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><tbody><tr><td>Pilocarpine hydrochloride</td><td>Sigma Aldrich</td><td>P6503</td><td>Pilocarpine</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-9</td><td>Spring Scissors for Tracheostomy</td></tr><tr><td>Sterile Saline Solution</td><td>Medline</td><td>RDI30296H</td><td>Saline</td></tr><tr><td>Dumont #7 Forceps</td><td>Fine Science Tools</td><td>11274-20</td><td>Curved Forceps</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>Straight Forceps</td></tr><tr><td>Standard Pattern Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Blunt Forceps</td></tr><tr><td>Fine Scissors- Tungsten Carbide</td><td>Fine Science Tools</td><td>14568-09</td><td>Dissection Scissors</td></tr><tr><td>Microhematocrit Heparinized Capillary Tubes</td><td>Fisher Scientific</td><td>22362566</td><td>Capillary tubes</td></tr><tr><td>Lubricant Eye Ointment</td><td>Refresh</td><td>N/A</td><td>Refresh Lacri-Lube</td></tr><tr><td>Goat polyclonal anti-Nkcc1</td><td>Santa Cruz Biotech</td><td>SC-21545</td><td>Nkcc1 Antibody</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>DAPI</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>ver6.0</td><td>Statistical Software</td></tr><tr><td>Cotton tipped applicator</td><td>Medline</td><td>MDS202000</td><td>Applicator for eye ointment</td></tr><tr><td>0.5cc Insulin Syringe, 29G x 1/2&quot;</td><td>BD</td><td>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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.1K 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

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

두개골 방사선 치료에 의한인지 감소를 Quantifying

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

초점 방사선 억제와 성인 시상 하부의 신경 발생의 기능 심문

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

신경과학

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

필로카핀 유도 간질 마우스에 있는 기억 기능의 평가

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

행동분석학

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

인간 Videofluoroscopic 삼키기 연구 방법을 적응하는 것은 검색 및 설치류 질병 모델에서 연하의 특성을

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

마우스 침샘에 줄기 세포의 분리

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

생물학

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Retroductal 나노 Murine Submandibular 동맥에 주입

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

생체공학

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. ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

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 ...

면역학 및 감염병학

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 ...