Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

참고 :이 프로토콜을 검토하고 피츠버그 기관 동물 관리 및 사용위원회의 대학에 의해 승인되었다. 수정 크렙스 솔루션 및 시험 약물 분취 1. 준비 <ol><li> 117.9의 NaCl, 4.7 KCl을 25의 NaHCO3, 1.3의 NaH 2 PO 4, 황산 1.2, 2.5 CaCl2를 11.1 D 글루코오스, 2 소듐 뷰티 레이트, (20), 아세트산 나트륨 (mm 단위)가 포함 개질 크렙스 용액 6 L 만들기 0.004 니페디핀 (자발적인 근육의 수축을 차단하기 위해), 0.003 인도 메타 신 (indomethacin)은 (내인성 프로스타글란딘의 합성을 차단하기 위해). 각각 조직의 절개 및 단일 섬유 녹음, 얼음처럼 차가운 따뜻한 크렙스 솔루션을 사용합니다. </li><li> 모든 화학 용액을 준비 (예를 들어, IS, 캅사이신, ATP) 원하는 농도로 분액. </li></ol> 대장 직장 - 신경 조직의 2 해부 <ol><li> 마취 및 수컷 마우스 안락사 (6-8주 이전, 20~30g)에서를10 변위 유량 CO 2 실 - 생쥐까지 분당 챔버 체적의 30 %는 가슴 움직임의 부재로 나타내는 바와 같이 호흡 완전히 멈춘다. </li><li> 즉시, 흉부 챔버를 개방 절단 우심방을 천공하고, 얼음처럼 차가운 크렙스의 충분한 볼륨 (~ 500 ml)에 마우스 시체를 침지 안락사,에게서 피를 뽑다 다음 (4 ° C) 솔루션은 carbogen (95 % O 2 버블 5 % CO 2). </li><li> 조심스럽게 모든 내장 그러나 콜론과 골반 장기를 제거합니다. 약간 횡격막 위 T12 척추 분절에 걸쳐 반 마우스 횡단면과 빙냉 함유 해부 챔버에 꼬리 절반을 전송하고, 크렙스 용액을 버블. </li><li> 실체 현미경 아래에서 요도에 자신의 접합에에 병행하여 방광 및 생식 기관을 제거하고 총장 골 동맥으로 분기합니다 때까지 하강 / 복부 대동맥을 제거합니다. PN 또는 surroundin에서 LSN 무료g의 무딘 절개하여 조직과 L6 및 S1 척추 (PN)에 열 또는 T13과 (LSN)에 L1 척추에 자사의 복부 진입 점까지 장골 외부에서 신경을 따릅니다. </li><li> 치골 치골 좌우 비구 관절을 잘라 및 장골 뼈를 제거합니다. 신경이 척추를 입력 어디까지 대장 직장 가까이에서 첨부 된 근육과 결합 조직에서 하나 PN 또는 LSN 조심스럽게 무료. </li><li> 조심스럽게 원위부 대장 직장을 노출 장골 뼈를 절제. 연속체에 부착 된 PN 또는 LSN과 원위부 대장 직장을 해부하다. </li><li> 티슈 챔버의 욕조에 부착 함과 함께 신경 대장 직장 전송. 더 해부 과도한 결합 조직을 제거하고 방지 장간막 국경을 따라 길이 방향으로 대장 직장을 엽니 다. </li><li> 이 위를 향하도록 점막면, chambe의 실리콘베이스에 기록 구획에 인접한 대장 직장의 장간막 가장자리를 고정R 및 힘은 액추에이터에만 연결 후크 긁어 대장 직장의 길이 antimesenteric 부착 (도 1에 도시되고도 2a에 촬영). </li><li> 마우스 홀 및 게이트에 의해 목욕 구획에 연결되는 기록 구획에 PN이나 LSN 연장. 부드럽게에 신경을 준수 할 수있는 친수성 ​​표면을 제공 녹화 실에서 작은 유리 거울에 신경 트렁크를하다. 따뜻한와 목욕 구획 Superfuse (30-32 ° C가), 크렙스 솔루션을 산소와 미네랄 오일과 기록 구획을 입력합니다. </li></ol> 3. 단일 섬유 녹음하고 받아들이는 필드의 현지화 <ol><li> 조심스럽게 높은 배율 (- 60X 50)에 입체 현미경 PN 또는 LSN에서 epineurium (신경초)를 벗겨. ~ 100 ㎛ 두께의 8 신경 다발 - 미세 집게를 사용하여, 5에 신경 트렁크를 애타게. </li><li> 백금 - 이리듐 참조를 배치티슈 챔버 크렙스 용액과 접촉하는 전극. 순차적으로 동일한 재료로 이루어진 기록 전극 상 개별 신경 다발을 배치. </li><li> 부드럽게 쓰다듬어에 의해 대장 표면 아래로 대장 구 심성에서 AP를 보여주고 부드러운 페인트 브러시를 사용합니다. 검출 AP (활동 전위) 녹음을 통해 콜론에 분포 신경 다발 (들)를 찾습니다. 참고 : PN과 LSN은 또한 방광과 골반 장기에 분포. </li><li> 추가로 10 μm의 두께 ~ 미세 필라멘트 다발로 신경 다발을 분할하고, 기록 전극 상 개별 필라멘트를 배치 30 G 바늘 팁의 쌍을 사용한다. </li><li> 전기적 현재 확산의 ~ 2mm 반경을 생산 초과 임계 자극 강도 (10mA 크기, 0.3 Hz에서 @ 0.5 밀리 초 시간)에서 구 심성 엔딩을 자극하는 점막 표면에 수직 라운드 팁 동심 전극을 배치합니다. 체계적 (~ 1.5 mm 단계를 전극 이동) 평평 대장 직장의 길이와 폭을 따라 수용 엔딩 현지화. </li><li> 구 심성 결말이 흥분 할 때, 최소한의 자극 강도 (자극 임계 값)이 필요 활성화 (수용 필드, RF)의 위치를​​ 정확히 파악할 수있는 전극의 위치를​​ 조정합니다. &gt; 3mA (19) 자극 임계 값 엔딩을 폐기하십시오. </li><li> 자극 인공물과 활동 전위의 발병 사이의 전도 속도 1 ~ (CV)) 수용 필드 (RF)에서, 자극 전극 및 상기 기록 위치, 2) 전도 지연 (예를 들어,도 2B의 간격)를 계산 . CV (m / 초) = 거리 (mm) / 전도 지연 (밀리 초). </li></ol> Mechanosensitive 대장 구 심성의 4 기능 분류 <ol><li> 전기 자극에 의​​한 RF를 찾은 후, RF에 다음과 같은 세 가지 기계적인 자극을 적용 <ol><li> CALIBRA의 끝을 눌러 프로빙 자극을 실시테드 프레이 같은 나일론 모노 필라멘트 (0.4 1g의 힘) 수직으로 평탄화 된 대장 직장에서 RF쪽으로. </li><li> 부드럽게 RF에 작은 표면 전단 응력을 생성하는 좋은 나일론 필라멘트 가닥 (10 mg의 힘)으로 대장 점막을 쓰다듬어하여 쓰다듬어 자극을 실시한다. </li><li> 경 스트레칭 력을 얻어 컴퓨터 제어 동력 액츄에이터를 이용하여 원주 연신을 실시 - 단계 2.8에 기재된 후크 퀴 통하여 대장 직장의 항 장간막 가장자리를 따라 원주 방향 (0~5 mN의 / 초에서 170 MN) . </li></ol></li><li> 장막으로, 점막을 (프로빙 둔 만 응답) 구 심성을 분류 근육 (원주 스트레칭에 대응하고 프로빙 둔) 근육 / 점막을 (원주 스트레칭, 점막 쓰다듬어에 대응하고 프로빙 둔), 또는 (쓰다듬어 점막 및 프로빙 둔 응답) MIA (세 기계적 자극의에 반응하지 않음). </li><li> 만에 (장간막 구 심성의 경우어려운 LS​​N의 신경 분포)은 전기 자극에 의​​해 선택적으로 활성화 장간막의 프로빙 / 기계 쓰다듬어하여 수용 엔딩을 찾습니다. </li><li> 스트레칭에 민감한 구 심성 (근육과 근육 점막)의 경우, 경 스트레칭 중 첫 번째 AP를 불러 일으키는 힘으로 정의 된 응답 임계 값을 결정합니다. </li><li> 장막 심성 들면, 컴퓨터 제어 동력 구동되는 액추에이터에 의해 수용 필드의 프로빙 점상의 수준을 상승 그들의 응답을 기록. </li></ol> 받아들이는 엔딩 5. 화학 응용 프로그램 / 변조 <ol><li> 기계적인 자극에 대한 기본 응답을 기록 (즉, 경 스트레칭, 프로빙 반점, 또는 점막 쓰다듬어에 응답). </li><li> 코트 바닥 튜브 (10mm 높고 4 × 4mm 2 정사각형 또는 4 황동 또는 스테인레스 스틸, - 5 mm 직경)의 조각의 가장자리에 바셀린과 대장 직장의 수용 필드 위에 놓습니다. </li><li> 제거화학 물질 (들)을 검사 할 함유하는 용액을 5 분에 170 μL - 크렙스 내부 튜브 용액 및 3에 대해 수용 결말을 노출. </li><li> 화학 응용 프로그램 중 심성의 반응을 모니터 (일부 구 심성은 chemosensitive 있습니다). </li><li> 화학 물질의 작용을 정지하고, 약액 튜브를 제거한다. 내 4 - 6 분, 기준선 응답과 동일한 기계적 자극에 대한 구 심성 응답을 테스트한다. </li><li> 워시 아웃의 충분한 기간 (&gt; 15 분) 후 다시 기계적인 자극을 다시 적용합니다. </li></ol> 6. 녹음 및 차별 AP 스파이크 <ol><li> 20 kHz에서 축삭에서 기록 된 전기 신호를 디지털화하여 컴퓨터에 데이터를 저장한다. 오디오 모니터에 의해 온라인 신호를 모니터링합니다. </li><li> 오프라인 AP 스파이크를 분석하고 개별 스파이크 파형 (25)의 주성분 분석을 기반으로 하나의 단위를 구별. 참고 : 하나의 기록이 일 더 이상 포함되어야합니다두 쉽게 식별 가능한 활성 단위. </li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unresponsive+afferent+nerve+fibres+in+the+sural+nerve+of+the+rat.">Unresponsive afferent nerve fibres in the sural nerve of the rat.</a> J Physiol. 435, 229-242 (1991).</li><li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum.">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300, G170-G180 (2011).</li><li>Feng, B., La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Irritable+bowel+syndrome:+methods,+mechanisms,+and+pathophysiology.+Neural+and+neuro-immune+mechanisms+of+visceral+hypersensitivity+in+irritable+bowel+syndrome.">Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome.</a> Am J Physiol Gastrointest Liver Physiol. 302, G1085-G1098 (2012).</li><li>Feng, B., 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=Long-term+sensitization+of+mechanosensitive+and+-insensitive+afferents+in+mice+with+persistent+colorectal+hypersensitivity.">Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 302, G676-G683 (2012).</li><li>Feng, B., 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=Altered+colorectal+afferent+function+associated+with+TNBS-induced+visceral+hypersensitivity+in+mice.">Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.</a> Am J Physiol Gastrointest Liver Physiol. 303, G817-G824 (2012).</li><li>Feng, B., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+roles+of+stretch-sensitive+pelvic+nerve+afferents+innervating+mouse+distal+colon+and+rectum.">Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum.</a> Am J Physiol Gastrointest Liver Physiol. 298, G402-G409 (2010).</li><li>Feng, B., 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=Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents.">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33, 9831-9839 (2013).</li><li>Jolliffe, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principal component analysis. , (2002).</li><li>La, J. H., Feng, B., Schwartz, E. S., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+hypertonicity+and+acidity+modulate+colorectal+afferents+and+induce+persistent+visceral+hypersensitivity.">Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 303, G802-G809 (2012).</li><li>Christianson, J. A., 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=plasticity+and+modulation+of+visceral+afferents.">plasticity and modulation of visceral afferents.</a> Brain Research Reviews. 60, 171-186 (2009).</li><li>Kiyatkin, M. E., Feng, B., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization.">Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization.</a> Am J Physiol Gastrointest Liver Physiol. 305, G638-G648 (2013).</li><li>Brumovsky, P. R., Feng, B., Xu, L., McCarthy, C. J., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystitis+increases+colorectal+afferent+sensitivity+in+the+mouse.">Cystitis increases colorectal afferent sensitivity in the mouse.</a> Am J Physiol Gastrointest Liver Physiol. 297, G1250-G1258 (2009).</li><li>Shinoda, M., Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+and+central+P2X+receptor+contributions+to+colon+mechanosensitivity+and+hypersensitivity+in+the+mouse.">Peripheral and central P2X receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse.</a> Gastroenterology. 137, 2096-2104 (2009).</li><li>Tanaka, T., Shinoda, M., Feng, B., Albers, K. M., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+&#945;-3+in+colorectal+afferents.">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor &#945;-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li></ol>

The authors have nothing to disclose.

여기에 설명 된 체외 대장 직장 - 신경 준비는 잘 내장 감각 신경에 다른 비 기능적 접근 방법 (예를 들면, 세포 분자, 조직 학적 연구)를 보완 개별 대장 구 심성 신경의 신경 인코딩 기능 (연구 할 수있는 강력한 방법이 될 입증되었습니다 ) 자세한 내용은 리뷰 27을 참조하십시오. 통각 및 장기 대장의 과민 반응에 기여하는 신경 메커니즘은 밝혀지지하고 약리 조작은 내장 통...

통증 과민 명백 병인 학적 원인이나 조직 손상의 유무에 존재 과민성 대장 증후군 (IBS)을 포함하여 위장 기능 장애로부터 고통받는 환자의 주된 불만이다. 예를 들어, IBS 환자 (복부의 촉진에 즉, 부드러움) 정상적인 장 기능 동안 직장 풍선 팽창 증가 감도 높게 응답을 포함하는 과민 반응,뿐만 아니라 체세포 추천의 과민 반응을 나타내는 일. 의 개선 이해, 대장 구 심성을 대상으로하는 IBS 환자에서 (구 아닐 레이트 사이 클라-C의 효능 linaclotide 4-6 경구 섭취 예를 들어, 국소 마취제 2,3의 내 직장 점안을) 통증과 과민을 완화에 효과적 일 수 있기 때문에 대장 직장의 구 심성 신경 분포가 중요합니다. 대장의 구 심성을 포함하여 내장 구 심성,/ nutrient- 화학적 및 열적 양상 (예를 들면, 7-9)에 응답 할 수있다. 그러나, 기계적 자극 (즉, mechanosensitive 구 심성)에 응답 내장 구 심성이되었습니다 기계적 자극 (예를 들어, 내강 팽만, 스트레칭) 일반적으로 불편과 고통 10-16을 포함하여 의식이 감각에 야기 할 것들이기 때문에 가장 철저하게 연구했다. 또한, 내장은 일반적으로, 기계적으로 문자를 구분 구 심성 (실종자)에 의해 신경 지배를 자동 또는 자고 통각 수용기 (17)이라고합니다. 정상적인 생리 조건에서, 실종자는 기계적인 자극에 응답하거나 매우 높은 응답이 18 임계 값이 있지만 활성화 및 병태 생리 학적 조건에서 mechanosensitivity를 획득 과민 반응에 공헌 할 수 없습니다. 여기에 설명 된 시험 관내 제조 방법 및 프로토콜을 이용하여, 우리는 개발 바다 전기 자극 전략을 채용<html> 대장 직장 (19) 모두 mechanosensitive 및 MIA 엔딩의 편견 식별을 가능하게 수용 엔딩에 대한 RCH. 대장 신경 분포는 요추 내장의 (LSN)과 골반 신경 (PN) 경로에서 파생 다섯 mechanosensitive 클래스 (장막, 점막, 근육, 근육 점막, 장간막) 한 MIA 클래스 (20)으로 분류 할 수있다 대장 구 심성을 포함한다. 이 체외 준비를 사용하여, 우리는 대장 실종자 인수 mechanosensitivity는 LSN 경로에 PN 경로에서 실종자의 71 %와 실종자의 23 %를 증감 염증 수프 (IS), 그들의 수용 필드의 짧은 노출 다음 (민감) 발견 19. 우리는 또한 오래 지속되는 행동 내장 과민 반응의 맥락에서 실종자의 장기 감작 (최대 28 일) 문서화 (모산 (21) 또는 2,4,6- 트리니트로 산으로 결장 내 치료를받은 쥐에서, 즉 (TNBS) 22) . mechanosensitive 구 심성 신경, 근육과 근육 점막 구 심성 중 jove_content은 &quot;&gt; (즉, 스트레칭 구분) 및 유해 대장 팽만 (23, 24)의 인코딩을 보조하다 카나비노이드 대장 직장의 원주 스트레치를 인코딩 할 수있는 유일한 클래스입니다. 컴퓨터 제어를 사용하여 동력 액츄에이터, 우리는 평면화 대장 암 조직의 원주 방향의 표준, 균질하고 재현성 램핑 신축성을인가하고 상기 스트레치 성 구 심성을 낮은 임계치와 높은 임계치 (23)를 분류. 또, 신축성 감작를 시간 경과에 따라 결장 내 모산 21 TNBS (22) 치료 후 과민 구 심성은 내장 통증 및 과민 반응의 스트레칭에 민감한 대장 구 심성의 역할을 제안, 행동 내장 과민 반응의 시작, 지속, 및 / 또는 복구에 해당합니다.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

이 비디오 상세히 마우스 대장 직장 - 신경 제제를 사용하여 시험 관내 전기 생리 단일 섬유 기록 프로토콜을 설명한다. 접근 방식은 편견 식별 및 개별 대장 구 심성의 기능을 특성화 할 수 있습니다. 하나 또는 몇 심성에서 발생하는 전파 활동 전위 (APS)의 세포 외 녹음 (즉, 단일 섬유) 대장 직장에서 수용 필드 (RI의)을 조롱 신경 섬유 다발로 만들어진다. 대장 직장은 골반 (PN) 또는 요추 내장의 (LSN) 신경 부착 길이 방향으로 열 중 하나를 제거한다. 조직 플랫 피닝 및 산소화 크렙스 용액으로 관류, 기록 챔버에 배치된다. 초점 전기 자극은 기능적으로 다섯 mechanosensi에 구 심성을 분류 (프로빙 점막 쓰다듬어 원주 스트레칭을 무디게) 더 세 가지 기계적인 자극에 의​​해 테스트 대장 구 심성 엔딩을 찾는 데 사용됩니다적인 클래스를 제공합니다. 이러한 기계적 자극 없음에 응답 엔딩 기계적으로 문자를 구분 구 심성 (실종자)으로 분류됩니다. 화학 물질에 대한 RI의 지역화 된 노출에 의해 mechanosensitive 및 실종자가 과민 반응 (즉, 향상된 응답 감소 임계 값 및 / 또는 mechanosensitivity의 취득)에 대한 평가 될 수는 모두 (예를 들어, 염증 수프 (IS), 캡사이신, 아데노신 삼인산 (ATP)). 우리는 장비와 대장 직장 - 신경 기록 준비, 부착 PN 또는 LSN, 신경 다발에서 대장 직장, 단일 섬유 녹음에 RI의 식별과 대장 직장의 수확 및 RF에 화학 물질의 지역화 된 응용 프로그램을 설명합니다. 또한, 표준화 된 제조 기계적 자극의 적용 과제도 논의된다.

설치는 그림 1에 예시되어있다. 그것은 실리콘 늘어선 목욕 구획 및 연속 미네랄 오일 충전 실에 첨부 된 신경의 대장 직장을 수용하는 맞춤형 조직 챔버를 포함한다. 두 챔버는 구획 CNC 기계에 의해 아크릴 수지의 고체 블록으로부터 기계 가공 하였다 두 구획의 바닥이어서 결장 조직 다운 핀을 쉽게 허용하도록 기업 실리콘 늘어서 하였다. 조롱 신경 다발에서 세포 외 AP는 공통 모드...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

마우스 대장 수입 성의 엔딩의 생체 기능 특성에

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

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Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K 조회수.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

비디오: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

의식 억제 마우스에 인슈린 과다 - euglycemic 클램프

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

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의학

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

파킨슨 병 분야에서 Mitochondrial Phenotypes을 모니터링하기위한 기본 인간 섬유 아세포의 사용

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

기본 마우스 대장 종양의 체외 Organoid의 문화

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

마우스 모델의 설립 및 요로 감염 및 CAUTI의 특성

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

심 초음파 이미징에 의해 가로 대동맥 수축의 마우스 모델에서 심장 형태 학적 평가 및 기능 변경

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

종<em&gt; 생체</em&gt;은 Cerebrovasculature의 이미징 : CNS 질환에 관련성

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

준비 및<em&gt; 체외</em&gt; 자화 특성의 내피 세포를 미르 변성

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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 ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.