연구는 국립 보건원, 국립 연구소의 치과 Craniofacial 연구 (DE025030)에서 교부 금에 의해 지원 되었다.

아래에 설명 된 프로토콜 기관 동물 관리 및 사용 위원회에 의해 승인 되었다 그리고 그것은 건강의 국가 학회에 의해 설립 하는 윤리적인 지침을 따릅니다. 이 보고서에 제공 된 모든 데이터는 10-12 주 나이의 여성 쥐를 사용 하 여 생성 되었습니다. 쥐의 다음 긴장 사용 했다: C57BL/6, BALB/c, DBA1, 129S, 및 (B6XA/J) F1. 모든 마우스 장에 장벽 (5 동물 케이지 당) 특정 병원 체 자유로운 조건에서 지 내게 되었고 먹이 물 ad libitum 제공.
1입니다. 준비
<ol><li>Pilocarpine 염의 재고 솔루션을 준비, 화합물의 10 밀리 그램의 무게 그리고 vortexing, 5.63 mg/mL의 재고 솔루션을 제공 하 여 살 균 isotonic 식 염 수의 1.776 mL에 녹이 지. 이 솔루션을 소독 아무 필요도 없다. 하지만 원하는 경우 0.2 µ M 필터를 통해 필터링. 최대 3 개월까지-80 oC 냉장고에 여러 aliquots에서이 재고 솔루션을 저장 합니다. 각 냉동된 주식은 단일 사용 됩니다. 일단 해 동, pilocarpine 솔루션을 refreeze 하지 마십시오.</li>
	<li>0.6 mL microfuge 관을 신중 하 게 온수 18 게이지 바늘으로 각 튜브의 하단에 작은 구멍을 펀치. 이러한 튜브 2 mL 튜브로 설정 합니다. 튜브 살 균 될 필요가 없습니다.</li>
	<li>샤 프, 메 마른 면도칼 블레이드를 사용 하 여 조각 길이 약 2 ㎝ 원통형 흡수 성 면봉 잘라. 2 원뿔 모양의 면봉에 게 대각선 각 2 cm 조각 잘라. 각 0.6 mL microfuge 관 한 면봉을 배치 합니다.</li>
	<li>무게 0.6 mL microfuge 관 마른 면봉을 포함 하는.</li>
	<li>전송으로 새로운 깨끗 한 물 병 공부 하 쥐. 수집 타 액 오염에서 음식 입자를 방지 하기 위해 타 액 컬렉션의 시작 이전에 적어도 2 시간에 대 한 음식 없이 마우스를 유지. 참고: 그것은이 절차에 대 한 감 금 소 당 1 마우스를 유지 하는 데 필요한 아니다. 그러나, 각 장에 배치 하는 쥐의 수는 특정 기관의 규칙 및 동물 사용에 대 한 규정에 따라 다릅니다.</li>
	<li>2 h의 끝의 바로 전에 멸 균 식 염 수에 재고 솔루션 100 배 희석 하 여 작업 pilocarpine 솔루션 (0.0563 mg/mL)을 준비 합니다. 추가 필터를 필요는 없습니다이 솔루션을 소독. 항상 작업 pilocarpine 솔루션 얼음에 보관 합니다. 주: 표 1 pilocarpine 마우스 몸 무게 줄 마지막 복용량 0.375 mg/kg의 체중의 범위에 대 한 주입을 금액을 제공 합니다.</li>
</ol>2입니다. 절차
<ol><li>각 마우스 무게 고 각 마우스를 사용 하 여 표 1에 대 한 마 취 혼합의 복용량을 결정 합니다. 복 경로 의해 마 취 혼합의 적절 한 볼륨으로 최초의 마우스를 주사. (대부분 마우스 긴장이이 프로토콜에서 테스트로 이동 자 2 분) 2 분 타이머를 설정 합니다. 마우스가 제대로 걷는 때 평평한 표면에 배치의 부족에 의해 마 취를 확인 합니다. 마우스는 여전히 걷고, 다음 단계로 진행 하기 전에 추가 2 분 동안 기다립니다. 건조를 방지 하기 위해 두 눈을 윤 활 유 안과 연 고의 한 방울을 적용 합니다. 참고: 테이블 1의 마 취 혼합의 복용량은 0.007 mL/g 몸 무게. 이 절차에 대 한 최적의 범위 0.006 0.008 mL/g 몸 무게는. 그러나, 4 분, 마우스는 여전히 산책을 하거나 육 포의 움직임을 전시 하는 경우 적절 하 게 증가 하는 마 취의 복용량에 대 한 제도적 수 의사와 상담 후.</li>
	<li>표 1 을 사용 하 여 마우스에 대 한 pilocarpine의 복용량을 결정 하 고 복 경로 의해 적절 한 금액을 투입. 2 분 타이머를 설정 하 고 머리와 귀 스틱 컷된 쪽으로 때까지 50 mL restrainer 튜브에 마우스를 삽입 합니다. 장소, 머리는 45도 각도로 튜브 및 위 복 부 표면. 이동에서 계속 절차 보드 튜브 테이프. 주: 표 1 제공 합니다 복용량을 17 g 보다는 마우스에 대 한이 절차가 쥐 17 g 아래의 무게에 시도 하지 않은.</li>
	<li>2 분의 끝에, 부드럽게 입으로 집게를 해 부 마이크로의 닫힌된 쌍을 삽입 하 고 입 열을 위쪽으로 아래 턱을 들어. 입, 혀는 집게의 최고 팔에 볼 다는 것을 확인에 집게를 1-2 m m 더를 밀어. 또 한 쌍의 좋은 집게, 원뿔 끝 가까이 미리 무게 마른 면봉 붙.</li>
	<li>부드럽게 입으로는 면봉의 원뿔 끝을 밀어. 구강에는 면봉의 끝 동안에 집게를 철회 한다.</li>
	<li>입 밖으로 확장 하는 면봉의 넓은 끝을 잡고 그것을 회전 입과의 접촉의 최대 영역을 허용 하도록. 15 분 동안이 자리에는 면봉을 유지. 참고: 이렇게 하면 면봉 컬렉션 동안 입안에 남아 있습니다. 면봉의 원뿔 끝 역할 심지와 면봉의 넓은 부분을 참고 입 밖으로 남아 있습니다.</li>
	<li>15 분의 끝에, 부드럽게 흡수 되지 어떤 타 액을 수집에 면봉을 회전 하 고 0.6 mL microfuge 관에서 젖은 면봉을 배치. 튜브를 닫고 얼음에 2 mL 튜브에 그것을 설정 합니다. 참고:이 시점에서 구강 점 막이 나타납니다 완전히 건조. 마우스 이제 이동할 수 있습니다 다시 그것의 감 금으로 마 취에서 회복을 위한.</li>
	<li>장에 마우스를 전송 합니다. 빠른 재 수 있도록 타 액 수집 후 장에 moistened 음식 펠 릿을 놓습니다. 참고: 쥐를 컬렉션의 끝 후 10 분 이내를 시작. 0.2 mL 미리 따뜻하게 isotonic 식 염 수의 피하 주사도 회복을 가속 화 하기 위해 주어진 수 있습니다.</li>
	<li>다음 마우스를 이동 합니다. 모든 그들은 완전히 복구 될 때까지 모니터링 해야 하는 쥐와는 보 행.</li>
</ol>3입니다. 측정
<ol><li>모든 컬렉션의 끝에, 젖은 면봉 0.6 mL 튜브 무게. 젖은 무게와 타 액 생산의 무게를 건조 무게의 차이 계산 합니다. 2 mL 튜브에 다시 타 액 0.6 mL 튜브를 놓습니다.</li>
	<li>2 ml 튜브의 뚜껑을 잘라. 그런 다음 복구는 타 액을 마이크로 원심 분리기에 4oC에서 7500 x g에서 2 분 동안 2 mL 튜브 원심. micropipette를 사용 하 여 얻은 타 액의 양을 측정 합니다.</li>
	<li>타 액 무게 (mg)10 15 분 이상 또는 타 액 무게 (mg)의 비율으로 결과 표현/몸 무게 (g) 마우스. 참고: 면봉에서 추출 하는 타 액의 양을 측정 하는 경우 타 액 복구 (mL)의 볼륨 또는 타 액의 양 (mL)의 비율으로 결과 표현 / 몸 무게 (g) 마우스.</li>
</ol>

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저자는 공개 관심의 없습니다 충돌 있다.

타 액 생산은 복잡 한 프로세스 이며 많은 요인에 의해 영향을 받습니다. 따라서, 실험실 동물에 침 샘 기능을 측정 하는 것은 도전 될 수 있습니다. 추가 도전 침 샘 기능 같은 동물에서의 반복된 측정은 질병의 발병을 설정 하거나 복구 치료 다음과 설명 필요입니다.
이 보고서에 설명 된 면봉 메서드는 데이터를 표현 하기 위해 여러 옵션을 수행할 수 기술적으로 간단 하다. ?...

침 샘 침 다양 한 신경 및 기계적 자극1에 대 한 응답을 생성합니다. 자극에 선 아드레날린 및 해 수용 체 교감 및 부 교감 신경 신경 시스템을 통해 수행 됩니다. Pilocarpine muscarinic 수용 체에 우위 하 게 작동 해, 파라 sympathomimetic 에이전트입니다. 침 샘에 그것은1M3 muscarinic 아 세 틸 콜린 수용 체에 행동에 의해 타 액의 생산을 유도합니다. Pilocarpine 관리에 따라 타 액 생산 자극에 응답 하는 침 샘의 능력의 지표 이며 침 샘 기능의 측정으로 상용 된다.
타 액 생산의 정확한 측정은 Sjögren의 증후군2 등 방사선 상해 다음 머리와 목 암 치료3침 샘 질환의 연구에 중요 합니다. 여러 가지 다른 방법은 설치류에 타 액 생산을 측정 하기 위해 개발 되었습니다. 4배설 침 덕트, 진공5, 아래 구강에서 타 액의 수집 그리고 유리 모 세관6 또는 micropipette7,8, 를 사용 하 여 컬렉션의 직접적인 cannulation 포함 됩니다. 9. 직접 cannulation 침 덕트의 가장 정확 하 고 순수한 타 액을 제공 합니다. 그러나, 이것은 기술적으로 도전적인 절차, 그리고 ductal 상해를 일으키는 원인이 되기를 위한 잠재력 같은 동물에서 반복적인 침 컬렉션 걸로 하겠습니다. 진공에서 타 액을 수집 튜브에서 타 액의 건조 변수 결과 발생할 수 있습니다. 이 감소는 감소 된 타 액 분 비와 쥐에 더 과장 된다. 그러나 유리 모 세관 후속 분석을 위해 타 액의 컬렉션, 병에 걸린 상태에서 분 비 하는 타 액의 점도 변화는 모 세관의 효율적인 충전을 방지 하는. 또한, 잔여 타 액을 수집 구강 청소 하려고 부상으로 이어질 수 있습니다. 피펫으로 메서드는, 완전 한 컬렉션에 대 한 수 있습니다 하 고 같은 연산자에 대 한 그것은 눈에 띄게 실험 사이. 그러나, 알 수 없는 이유로이 방법은 다른 연산자 사이의 중요 한 변화를 보여줍니다. 따라서, 적절 한 비교를 허용 하려면 동일한 연산자는 특정 프로젝트와 관련 된 모든 실험을 수행 필수적 된다. 명확 하 게,이 실험실을 위한 중요 한 불리를 나타냅니다.
이러한 문제를 해결 하려면 프로시저 결합 타 액 수집 방법 인간과 설치류에 대 한 사용 개발 되었다. Pilocarpine 유도 침 볼륨 측정에 대 한 아래 설명 하는 면봉 방법을 간단 하 고, 재현성, 연산자에 의해 영향을 받지 이며 같은 동물에 반복적으로 수행할 수 있습니다. 또한, 단백질, 면역 글로불린, 또는 다른 생체의 후속 분석을 위해 타 액을 수집 하는 수 있습니다.

<table>
	<tbody>
		<tr>
			<td>Chemicals for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pilocarpine hydrochloride</td>
			<td>Alfa Aesar, Tewksbury, MA, USA</td>
			<td>B21410</td>
			<td>Dilute in sterile isotonic saline. Store single use aliquots of 100X stock at -80oC</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Anesthesia Mix solution</td>
			<td></td>
			<td></td>
			<td>Mix 1 mL ketamine hydrochloride + 0.5 mL xylazine + 8.5 mL sterile isotonic saline in a sterile vial. Can be used for 3 months.</td>
		</tr>
		<tr>
			<td>Zetamine (Ketamine hydrochloride)</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>C3N VT1</td>
			<td>Stock is 100 mg/mL</td>
		</tr>
		<tr>
			<td>Anased (Xylazine)</td>
			<td>Med-Vet International, Mettawa, IL, USA</td>
			<td>RXANASED-20</td>
			<td>Stock is 20 mg/mL</td>
		</tr>
		<tr>
			<td>Isotonic sterile saline</td>
			<td>Vet One, Boise, Idaho, USA</td>
			<td>501032</td>
			<td>Used as diluent</td>
		</tr>
		<tr>
			<td>Artificial tears (lubricant ophthalmic ointment)</td>
			<td>Henry Schien, Dublin, OH, USA</td>
			<td>48272</td>
			<td>Used to prevent eyes from drying during the procedure</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials for saliva collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SalivaBio Children&#39;s swab</td>
			<td>Salimetrics, LLC Carlsbad, CA, USA</td>
			<td>N/A</td>
			<td>Individually wrapped swabs</td>
		</tr>
		<tr>
			<td>50 mL polypropylene tubes</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>89004-364</td>
			<td>Cut off the bottom 1 cm of the tube. Make sure that the cut edge is smooth.</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 0.6 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-290</td>
			<td>Make holes in the bottom of tube with heated 18 gauge needles</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2 mL</td>
			<td>VWR, Radnor, PA, USA</td>
			<td>87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes with permanently attached needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge regular bevel needles</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scalpel blade #11</td>
			<td>Integra York Inc PA, USA</td>
			<td>4-311</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting forceps</td>
			<td>Roboz, Gaithersburg, MD, USA</td>
			<td>RS-5139</td>
			<td>Serrated angular 0.8 mm tip, 4&#34; length</td>
		</tr>
		<tr>
			<td>Label tape</td>
			<td>Santa Cruz Biotechnology, Dallas, TX, USA</td>
			<td>sc-224487</td>
			<td></td>
		</tr>
		<tr>
			<td>3 channel timer</td>
			<td>Amazon.com, Seattle, WA, USA</td>
			<td>B06W2KCYVN</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Balance</td>
			<td>Mettler Toledo, Columbus OH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ reagents for inducing salivary dysfunction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Invivogen, San Deigo, CA, USA</td>
			<td>tlrl-b5lps</td>
			<td>Dissolve in endotoxin free water, and store stock solution at 5 mg/mL . dilute in sterile HBSS 100 ug/mL - inject 100 uL/ moue ip</td>
		</tr>
		<tr>
			<td>Imject Alum adjuvant</td>
			<td>Thermo Scientific</td>
			<td>77161</td>
			<td>Dilute 1:1 in sterile saline. Inject intraperitoneally 0.1 mL/mouse</td>
		</tr>
		<tr>
			<td>Rabbit anti-Ro52 antiserum</td>
			<td>Generated in lab</td>
			<td></td>
			<td>Immunization of rabbits with recombinant mouse Ro52</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals/ Kits for saliva analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Salivary lysozyme estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EnzChek Lysozyme Assay Kit</td>
			<td>Molecular Probes, Eugene, OR, USA</td>
			<td>E-22013</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary IgA estimation by sandwich ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgA</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>0106-01</td>
			<td>Standards for sandwich ELISA - range 30 ng/mL to 0.5 ng/mL</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA unlabeled</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-01</td>
			<td>Coat at 1 ug/mL in bicarbonate buffer</td>
		</tr>
		<tr>
			<td>Goat anti- mouse IgA HRP</td>
			<td>Southern Biotech, Birmingham, AL, USA</td>
			<td>1040-05</td>
			<td>Detection antibody used at 1:4000 dilution</td>
		</tr>
		<tr>
			<td>TMB substrate</td>
			<td>Becton Dickinson and Company, Franklin Lakes, NJ, USA</td>
			<td>555214</td>
			<td>Used as per manufacturer&#39;s instructions</td>
		</tr>
		<tr>
			<td>Immulon 4HBX Microtiter 96 well plates</td>
			<td>Thermo Scientific, Rochester, NY, USA</td>
			<td>3855</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Salivary protein estimation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Dye Reagent Concentrate</td>
			<td>BioRad, Hercules, CA, USA</td>
			<td>5000006</td>
			<td>For protein estimation as per manufacturer&#39;s instructions</td>
		</tr>
	</tbody>
</table>

a method for the measurement of salivary gland function in mice

Sjögren의 증후군, 전 분 비에 영향을 미치는 면역 질환 환자는 침 샘 염증을 개발 하 고 타 액 생산을 감소 했습니다. 마찬가지로 타 액 생산 머리와 목 암에 대 한 방사선 치료를 받은 환자에서 심각 하 게 손상 됩니다. 설치류 모델, 이러한 임상 조건 모방 개발 질병 pathogenesis의 이해를 촉진 하 고 새로운 치료 전략의 개발에 대 한 허용. 따라서, 정확 하 게, reproducibly, 그리고 반복적으로 동물 모델에서 침 샘 기능을 측정 하는 능력은 중요 합니다. 문학에서 설명한 이전 절차에 건물, 메서드는 이러한 기준을 충족 하 고 쥐에 있는 침 샘 기능을 평가 하는 데 사용 했다 개발 되었다. 이 새로운 방법의 추가적인 이점은 그것 쉽게 마스터 하 고는 작은 간 연산자 변화입니다. 침 샘 기능 (무게 또는 양) 또는 pilocarpine 자극에 대 한 응답에서 생산 하는 타 액의 속도 (mL/min)로 평가 됩니다. 수집 된 타 액 단백질 콘텐츠, 면역 글로불린 농도, 및 다른 생체 분석에 대 한 좋은 소스입니다.

실험 마우스 모델 시스템에서 응답 pilocarpine 자극으로 타 액을 생산 하는 능력은 침 샘 기능의 지표로 서 사용 됩니다. 타 액 생산 면봉 메서드에서 11 주 된 C57BL/6 여성 쥐에서 측정 했다. 그림 1A, 결과 타 액 (mg) 수집된 pilocarpine의 증가 복용량을 다음의 금액으로 표시 됩니다. 체중 1 mg/kg의 복용량에 쥐의 일부 (무의식적인 떨고 고 떨고) 조 난 전시 시?...

Watch this Scientific Journal Video about A Method for the Measurement of Salivary Gland Function in Mice at JoVE.com

A Method for the Measurement of Salivary Gland Function in Mice

침 샘 hypofunction는 자가 면역 질환과 방사선 치료의 빈번한 결과 이다. 이러한 질병 마우스 모델에서 침 샘 기능의 재현 평가 기술 도전 이다. 여기, 쥐에 타 액 생산의 정확 하 고 재현 가능한 측정을 위한 간단한 방법을 설명 합니다.

쥐에 있는 침 샘 기능의 측정 하는 방법

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

a-method-for-the-measurement-of-salivary-gland-function-in-mice

Research

JoVE Journal

Immunology and Infection

18.3K 조회수.  Oklahoma Medical Research Foundation. 침 샘 hypofunction는 자가 면역 질환과 방사선 치료의 빈번한 결과 이다. 이러한 질병 마우스 모델에서 침 샘 기능의 재현 평가 기술 도전 이다. 여기, 쥐에 타 액 생산의 정확 하 고 재현 가능한 측정을 위한 간단한 방법을 설명 합니다.

비디오: A Method for the Measurement of Salivary Gland Function in Mice

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

SJL 마우스의 실험적 면역 Hypophysitis의 유도

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

연구

면역학 및 감염병학

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

배아 마우스에 Vasculature을 라벨링하는 방법

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

마우스에 γHV68 감염의 측정

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

이종 교잡<em&gt; 현장에서</em&gt; 침샘, 난소, 그리고 벡터 모기의 태아의

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

면역 반응과 수면에서의 양적 측정<em&gt; Drosophila</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

똑바로 Intravital 현미경 Murine Submandibular 침 샘의 준비

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

쥐에서 담 즙이 폐쇄 증 증후군 장황 하는 실버 나노 방법

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

기능 리포터 시스템으로 특정 진 균의 오 번역 비율 측정

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...