이 작품은 벨기에 연방 정부 (IUAP P6/28), 학술 진흥 재단 - 플랑드르 (FWO) (G.0565.07 및 G.0686.09), Astellas 유럽 재단 상 수상 2009 구 Leuven의 연구위원회에서 보조금에 의해 지원되었다 (고아 2009 / 07, EF/95/010 및 PFV/10/006). PU 우리는 학술 진흥 재단 - 플랑드르 (FWO)의 박사 동문입니다. 메가바이트은 마리 퀴리의 동료입니다. FWO의 DDR은 기본 - 임상 동료.

1. 실험실 동물 <ol><li> 동물 (마우스, 쥐)이 12 시간 빛 어두운주기 물과 표준 식품 펠릿에 광고 libitum 접속 설비를 갖춘 전문 동물 시설에 자리 잡고 있습니다. 동물의 연령 및 성별 모두 필요에 따라 표준화해야합니다 중요한 매개 변수입니다. 12주 세 여성 동물 5,6 - 우리가 일반적으로 10 cystometry을 수행합니다. </li><li> 모든 동물 실험은 유럽 연합 (EU) 지역위원회 지침에 따라 수행과 지역 윤리위원회에 의해 승인됩니다. </li></ol> 2. 마취 <ol><li> 그 반감기는 짧은으로 인해 복용에 쉽게이기 때문에 Isoflurane 마취는 작은 수술 절차를 수행하는 데 사용됩니다. isoflurane을 사용하면 소기 적절한 폐기물 가스가 필요합니다. <ol class="lalpha"><li> 동물 순수 산소 isoflurane 5퍼센트 (1 L / 분)와 가스에 폐쇄 상자에 배치됩니다. </li><li> 해당 리터의 유도, 마취 후1리터 / 1 분 산소 - - isoflurane 1.5 % 거시기는 0.8의 작은 마스크 (컷 10 ML의 주사기)에 의해 유지됩니다. </li></ol></li><li> 마우스의 경우, 우레탄 마취는 기능 녹음 (cfr. 인프라)에 사용됩니다. 대부분의 다른 anesthetics는 대조적으로, 방뇨 반사이 마취 에이전트 사에 의해 억압되지 때문에 우레탄 마취는 기능 녹음하는 동안 사용됩니다. 우레탄 1.2 g / kg의 체중은 (SC) 60 분 녹음이 시작되기 전에 피하 관리합니다. 필요한 경우, 적절한 마취를 얻기 위해 필요한 우레탄의 복용은 피하에 추가 용량을 (0.1 g / kg) 관리에 의해 titrated 할 수 있습니다. 이것은 주목해야하지만, 그 추가 마취 voiding 효율성과 결과적으로 방뇨 주파수 7,8을 변경할 수 있습니다. 따라서, cystometric 녹음 면밀히 모니터하고 제어 동작에서 상당한 편차가 관찰 될 때마다 폐기해야합니다. 동안 Anesthetics가 관리하지 않아야 테스트화합물은 데이터의 오해를 피하기 위해 적용됩니다. </li></ol> 3. 수술 - 방광 카테터에 주입 <ol><li> 수술기구는 사용하기 전에 압력솥에 소독되어 있습니다. , 동물의 복부를 면도 에탄올 70%로 소독하고 방광을 노출 할 수있는 낮은 중간 선을 개복술을 수행합니다. </li><li> 수술 현미경에서 비 흡수성, 모노 필라멘트의 봉합을 (크기 6-0)를 사용하여 방광 돔에서 지갑 문자열 봉합을 배치합니다. </li><li> 지갑 문자열 안에 작은 cystostomy을 (우리는 우선적으로 18 G 바늘을 사용)을 수행하고이 구멍을 통해 끝 부분에 작은 커프 (조심스럽게 튜브를 가열하여 얻은)로, PE 50 폴리에틸렌 카테 테르를 삽입합니다. 손에 얇은 튜브 (PE 10) 높은 더 많은 변수 저항이 있습니다. 튜브는 에탄올 70 % 용액을 넣어하거나 압력솥에 소독하여 소독 할 수 있습니다. 주입하기 전에 튜브는 식염수에 플러시해야합니다. </li><li> 지갑으로 str을 확보외과 의사의 매듭과 관 주위에 들죠. 종 모양의 팁까지 부드럽게 바깥쪽으로는 바로 봉합 아래 위치 카테터를 당겨. 누출 방지 할 수있는 봉합에 대한 18 G 카테터의 일부를 밀어 넣습니다. </li><li> 부드럽게 누수를 확인하기 위해 - 생리의 작은 볼륨 (200 μl 100) 달이다. 누수가 존재하면, 추가로 봉합을 배치해야합니다. </li><li> 절개의 위쪽 부분에있는 카테 테르를위한 통로를두고, 모노 필라멘트의 봉합을 사용하여 복부 근육을 닫습니다. </li><li> 터널 속이 빈 금속 막대를 사용하여 interscapular 지역 관​​은,이를 실험이 진행되는 동안 튜브를 물에 동물을 방지한다. </li><li> 피부는 비 흡수성, 모노 필라멘트의 봉합을 사용하여 문을 닫습니다. 주입 카테터은 그 투자율을 확인하는 식염수 플러싱된다. </li><li> 동물 각성하기 전에, 진통제는 (buprenorphine, 0.05 밀리그램 / kg) 피하 주어집니다. </li></ol> 4. 설치 및 Cystometry <ol><li> 실험 questi에 따라(그리고 종 사용)에 cystometry가 깨어 (구속 또는 무제한) 또는 우레탄 - anesthetized 동물에 수행됩니다. cystometry은 절제된 환경에서 침착하는 경향이 의식 쥐에 완벽하게 가능한 반면 마우스 3,5,6,9를 사용할 때, 우리는 일반적으로 우레탄 마취 (피하 1.2 밀리그램 / kg)에서 cystometry을 수행합니다. </li><li> anesthetized 마우스에서 몸의 온도가 지속적으로 모니터링하고 36.5에 보관됩니다 ° C ± 0.2 ° C의 온도 프로브 및 가열 램프에 의해. </li><li> 설정은 제조업체의 지시에 따라 모든 실험을하기 전에 교정된다. </li><li> 주입 카테터는 한쪽과 (하버드 장치), 다른 한편으로는 주입 펌프에 압력 변환기 (에드워즈 Lifesciences)에 연결되는 3 방향 탭에 luer 스텁에 의해 연결되어 있습니다. 압력 변환기는 데이터 수집 시스템 (DI-730-USB, Dataq 악기)에 증폭기 (78534c 모니터, 휴렛 패커드)과 공동 통해 연결되어mputer. Windaq / 라이트 소프트웨어는 녹음에 사용할 수 있습니다. </li><li> 주입 펌프 예를 들어,에서 염분이나 PBS의 주입을 가능하게 시작, 100 μl / 분 (쥐)을 20 μl / 분 (쥐). intravesical 압력이 녹화되는 동안 같은 방광은 일정한 속도로 입력됩니다. </li><li> 그림 5는 일반적인 압력 추적 묘사 : 특정 볼륨 / 압력 도달 할 때까지 유체 주입하는 동안, 방광의 느린 압력 축적은 방뇨 기준 액에 도달이 있습니다. 일단이 기준 액은 방광이 축소됩니다에 도달하고 이후 요도 조임근이 열립니다, 요도를 통해 소변의 흐름을 수 있도록합니다. 따라서, 방광이납니다, 수축이 중지되고 압력은 &#39;기초&#39;수준으로 다시 떨어질 것이다. </li><li> 쥐가 신진 대사 케이지와 디지털 균형을 위의 위치에 따라, voided 볼륨이 voided 볼륨에 대한 정보를 제공 동시에 측정 할 수 있습니다. 마일의 작은 voided 볼륨으로 인해CE는, 그것은이 시스템을 사용하여 매우 도전이 될 수 있습니다. 따라서 voided 볼륨이 voiding 전후 무게가 작은 여과지를 사용하여 결정됩니다. </li><li> 일반적으로, 우리는 시스템이 30 분의 녹화 기간이 다음 30 분에 대한 평형 보자. 그런 다음, 약물이 체계적으로 또는 intravesically 관리 할 수​​ 있으며, 또 다른 30 ​​분이 기록되어 있습니다. </li><li> 기록 된 데이터는 정량 분석​​을위한 전문 소프트웨어로 가져올 수 있습니다. csv 파일로 내보낼 수 있습니다. 우리는 일반적으로 (OriginLab 공사, MA, USA) 오리진을 사용합니다. </li><li> 실험 후, 동물 자궁 경부 전위 (마우스) 또는 CO 2 중독증 (쥐)에 의해 희생되어 있습니다. </li></ol> 5. 대표 결과 <img alt="그림 1" src="/files/ftp_upload/3869/3869fig1.jpg" /> 그림 1. 개복술 개요. A) 위로 향한 위치에 쥐를 놓습니다. B) 워시와 antiseptically 외과 사이트를 준비합니다. 피부의 C) 절개. D) 절개 OF 복부 근육과 방광 노출. <img alt="그림 2" src="/files/ftp_upload/3869/3869fig2.jpg" /> 그림 2. 지갑 문자열 봉합. <img alt="그림 3" src="/files/ftp_upload/3869/3869fig3.jpg" /> 3. 카테 테르의 주입을 그림. <img alt="그림 4" src="/files/ftp_upload/3869/3869fig4.jpg" /> 그림 4. 카테 테르의 Tunnelling. 의식 쥐와 anesthetized 마우스 생리의 intravesical 재관류 동안 얻은 압력 측정치의 예는 그림 5에 표시됩니다. 여러 개의 매개 변수는 압력 신호 (intercontractile 간격, 기준 압력과 임계 압력을 예를 들어)에서 추출 할 수 있습니다. 이 매개 변수의 포괄적 인 설명을 보려면 앤더슨 외. (심판 4) 및 Yoshiyama 외를 참조하시기 바랍니다. (<html> 심판이 10). 우리는 최근에 겨자 오일의 분자 타겟 (MO), 긴 염증과 같은 방광 11,12 등의 내장 기관의 hyperalgesia의 실험 모델에 사용 된 반응성이 매우 높은 화합물을 식별 할 수 cystometry를 사용하고 있습니다. 10 MM MO의 Intravesical 주입은 야생 형 생쥐의 voiding 주파수에 강한 증가 (intercontractile 간격의 감소) (그림 6A, B)와 voided 볼륨 6 감소 유도. 흥미롭게도, MO는 MO 수용체 TRPA1의 결핍 쥐에서 유사한 변화를 유도. 반면에, MO는 WT 마우스에 비해 Trpv1 KO 마우스의 cystometric 매개 변수의 많은 약한 변화를 유도하고 Trpa1/Trpv1 KO 마우스에 영향없이했습니다. 함께 펩타이드 (CGRP) 6 관련 칼시토닌 유전자의 릴리스 측정이 데이터는 TRPV1은 MO에 의해 유도 내장 염증에 중요한 역할을 할 수 있다는 것을 입증 나타냅니다. ve_content &quot;&gt; <img alt="그림 5" src="/files/ftp_upload/3869/3869fig5.jpg" /> 그림 5. 의식 좋아하지 (A)와 anesthetized 여성 마우스 (B)에 기록 intravesical 압력의 대표 흔적. 낮은 압력 &quot;기준 압력&quot;(빨간색 화살표)로 정의됩니다. 충전 단계의 끝에서 압력이 파란색 화살표로 표시되어있다. 압력 차이에 의해 구분이 점, 사이에 주입 유체의 볼륨 수있는 방광 벽의 준수 (컴플라이언스는 = 주입 볼륨 / (임계 압력의 계산 -. 기준 압력) &quot;intercontractile 간격&quot;(- 이리)이 사이의 시간 이 voiding 수축. <img alt="그림 6" src="/files/ftp_upload/3869/3869fig6.jpg" /> 야생 유형과 Trpa1, Trpv1 및 Trpa1/Trpv1 녹아웃 마우스에서 cystometry 패턴에 겨자 기름의 intravesical 응용 프로그램의 그림 6. 효과.(A) 생리와 10 MM MO의 주입에 대응 WT, Trpa1 KO, Trpv1 KO와 Trpa1/Trpv1 KO 마우스에 기록 intravesical 압력 변화를 대표 예. 전, MO의 intravesical 주입 과정에서의 평균 순간 voiding 주파수의 (B) 시간 코스입니다. 모든 마우스의 경우, 데이터는 생리 주입하는 동안 얻은 평균 주파수로 표준화되었다. 이러한 데이터는 Elsevier의 허가와 함께 Everaerts 외. (심판 6)에서 구성된다.

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B., Abel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+the+study+of+visceral+pain+states:+chronic+inflammation+of+the+chronic+decerebrate+rat+urinary+bladder+by+irritant+chemicals.">A model for the study of visceral pain states: chronic inflammation of the chronic decerebrate rat urinary bladder by irritant chemicals.</a> Pain. 28, 109-127 (1987).</li><li>Du, S., Araki, I., Yoshiyama, M., Nomura, T., Takeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+receptor+potential+channel+A1+involved+in+sensory+transduction+of+rat+urinary+bladder+through+C-fiber+pathway.">Transient receptor potential channel A1 involved in sensory transduction of rat urinary bladder through C-fiber pathway.</a> Urology. 70, 826-831 (2007).</li><li>Streng, T., Santti, R., Talo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+female+and+male+rat+voiding.">Similarities and differences in female and male rat voiding.</a> Neurourol. Urodyn. 21, 136-141 (2002).</li><li>Igawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+findings+in+mice+lacking+muscarinic+M2+or+M3+receptors.">Cystometric findings in mice lacking muscarinic M2 or M3 receptors.</a> J. Urol. 172, 2460-2464 (2004).</li><li>Schroder, A., Newgreen, D., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detrusor+responses+to+prostaglandin+E2+and+bladder+outlet+obstruction+in+wild-type+and+Ep1+receptor+knockout+mice.">Detrusor responses to prostaglandin E2 and bladder outlet obstruction in wild-type and Ep1 receptor knockout mice.</a> J. Urol. 172, 1166-1170 (2004).</li><li>Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+lower+urinary+tract+in+mice+lacking+alpha1d-adrenoceptor.">Function of the lower urinary tract in mice lacking alpha1d-adrenoceptor.</a> J. Urol. 174, 370-374 (2005).</li><li>May, V., Vizzard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+and+altered+somatic+sensitivity+in+PACAP-/-+mice.">Bladder dysfunction and altered somatic sensitivity in PACAP-/- mice.</a> J. Urol. 183, 772-779 (2010).</li><li>Thorneloe, K. S., Meredith, A. L., Knorn, A. M., Aldrich, R. W., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urodynamic+properties+and+neurotransmitter+dependence+of+urinary+bladder+contractility+in+the+BK+channel+deletion+model+of+overactive+bladder.">Urodynamic properties and neurotransmitter dependence of urinary bladder contractility in the BK channel deletion model of overactive bladder.</a> Am. J. Physiol. Renal. Physiol. 289, 604-610 (2005).</li></ol>

관심 없음 충돌이 선언 없습니다.

여기에 제시된 cystometry 기술은 동물 모델에서 방광 기능의 생체 측정에 수행 할 수 있습니다. 쥐가 아마도 가장 많이 사용되는 동물 모델이다. 마우스가 처리 할 더 어렵습니다하지만, 유전자 조작 동물을 사용하는 이점을 제공합니다. 때문에 주입 카테터와 intravesical 압력에 영향을 미칠 수있는 내부 - 복부 압력의 변화 느슨해의 결과로 매우 활성화하는 경향이 의식 마우스를 사용할 수있?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 상업 이름, 회사, </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 우레탄 </td><td> 우레탄, 시그마 - 알드리치 </td><td> 315419 </td><td> 그룹 2B 발암 물질 </td></tr><tr><td> isoflurane </td><td> Isoba, Schering-북두칠성 동물 건강 </td><td></td><td></td></tr><tr><td> 폴리에틸렌 카테 테르 </td><td> Intramedic 폴리에틸렌 튜브 PE50, Becton 디킨슨 </td><td> 427411 </td><td></td></tr><tr><td> 수술 현미경 </td><td> 사설 미 6, 칼 Zeiss </td><td> 사설 미 6 </td><td></td></tr><tr><td> 지갑 문자열 봉합 </td><td> Prolene 6 / 0, Ethicon </td><td> 8610H </td><td></td></tr><tr><td> 근막과 피부 봉합 </td><td> Ethilon 4 / 0 또는 5 / 0, Ethicon </td><td> 662G 또는 661G </td><td></td></tr><tr><td> 수술 후 진통제 </td><td> Temgesic, Schering-북두칠성 동물 건강 </td><td></td><td> 쥐에 대한 투여 : 0.05 밀리그램 / kg </td></tr><tr><td> 증폭기 </td><td> 78534c 모니터, 휴렛 패커드 </td><td></td><td></td></tr><tr><td> 분석 균형과 밸런스 데이터 수집 소프트웨어 </td><td> FZ 300i, A &amp; D </td><td> FZ-300i </td><td></td></tr><tr><td> 주입 펌프 </td><td> 펌프 33, 하버드 장치 </td><td> HA33 </td><td></td></tr><tr><td> cystometry 기록 시스템 </td><td> Dataq 악기, DI-730 시리즈와 Windaq / 라이트 </td><td> DI-730-USB Windaq / 라이트 </td><td></td></tr><tr><td> 온도 등록 </td><td> 재수 52 KJ 온도계 </td><td> 52 KJ </td><td></td></tr><tr><td> 압력 트랜스 듀서 </td><td> 에드워즈 Lifesciences, 압력 모니터링 세트 </td><td> T322247A </td><td></td></tr></tbody></table>

the use of cystometry in small rodents: a study of bladder chemosensation

<p class="jove_content"&gt; 소변을 저장하고 효율적으로 편리한 시간에 쫓아 할 수있는 동적 저수지로 낮은 비뇨기 기관 (LUT) 함수. 소변을 저장하는 동안 그러나, 방광은 폐기물 제품에 장시간 동안 노출됩니다. 꼭 장벽 역할을함으로써, LUT, urothelium의 상피 안감은 유해 물질의 재 흡수를 방지합니다. 또한, 유해 화학 물질은 방광의 nociceptive innervation을 자극하고 방광의 내용을 추방 수축을 voiding 시작합니다. 흥미롭게도, 유해 화학 물질에 대한 방광의 감도는 intravesically neurogenic 방광 overactivity을 치료하는 TRPV1 작용제의 capsaicin을 기적, 임상 연습에서 성공적으로 사용되었습니다<sup&gt; 1</sup&gt;. 이 chemosensory 기관으로 방광을 볼 수있는 장점을 강조하고 추가 임상 연구하라는 메시지가 나타납니다. 그러나 윤리적 문제가 심각하게 인간의 과목 LUT 임상 약리학의 분자 기지를 해명하는 데 필요한 침습적 측정에서 수행 할 수있는 가능성을 제한 할 수 있습니다. 이러한 한계를 극복하기위한 방법은 여러 가지 동물 모델을 사용하는 것입니다<sup&gt; 2</sup&gt;. 여기 마우스 및 쥐의 cystometry, 제어 방광 관류의 조건에 intravesical 압력을 측정 할 수있는 기술의 구현을 설명합니다.</p&gt;<p class="jove_content"&gt; 개복술 후, 카테터가 방광 돔에 이식하고 interscapular 지역에 피하 터널링되어 있습니다. 요도가 방뇨를위한 무료 남아있는 동안 다음 방광은 통제 된 속도로 채워 할 수 있습니다. 충전 및 voiding의 반복주기 동안, intravesical 압력은 주입 카테터를 통해 측정 할 수 있습니다. 따라서, 압력 변화 정량화하고 분석 할 수 있습니다. 또한, voided 볼륨의 동시 측정 할 수 있습니다 비 voiding 수축의 구별 voiding 수축<sup&gt; 3</sup&gt;.</p&gt;<p class="jove_content"&gt; 중요한 것은, 동물과 인간 사이의 방뇨 제어의 차이로 인해,이 동물 cystometric 측정은 제한된 병진 가치가<sup&gt; 4</sup&gt;. 그럼에도 불구하고, 그들은 실험 사전 임상 설정에서 방광 pathophysiology과 약리학의 연구에 매우 중요한 역할을합니다. 이 기술을 사용하여 최근의 연구는 방광의 메카 및 화학 감각 속성에서 소설 분자 플레이어의 핵심 역할을 밝혔다.</p

Watch this Scientific Journal Video about The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation at JoVE.com

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Cystometry 작은 동물의 방광 기능을 측정 할 수있는 효과적인 방법입니다<em&gt; 생체 내</em&gt;. 방광은 지속적으로 요도가 방뇨를위한 무료 남지 반면, intravesical 카테터를 통해 제어 속도로 주입된다. 이 intravesical 압력과 voided 볼륨이 기록되는 동안, 반복에 입력하고 방광의 비우는 수 있습니다.

작은 쥐의 Cystometry의 사용 : 방광 Chemosensation의 연구

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

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22.3K 조회수.  KU Leuven, Belgium. Cystometry 작은 동물의 방광 기능을 측정 할 수있는 효과적인 방법입니다 생체 내. 방광은 지속적으로 요도가 방뇨를위한 무료 남지 반면, intravesical 카테터를 통해 제어 속도로 주입된다. 이 intravesical 압력과 voided 볼륨이 기록되는 동안, 반복에 입력하고 방광의 비우는 수 있습니다.

비디오: The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

마우스 블레더 월 주입

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

연구

의학

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

쥐의 방광 통증 모델로 방광 팽창 유발 내장 운동 응답

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb &#8211; specifically the hind limb of a rat &#8211; ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

원격 사지 허혈 양상 : 설치류에 신경 기술

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

서스펜션에 망막 색소 상피 세포를 제공하기 위해 설치류에서 망막 주사를 수행

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

효능 평가<em&gt; H. 파이로리</em동소 이식 쥐 모델에서 방광암의 치료를위한 치료 도구로&gt; 단백질 HP-NAP

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

대한 다낭성 신장 질환의 작은 설치류 모델에서 매우 높은 필드 MRI의 사용<em&gt; 생체</em&gt; 표현형 의약품 모니터링

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity (AIDN): A Translational Neuroscience Approach

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children. Recent investigations in animals have raised concerns that anesthesia exposure may lead to neuronal apoptosis, known as anesthesia-induced developmental neurotoxicity (AIDN). Furthermore, some clinical studies in children have suggested that anesthesia exposure may lead to neurodevelopmental deficits later in life. Nonetheless, an ideal animal model for preclinical study has yet to be developed. The neonatal piglet represents a valuable model for preclinical study, as they share a striking number of developmental similarities with humans.
The anatomy and physiology of piglets allow for implementation of rigorous human perioperative conditions in both survival and non-survival procedures. Femoral artery catheterization allows for close monitoring, thus enabling prompt correction of any deviation of the piglet's vital signs and chemistries. In addition, there are multiple developmental similarities between piglets and human neonates. The techniques required to use piglets for experimentation will require experience to master. A pediatric anesthesiologist is a critical member of the investigative team. We describe, in a general sense, the appropriate use of a piglet model for neurodevelopmental study.

Use of a Piglet Model for the Study of Anesthetic-induced Developmental Neurotoxicity AIDN: A Translational Neuroscience Approach

Anesthesia-induced developmental neurotoxicity (AIDN) research has focused on rodents, which are not broadly applicable to humans. Non-human primate models are more relevant, but are cost-prohibitive and difficult to use for experimentation. The piglet, in contrast, is a clinically relevant, practical animal model ideal for the study of anesthetic neurotoxicity.

마취 유발 신경 독성 (AIDN) 연구를위한 돼지 모델의 사용 : 번역 신경 과학적 접근법

Anesthesia cannot be avoided in many cases when surgery is required, particularly in children.  Recent investigations in animals have raised concerns that ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

여성 작은 설치류에 신경을 살려주는 중간 요도 폐색 (네모)

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...