 조사하는 강력한 도구입니다.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Sterile saline</td>
			<td>Baxter healthcare</td>
			<td>AHB 1307</td>
			<td>Any brand can be used. Example here is Baxter 100 ml saline bags, box of 50</td>
		</tr>
		<tr >
			<td >70 % ethanol in water</td>
			<td>Any</td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Chlorhexidine gluconate solution (Microshield 4)</td>
			<td>Livingstone International</td>
			<td>JJ60243L</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=JJ60243L</td>
		</tr>
		<tr >
			<td >Betadine solution</td>
			<td>Livingstone International</td>
			<td>BU0510</td>
			<td>Any brand can be used. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=BU0510</td>
		</tr>
		<tr >
			<td >Ilium Ketamil (Ketamine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >KETA I 1</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Ilium Xylazil (Xylazine 100 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >TRO-3828</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >ACP 10 Injection (Acepromazine 10 mg/ml)</td>
			<td >PROVET VICTORIA&#160;</td>
			<td >VTG-DACP010020</td>
			<td>http://www.provet.com.au/</td>
		</tr>
		<tr >
			<td >Sodium pentobarbitone</td>
			<td>PROVET VICTORIA&#160;</td>
			<td>24529</td>
			<td>Any brand can be used. Example here is Lethabarb&#174; 325 mg/ml sodium pentobarbitone, Virbac Animal Health. http://www.provet.com.au</td>
		</tr>
		<tr >
			<td >Heparin (35000I.U. in 35 mL)</td>
			<td>Sigma Pharmaceuticals</td>
			<td>337220</td>
			<td>http://sigmaco.com.au/</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td >Sigma-Aldrich</td>
			<td >E1644</td>
			<td>Any brand can be used. Example here is disodium salt of EDTA from Sigma.&#160;</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.96 mm x i.d. 0.58 mm</td>
			<td>Microtube extensions</td>
			<td>PE8050</td>
			<td>Any brand can be used. Example here is PE tubing 0.8x0.5 mm, 30 m</td>
		</tr>
		<tr >
			<td >Polyethylene (PE) cannula o.d. 0.8 mm x i.d. 0.5 mm</td>
			<td>Microtube extensions</td>
			<td>PE9658</td>
			<td>Any brand can be used. Example here is PE tubing 0.96x0.58 mm, 30 m</td>
		</tr>
		<tr >
			<td >Ruler</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Markers</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cigarette lighter</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >Cyanoacrylate glue</td>
			<td>Any</td>
			<td ></td>
			<td>Any brand can be used</td>
		</tr>
		<tr >
			<td >23 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN23GX0.75LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 23GX0.75inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT= 
			6&#38;search=DN23GX0.75LV</td>
		</tr>
		<tr >
			<td >25 gauge needles</td>
			<td>Livingstone International</td>
			<td>DN25GX1.0LV</td>
			<td>Any brand can be used. Example here is Livingstone Disposable Needle, Sterile, 25GX1.0inch, 100/BOX. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search= 
			DN25GX1.0LV</td>
		</tr>
		<tr >
			<td >1 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS01TA</td>
			<td>Any brand can be used. Example here is Terumo syringe 1 ml Slip Tuberculin 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS01TA</td>
		</tr>
		<tr >
			<td >10 ml syringe</td>
			<td>Livingstone International</td>
			<td>T3SS10SA</td>
			<td>Any brand can be used. Example here is Terumo syringe 10 ml Slip 100/Box. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=T3SS10SA</td>
		</tr>
		<tr >
			<td >Gauze swabs</td>
			<td>Livingstone International</td>
			<td>GSC075</td>
			<td>Any brand can be used and cut to required size. Example here is gauze swabs cotton filled 7.5x7.5 cm, 8 ply. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=GSC075</td>
		</tr>
		<tr >
			<td >Cotton buds</td>
			<td>Livingstone International</td>
			<td>CTAST075DP</td>
			<td>Any brand can be used. Example here is Livingstone cotton applicator plastic double tipped. 75MM. 100/PK. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=CTAST075DP</td>
		</tr>
		<tr >
			<td >Heating pad</td>
			<td>Ratek</td>
			<td>WT1</td>
			<td>Any brand that keeps temperature at 37C can be used. Example here is Ratek warming tray.</td>
		</tr>
		<tr >
			<td >Surgical light</td>
			<td>Harvard Apparatus</td>
			<td>72-0215 with 72-0267</td>
			<td>Any brand can be used. Example here is Harvard apparatus V-Lux 1000 Cold Light Source with Bifurcated Gooseneck Light Guide, Black, 4.7 mm fiber diameter (each arm). http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_50601_ 
			-1_HAI_ProductDetail and&#160; http://www.harvardapparatus.com/webapp/wcs/stores/servlet/product_11051_10001_35487_ 
			-1_HAI_ProductDetail___</td>
		</tr>
		<tr >
			<td >Surgical microscope</td>
			<td>Zeiss</td>
			<td>495005-0014-000</td>
			<td>Any brand can be used. Example here is Zeiss Stereomicroscope Stemi 2000-C with Stand S Double Spot and KL 300 LED. https://www.micro-shop.zeiss.com/?l=en&#38;p=us&#38;f=e&#38;i=10143</td>
		</tr>
		<tr >
			<td >Silk suture</td>
			<td>Livingstone International</td>
			<td>DTSK163019F4</td>
			<td>Any brand can be used. Example here is *&#160; 
			Email this item to my friend 
			3/8 Circle Reverse Cut Silk Suture 3/0 Thread 19mm. http://www.livingstone.com.au/?PG=search_result&#38;CAT=6&#38;search 
			=DTSK163019F4</td>
		</tr>
		<tr >
			<td >Scalpel blades</td>
			<td >Fine Science Tools (FST)</td>
			<td >10020-00</td>
			<td>Any brand can be used. Example here is FST Scalpel Blade #20. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=191</td>
		</tr>
		<tr >
			<td >Scalpel handle</td>
			<td >Fine Science Tools (FST)</td>
			<td>10004-13</td>
			<td>Any brand can be used. Example here is FST Scalpel Handle #4. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=298&#38;CategoryId=51</td>
		</tr>
		<tr >
			<td >1 x Small surgical scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>14060-09</td>
			<td>Any brand can be used. Example here is FST Fine Scissors, 9 cm with 21 mm cutting edge, sharp, straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=40&#38;CategoryId=17</td>
		</tr>
		<tr >
			<td >2 x Forceps with serrated curved tip</td>
			<td >Fine Science Tools (FST)</td>
			<td >11001-13</td>
			<td>Any brand can be used. Example here is FST 13 cm standard pattern forceps with curved 2.8x1.4 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=405&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Iridectomy scissors</td>
			<td >Fine Science Tools (FST)</td>
			<td>15000-08</td>
			<td>Any brand can be used. Example here is FST Vannas Spring Scissors - 2.5mm Cutting Edge, Straight. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=17&#38;CategoryId=16&#160;</td>
		</tr>
		<tr >
			<td >1 x Forceps with straight serated tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11650-10</td>
			<td>Any brand can be used. Example here is FST Graefe 10 cm straight with serrated 1 x 0.99 mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=390&#38;CategoryId=32</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth sharp straight fine tip</td>
			<td >Fine Science Tools (FST)</td>
			<td>11251-10</td>
			<td>Any brand can be used. Example here is FST Dumont #5 forceps straight 11cm with 0.08 x 0.04mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=335&#38;CategoryId=29</td>
		</tr>
		<tr >
			<td >1 x Forceps with smooth fine curved forceps</td>
			<td >Fine Science Tools (FST)</td>
			<td>11063-07</td>
			<td>Any brand can be used. Example here is FST Delicate Forceps 9 cm with smooth 0.4 x 0.3mm tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=360</td>
		</tr>
		<tr >
			<td >2 x Hemostats</td>
			<td >Fine Science Tools (FST)</td>
			<td >13010-12</td>
			<td>Any brand can be used. Not all operators use the hemostats. Example here is FST 12 cm Micro-Mosquito Hemostats with 20 mm length x 1.3 mm width serrated, straight tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=377&#38;CategoryId=33</td>
		</tr>
		<tr >
			<td >1 x Suture needle holder</td>
			<td >Fine Science Tools (FST)</td>
			<td>12001-13</td>
			<td>Any brand can be used. Example here is FST 13cm Hasley Needle Holder with 16 mm length x 1.9 mm width tip. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=254&#38;CategoryId=70</td>
		</tr>
		<tr >
			<td >1 x Artery clamp</td>
			<td >Fine Science Tools (FST)</td>
			<td>18050-28</td>
			<td>Any brand can be used. Example here is FST Bulldog Serrefines straight, 28 mm long, 9x1.6 mm jaw dimension with medium clamp press. http://www.finescience.ca/Special-Pages/Products.aspx?ProductId=270&#38;CategoryId=82</td>
		</tr>
		<tr >
			<td >Oleic acid</td>
			<td >Sigma Aldrich</td>
			<td >O1008</td>
			<td>When required, any brand can be used. Example here is 99% pure oleic acid. http://www.sigmaaldrich.com/catalog/product/sial/o1008?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >14C-oleic acid</td>
			<td >Perkin&#160;</td>
			<td>NEC317050UC&#160;</td>
			<td>Any brand can be used. Example here is Oleic Acid, [1-14C]-, 50&#181;Ci (1.85MBq). http://www.perkinelmer.com/Catalog/Product/ID/NEC317050UC</td>
		</tr>
		<tr >
			<td >Sodium taurocholate</td>
			<td >Sigma Aldrich</td>
			<td >T4009</td>
			<td>Any brand can be used. Example here is taurocholic acid sodium salt hydrate &#8805;95% (TLC) . http://www.sigmaaldrich.com/catalog/product/sigma/t4009?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Halofantrine</td>
			<td >Glaxo Smith Kline</td>
			<td ></td>
			<td>Halofantrine was kindly provided as a gift from Glaxo Smith Kline</td>
		</tr>
		<tr >
			<td >Sodium phosphate monobasic</td>
			<td >Sigma Aldrich</td>
			<td >71507</td>
			<td >Any brand can be used. Example here is sodium phosphate monobasic monohydrate, BioXtra, for molecular biology, &#62;99.5%. http://www.sigmaaldrich.com/catalog/product/sigma/71643?lang=en&#38;region=AU</td>
		</tr>
		<tr >
			<td >Sodium phosphate dibasic</td>
			<td >Sigma Aldrich</td>
			<td >71643</td>
			<td >Any brand can be used. Example here is sodium phosphate dibasic dihydrate, BioUltra, for molecular biology, &#62;99%. http://www.sigmaaldrich.com/catalog/product/sigma/71507?lang=en&#38;region=AU</td>
		</tr>
	</tbody>
</table>

the mesenteric lymph duct cannulated rat model: application to the assessment of intestinal lymphatic drug transport

장 림프 시스템은 유체 수송, 지질의 흡수와 면역 기능에 중요한 역할을한다. 림프 장간막 림프절 덕트 수렴 림프관 및 일련의 노드를 통해 소장에서 직접 흐른다. 장간막 림프절 덕트의 캐 뉼러 따라서 소장에서 흐르는 장간막 림프절의 수집을 할 수 있습니다. 장간막 림프 및 지단백 분획 (지질, 친 유성 분자와 아포 단백질) (사이토 카인과 장 호르몬 등의 유체, 펩타이드 및 단백질) 면역 세포 (99 % 림프구), 수성 부분의 세포 부분으로 구성되어 있습니다. 장간막 림프절 덕트 삽관 모델 따라서 림프계를 통해 농도 및 소장에서 다양한 요인의 이송 속도를 측정하는데 사용될 수있다. 다른 도전에 대한 응답에서 이러한 요소의 변경 (예를 들면, 다이어트, 항원, 약물)과 질병 (예를 들어, 염증성 장 질환, HIV는 당뇨병)도 수 b전자는 결정. 관심을 확대 영역은 장 지질 흡수 경로와 연관 경구 투여 친 유성 약물과 전구 약물의 흡수에 림프 수송의 역할이다. 여기에 우리가 설명, 세부 사항, 장 배달 다음 몇 시간 동안 림프 시스템을 통해 속도와 지질 및 약물 수송의 정도의 평가를 할 수있는 장간막 림프절 덕트 유관 쥐 모델. 방법은 림프 다른 파라미터의 측정에 용이하게 적응할 수있다. 우리는 실험 성공을 확인하고 얻은 데이터를 해석하는 방법에 대한 교육을 제공하기 위해이 복잡한 수술 방법을 설정할 때 발생할 수있는 문제에 대한 자세한 설명뿐만 아니라, 실패와 성공의 실험에서 대표 데이터를 제공합니다.

대표적인 실험의 결과는도 4 및도 5에 도시되어 장간막 림프절 삽관 모델을 이용하여 장내 전달 다음 림프계를 통해 누적 범위와 지질 및 약물 수송 속도를 정량화한다.이 실험에서, 모델 친 유성 200 μg의를 포스페이트 완충에 5.6 ㎖의 5 mM의 나트륨 타우로 콜산에 분산 및 7.1 mg의 2 모노 올레 인 - 약물 할로 판 트린 40 mg의 올레산 (5 μCi를 14 C-올레산 2 포함) 아세트산 함유 제제?...

Watch this Scientific Journal Video about The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport at JoVE.com

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

장간막 림프 덕트 유관 쥐 모델 : 창자 림프 약물 운송의 평가에 응용

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

the-mesenteric-lymph-duct-cannulated-rat-model-application-to

Research

JoVE Journal

Immunology and Infection

28.3K 조회수.  Monash University (Parkville Campus). Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

비디오: The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune response and therefore, the development of effector cells. As such, LNs are key to fighting invading pathogens and maintaining health. The choice of LN to study is dictated by accessibility and the desired model; the inguinal lymph node is well situated and easily supports studies of biologically relevant models of skin and genital mucosal infection.
The inguinal LN, like all LNs, has an extensive microvascular network supplying it with blood. In general, this microvascular network includes the main feed arteriole of the LN that subsequently branches and feeds high endothelial venules (HEVs). HEVs are specialized for facilitating the trafficking of immune cells into the LN during both homeostasis and infection. How HEVs regulate trafficking into the LN under both of these circumstances is an area of intense exploration. The LN feed arteriole, has direct upstream influence on the HEVs and is the main supply of nutrients and cell rich blood into the LN. Furthermore, changes in the feed arteriole are implicated in facilitating induction of adaptive immune response. The LN microvasculature has obvious importance in maintaining an optimal blood supply to the LN and regulating immune cell influx into the LN, which are crucial elements in proper LN function and subsequently immune response. 
The ability to study the LN microvasculature in vivo is key to elucidating how the immune system and the microvasculature interact and influence one another within the LN. Here, we present a method for in vivo imaging of the inguinal lymph node. We focus on imaging of the microvasculature of the LN, paying particular attention to methods that ensure the study of healthy vessels, the ability to maintain imaging of viable vessels over a number of hours, and quantification of vessel magnitude. Methods for perfusion of the microvasculature with vasoactive drugs as well as the potential to trace and quantify cellular traffic are also presented. 
Intravital microscopy of the inguinal LN allows direct evaluation of microvascular functionality and real-time interface of the direct interface between immune cells, the LN, and the microcirculation. This technique potential to be combined with many immunological techniques and fluorescent cell labelling as well as manipulated to study vasculature of other LNs.

A technique for performing intravital microscopy of the inguinal lymph node (LN) is outlined. Such technique allows for real-time, in vivo study of the lymph node microvasculature and structure both during homeostasis and infection. This technique can be adapted to cell trafficking studies and to other lymph node sites.

사타구니 림프 노드의 Intravital 현미경

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

연구

면역학 및 감염병학

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

표준 임상 Assays에 의해 탐지의 한도 이하 바이러스성로드와 HIV 감염된 개인의 확장 및 HIV - 1 RNA를 Quantifying

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

마우스 오금 림프절의 Intravital 이미징

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

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 ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

의 검출을위한 자석 교반기 방법<em&gt; 선모충</em근육 샘플에서&gt; 애벌레

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

인간의 과발현과 정제<em&gt; 시스</em&gt; - 프레 닐 트랜스퍼 라제<em&gt; 대장균</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

소설 급지대 무료 시스템 대량 생산을 위한 생체 외에서 Murine 자연 킬러 세포의

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

구두 항생제 치료 후 Murine 장 Microbiota의 정량적 중 합 효소 연쇄 반응-기반 분석

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

만성 살모넬라 감염 유도 장 섬유증

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...