이 간행물에 보고 된 연구는 국립 연구소의 치과 Craniofacial 연구 (NIDCR)와 보너스 번호 R56 DE025098, UG3 DE027695, 및 CA206296 F30 국립 보건원의 국립 암 연구소 (NCI)에 의해 지원 되었다. 내용은 전적으로 저자의 책임 이며 반드시 국립 보건원의 공식 의견을 대표 하지 않는다. 이 작품 또한 NSF DMR 1206219와 IADR 혁신 구강 케어 상 (2016)에 의해 지원 되었다.
우리는 박사에 리 마루야마와 앤드류 Hollomon 타 액 컬렉션과 함께 그들의 도움에 감사 하 고 싶습니다. 우리는 동맥 해 부와 그의 지원에 대 한 페이 Lun Weng 감사 하 고 싶습니다. 우리는 매튜 잉 그림 준비에 그의 원조에 대 한 감사 하 고 싶습니다. 우리는이 논문의 중요 한 읽기 박사 Elaine Smolock, 에밀리 우 감사 하 고 싶습니다.

아래에 설명 된 모든 vivo에서 절차는 로체스터, 로체스터의 대학에서 동물 자원에 대학 위원회에 의해 승인 되었다. NY입니다.
1입니다. 준비
<ol><li>분석 균형을 사용 하 여, pilocarpine의 20 밀리 그램 무게. Microcentrifuge 튜브에서 메 마른 염 분 2 mL에 녹. 참고: pilocarpine 빛에 민감한 이며 시간이 지남에 따라 활동을 잃는다, 때문에이 솔루션 주입의 날 준비와 있어야 관리까지 빛 으로부터 보호 합니다.</li>
	<li>분석 균형을 사용 하 여, 무게 고 모든 컬렉션 튜브와 유리 모 세관을 식별 합니다. 컬렉션 튜브와 유리 모 세관의 수 생쥐의 수를 일치 해야 합니다.</li>
</ol>2입니다. tracheostomy
<ol><li>C57/BL6 마우스 사용 하 여 분석 균형 무게.</li>
	<li>29G x ½"바늘 0.5 mL 주사기를 사용 하 여, 100 mg/kg 케 타 민 고 10 mg/kg xylazine의 intraperitoneally 주입 된 살 균 염 분 솔루션 마우스 anesthetize. 마우스 이상에 응답 자극 (즉 페달 철수 반사의 부재), 일반적으로 주입 다음 5 ~ 10 분 이내 발생 하는 경우 다음 단계로 진행 합니다.</li>
	<li>솜 밀고 주걱에 윤활제를 분배 후 부드럽게 눈에 적용 하 고 무대에서 부정사 위치에 마우스를 놓습니다. 마 취 동물 따뜻한 표면에 유지 되어야 한다. 참고: 모든 단계는 상 온에서 수행 됩니다.</li>
	<li>보안 코, 팔, 다리 및 꼬리 수술 테이프를 사용 하 여 단계를.</li>
	<li>깨끗 하 고 젖은 알코올 지우기와 목. 참고:이 입력 이후 절에서 모피를 방지 도움이 됩니다.</li>
	<li>집게와 복 부 중간 목 피부를 올리는, 작은, 표면 커트 해가 위를 사용 하 여 확인 합니다. 오프닝으로는 위를 안내 하 고 천천히 피하 조직 비행기를 분리 하는 블레이드를 엽니다.</li>
	<li>입 아래 1cm를 잘라 신중 하 게 발생 하는 피부를 유지.</li>
	<li>첫 번째 컷의 열 등 하 고 우량한 측면에 두 개의 측면 절 개를 확인 합니다. 집게를 사용 하 여 부드럽게 반영 멀리 머리 및 목 구조에 액세스할 수 있도록 피부.</li>
	<li>8 X 확대 해 현미경을 사용 하 여 시각화 submandibular 동맥 (중간: 그림 1).</li>
	<li>기도 overlying 4 infrahyoid (스트랩) 근육을 노출 하는 땀 샘을 리프트 부드럽게 미세 집게를 사용 하 여. 찢 어 또는 주변 맥 관 구조 또는 배설 덕트 방해 하지 마십시오.</li>
	<li>둘 이상의 마우스에서 타 액을 수집 하는 경우 계속 하기 전에 단계 2.1-2.10 각 마우스를 반복 합니다. 참고:이 절차를 수행할 수 있습니다 안전 하 게에 5 쥐까지 동시에.</li>
	<li>작은 해가 위, 가능한 중간 선 가까이 머물면서 스트랩 근육의 중간 부분을 제거 합니다. 기도 시각화 하 고 절차 동안 방식의 스트랩 근육을 유지 하는 필요한 만큼만 잘라. 상당한 출혈을 보조 볼륨 고갈 경우 pilocarpine 됩니다 효과적으로 분 비를 유도 하기 때문에 어떤 가까운 선박 nicking 하지 마십시오.</li>
	<li>기도에서 결박 근육을 반영 합니다.</li>
	<li>갑 상선, 기관지, 후 두를 시각화 합니다. 기관 조직 overlying의 명확한 인지 확인 합니다.</li>
	<li>게는 가로 절 개 기도에 열 등/후부 작은 해가 위를 사용 하 여 갑 상선. 특허 및 액체의 명확한 기도 인지 확인 합니다.</li>
</ol>3. 타 액 컬렉션
<ol><li>입과 각도 해 부 무대 아래로 근처 테이프 제거 (45 °, cranially) 타 액 흐름을 지원 하기 위해.</li>
	<li>29G x ½"바늘 0.5 mL 주사기를 사용 하 여, 100 mg/kg의 총계 복용량에 대 한 10 µ L/g 몸 무게 pilocarpine의 복 주사를 수행 합니다.</li>
	<li>타이머를 시작 합니다. 여러 생쥐를 먹이 지, 신속 하 게 이동 하 여 및 보조 나머지 마우스를 동시에 주입 함으로써 리드 타임을 줄일.</li>
	<li>표준 집게를 사용 하 여, 모 세관 튜브 액세스에 대 한 입을 엽니다.</li>
	<li>타 액의 구슬을 입에 관찰 되 면 선단부 수집 관 해 단계 (사전 무게 두 튜브) 아래 위치에 배치와 함께 액체에 모 세관 튜브의 근 위 끝을 놓습니다. 참고: C57/BL6 여성 쥐에 있는 나이의 6-10 주 심한 타 액 분 비 발생 합니다 pilocarpine 주입의 2 분 이내.</li>
	<li>경우 타 액은 건물 입에 하지 튜브를 통해 흐르는, 이전 단계를 다시 시도 합니다. 튜브 입구를 저지할 수 있는 점 막 표면에 직접 배치 되지 않습니다 확인 하십시오.</li>
	<li>Pilocarpine 자극 다음 12 분의 총에 대 한 타 액을 수집 합니다. stoma 명확 남아 확인 하십시오. 참고: 증가 해 일 볼륨 및 분 비 물 (침, 오 줌, 배설물)은 보통이 시간 동안.</li>
	<li>마우스는 침 함수를 우울 하 게 하는 경우 (예., 부상 또는 개입), P200 micropipette를 사용 하 여 컬렉션 튜브를 입에서 나머지 액체를 전송. 여러 마우스에서 수집 하는 경우 팁 변경 됩니다 확인 합니다.</li>
	<li>기록 수집된 타 액 플러스 튜브 pilocarpine 주사 후 12 분의 무게.</li>
</ol>4. 동맥 해 부
<ol><li>CO2 안락사로 마우스를 안락사 (동물의 안락사에 AVMA 지침에 설명 된 대로: 2013 판) 이어서 thoracotomy, 머리와 목의 구조를 보존 하도록. 이러한 이유로, 안락사의 방법으로 자 궁 경부 전위를 하지 마십시오.</li>
	<li>무대에서 부정사 위치에 마우스를 놓습니다. 기도 (그림 1)을 통해 원래 사이트에 샘을 재조 정할.</li>
	<li>8 배 확대, 아래 해 현미경을 사용 하 여 시각화 주요 동맥: PG, SMG, SLG. 해 부 동안 동맥을 구별 하는 PG는 확산, SMG와 SLG 측면 거짓말 식별 합니다. 참고: 경우 PG, 해 부 한다 이렇게 먼저 이상 정의 하 고 가장 찢 길 것 이기 때문에.</li>
	<li>기본 구조에서 당기고 집게와 PG의 꼬리를 잡고 하 고 부드럽게 해 부가 위와 PG의 머리를 절단 하기 전에 긴장을 적용 합니다.</li>
	<li>위반 주변 SMGs 집게를 사용 하 여 캡슐. 부드럽게 왼쪽 및 오른쪽 SMG를 구분 합니다.</li>
	<li>집게를 사용 하 여 준수 nonglandular 조직에서 서울시의 등 쪽 부분을 무료.</li>
	<li>집게와 긴장 된 왓슨의 덕트 절단가 위 해 부와 덕트 여 목에서 서울시를 제거 합니다.</li>
	<li>부드럽게 집게를 사용 하 여 서울시에서는 SLG를 구분 합니다. 참고:는 SLG는 SMG의 superolateral 부분에는.</li>
	<li>분석 균형을 사용 하 여, SMG 무게 기록.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotype+analysis+of+aquaporin-8+null+mice.">Phenotype analysis of aquaporin-8 null mice.</a> American Journal of Physiology-Cell Physiology. 288 (5), C1161-C1170 (2005).</li><li>Kamiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-Ray-Induced+Damage+to+the+Submandibular+Salivary+Glands+in+Mice:+An+Analysis+of+Strain-Specific+Responses.">X-Ray-Induced Damage to the Submandibular Salivary Glands in Mice: An Analysis of Strain-Specific Responses.</a> BioResearch Open Access. 4 (1), 307-318 (2015).</li><li>Patel, R. M., Varma, S., Suragimath, G., Zope, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+and+Comparison+of+Salivary+Calcium,+Phosphorous,+Alkaline+Phosphatase+and+pH+Levels+in+Periodontal+Health+and+Disease:+A+Cross-sectional+Biochemical+Study.">Estimation and Comparison of Salivary Calcium, Phosphorous, Alkaline Phosphatase and pH Levels in Periodontal Health and Disease: A Cross-sectional Biochemical Study.</a> Journal of Clinical and Diagnostic Research. 10 (7), ZC58-ZC61 (2016).</li><li>Droebner, K., Sandner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+salivary+secretion+assay+in+F508del+mice--the+murine+equivalent+of+the+human+sweat+test.">Modification of the salivary secretion assay in F508del mice--the murine equivalent of the human sweat test.</a> Journal of Cystic Fibrosis. 12 (6), 630-637 (2013).</li><li>Lamy, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+mouse+whole+saliva+soluble+proteome+induced+by+tannin-enriched+diet.">Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet.</a> Proteome Science. 8 (1), 65 (2010).</li><li>Mahomed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+mucin+immunohistochemistry+in+salivary+gland+tumors+and+head+and+neck+squamous+cell+carcinoma.">Recent advances in mucin immunohistochemistry in salivary gland tumors and head and neck squamous cell carcinoma.</a> Oral Oncology. 47 (9), 797-803 (2011).</li><li>Kohlgraf, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+SPLUNC1+in+saliva+with+an+xMAP+particle-based+antibody+capture+and+detection+immunoassay.">Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay.</a> Archives of Oral Biology. 57 (2), 197-204 (2012).</li><li>Maimets, M., Bron, R., de Haan, G., van Os, R., Coppes, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+ex+vivo+expansion+and+post-irradiation+regenerative+potential+of+juvenile+and+aged+salivary+gland+stem+cells.">Similar ex vivo expansion and post-irradiation regenerative potential of juvenile and aged salivary gland stem cells.</a> Radiotherapy and Oncology. , (2015).</li><li>Lombaert, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+salivary+gland+function+after+stem+cell+transplantation+in+irradiated+glands.">Rescue of salivary gland function after stem cell transplantation in irradiated glands.</a> PLoS One. 3 (4), e2063 (2008).</li></ol>

저자는 공개 없다.

우리 동맥 상해 및 치료제 연구에 적용 될 수 있는 침 샘 기능을 평가 하기 위해 다단계 방법 제시. 우리의 절차 tracheostomy, 타 액, 수집과 동맥 해 부, 각각의 침 샘 생물학의 통합된 연구를 지원할 수 있는 실험적인 응용 프로그램은 포함 한다. 예를 들어 murine tracheostomy 구강 방해 절차 동안 일반 기도 관리를 위해 사용 되었습니다.
Pilocarpine에 필요한 적절 한 해 부 및 tracheal 절 ...

침 샘 장애 증후군 dysregulated 또는 장애인된 분 비 과잉 (sialorrhea) 또는 타 액1의 이용률 (xerostomia 및 hyposalivation)를 선도 포함 됩니다. 두 경우 모두에서 치료 개발2의 최종 목표를 향해 침 샘 생물학의 우리의 이해를 향상에 관심이 있다.
침 샘 매우 radiosensitive 기관, 그리고 종종 머리와 목 암 방사선 치료, 영구적인 구강 건조 증 (xerostomia)3,4로 이어지는 동안 손상 된. 그러나 달리 다른 radiosensitive 조직,, 침 샘 이직 률 상대적으로 낮은 이며 분 비 손실의 메커니즘은 불완전 하 게 이해5,6. 이 독특한 부상 설정에서 조직 재생과 radioprotection 전략 침 기능 평가 요구 한다. 실험적으로, murine 침 컬렉션은 방사선을 치료제 선 응답을 평가에 특히 유용한 도구입니다.
여기, 우리가 수행 및 pilocarpine, 강력한 muscarinic 주 작동 근7를 사용 하 여 자극된 타 액 분 비를 측정 하는 방법 제시. Pilocarpine 동맥 분 비8,9를 유도 하는 자율 신 경계를 자극. 이 테스트를 적절 하 게 완료 한 tracheostomy는 마우스는 절차를 통해 특허 기도 유지 하 고 질 식와 구강10에서 풀링된 분 비에서 소망의 위험을 줄이기 위해 필요 합니다.
이것은 3 개의 주요 침 샘의 제거에 culminating 터미널 절차: 귀 밑 (PG), submandibular (서울시), 그리고 삶의 (SLG). 기능 연구에 대 한 선 무게 기록 됩니다 하 고 타 액 측정11,,1213정상화에 자주 사용 됩니다. 이 데이터는 어떤 점에서 선 위축 예상된 결과14,15 는 방사선 연구에서 특히 중요 하다
어떻게 자극된 타 액 분 비에 관하여 문학에 있는 가변성 수행 되 고16보고 있다. 예를 들어 문학 범위 적어도 3 개의 크기 순서17,18,19,20,,2122,23내 pilocarpine 복용량. 여기, 메서드 실행, 뿐만 아니라 기술 (tracheostomy, 타 액, 수집과 동맥 해 부)으로 적응 될 수 있는 모듈형 플랫폼을 제공 하는 향상 된 재현성의 목적으로 최적화 된 높은 복용량 pilocarpine 프로토콜 선물이 필요합니다.
프로토콜 데모, 뿐만 아니라 우리는 서울시 지역에 분류 한 방사선 (6.85 Gy의 4 복용량) 다음 2 주에 타 액 흐름의 대표 기능 데이터를 포함 합니다.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11000-12</td>
			<td>Blunt Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
		</tr>
	</tbody>
</table>

murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

Hyposalivation는 Sjögren의 증후군 또는 주요 침 샘을 다음 방사선 상해의 면역 반응에서 일반적으로 관찰 된다. 이러한 경우에 질문은 질병 병 인 및 효과적인 개입에 관한 남아 있다. 침 샘의 기능 평가 허용 하는 최적화 기법 조사 전 선 생물학, 부전, 및 치료제에 대 한 소중한 있다. 여기, 우리가 pilocarpine 수행 하는 단계 접근 방식을 제시 tracheostomy 등 세 가지 주요 murine 침 샘의 해 부, 타 액 분 비를 자극. 우리는 또한 적절 한 murine 머리와 목 해부학이이 기술 중 액세스를 선발. 이 방식은 확장성, 따라서 작업 흐름의 효율성을 향상 시키는 여러 마우스를 동시에 처리 될 수 있도록 합니다. 우리는 각각의 추가 필드 내에서 응용 프로그램은이 방법의 재현성을 향상을 목표로. 타 액 컬렉션, 이외에 우리는 메트릭을 측정 하 고이 조직의 기능 능력을 정상화 논의. 대표적인 데이터 분류 한 방사선 (6.85 Gy의 4 복용량) 다음 2 주 우울된 침 샘 기능 submandibular 동맥에서 포함 되어 있습니다.

높은 복용량 pilocarpine 수행 자극 타 액 컬렉션, 그것은 흡 인을 방지 하기 위해 기도 유지 하는 것이 중요 또는 구강에 분 비 물에서 질 식. tracheostomy 회로도 (그림 1)를 제공 됩니다. Tracheal 절 개, 다음은 stoma 조직과 체액의 명확한 유지 되어야 합니다.
타 액 수집 중 모 세관 작용을 향상, 마우스 아래쪽 45 ?...

Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Pilocarpine 자극 다음 통해 murine 침 기능 평가 분류 한 방사선

선물이 murine tracheostomy 등 3 개의 주요 침 샘의 침 컬렉션을 수행 하는 자세한 접근.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

murine-salivary-functional-assessment-via-pilocarpine-stimulation

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11.0K 조회수.  University of Rochester. 선물이 murine tracheostomy 등 3 개의 주요 침 샘의 침 컬렉션을 수행 하는 자세한 접근.

비디오: Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

와튼의 덕트를 통해 마우스 턱밑 침샘의 Cannulation

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

연구

의학

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

돼지의 심근 경색 및 기능적 결과의 평가

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

적응 깊은 뇌 자극으로 파킨슨 병을 제어

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

마우스 모델을 사용하여 급성 이온화 방사선 유발 피부 독성의 정량적 평가를위한 광학 분광학 확산

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

타액 기능 저하의 쥐 모델에서 Retroductal 턱밑 글 랜드 점적 및 현지화 된 분획 조사

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

고해상도 광학 매핑 Murine Atria electrophysiological 평가

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

개발 및 Murine Tolerogenic 수지상 세포의 기능적 특성

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

하 악 및 파 타 액 땀 샘에서 유기 유형 배양의 방사선 치료는 생체 내 특성의 핵심

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

Pilocarpine 자극 다음 통해 murine 침 기능 평가 분류 한 방사선

요약

더 많은 비디오 탐색

이 비디오의 챕터

와튼의 덕트를 통해 마우스 턱밑 침샘의 Cannulation

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

돼지의 심근 경색 및 기능적 결과의 평가

적응 깊은 뇌 자극으로 파킨슨 병을 제어

마우스 모델을 사용하여 급성 이온화 방사선 유발 피부 독성의 정량적 평가를위한 광학 분광학 확산

타액 기능 저하의 쥐 모델에서 Retroductal 턱밑 글 랜드 점적 및 현지화 된 분획 조사

고해상도 광학 매핑 Murine Atria electrophysiological 평가

개발 및 Murine Tolerogenic 수지상 세포의 기능적 특성

하 악 및 파 타 액 땀 샘에서 유기 유형 배양의 방사선 치료는 생체 내 특성의 핵심

Pilocarpine 자극 다음 통해 murine 침 기능 평가 분류 한 방사선

요약

더 많은 비디오 탐색

이 비디오의 챕터

와튼의 덕트를 통해 마우스 턱밑 침샘의 Cannulation

신경근육학 전기적 자극에 의​​해 근육 트레이닝의 Murine 모델

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

돼지의 심근 경색 및 기능적 결과의 평가

적응 깊은 뇌 자극으로 파킨슨 병을 제어

마우스 모델을 사용하여 급성 이온화 방사선 유발 피부 독성의 정량적 평가를위한 광학 분광학 확산

타액 기능 저하의 쥐 모델에서 Retroductal 턱밑 글 랜드 점적 및 현지화 된 분획 조사

고해상도 광학 매핑 Murine Atria electrophysiological 평가

개발 및 Murine Tolerogenic 수지상 세포의 기능적 특성

하 악 및 파 타 액 땀 샘에서 유기 유형 배양의 방사선 치료는 생체 내 특성의 핵심

신경근육학 전기적 자극에 의해 근육 트레이닝의 Murine 모델