저자는 친절 하 게 제공 하 고 유전자 변형 쥐 리 첸 조직학 지원에 대 한 박사 데이비드 로우를 감사 하 고 싶습니다.
이 간행물에 보고 된 연구는 국립 연구소의 치과 Craniofacial 연구의 보너스 번호 K08DE025914에서 건강의 국가 학회 및 미국 협회의 교정 기초 Sumit Yadav에 의해 지원 되었다.

<ol>
	<li>LeResche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Temporomandibular+Disorders:+Implications+for+the+Investigation+of+Etiologic+Factors.">Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors.</a> Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).</li><li>Chen, J., 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+functional+loading+causes+differential+effects+in+the+subchondral+bone+and+condylar+cartilage+in+the+temporomandibular+joint+from+young+mice.">Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice.</a> Osteoarthr Cartil. 17 (3), 354-361 (2009).</li><li>Pirttiniemi, P., Kantomaa, T., Sorsa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decreased+loading+on+the+metabolic+activity+of+the+mandibular+condylar+cartilage+in+the+rat.">Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat.</a> Eur J Orthod. 26 (1), 1-5 (2004).</li><li>Chavan, S. J., Bhad, W. A., Doshi, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+temporomandibular+joint+changes+in+Twin+Block+and+Bionator+appliance+therapy:+a+magnetic+resonance+imaging+study.">Comparison of temporomandibular joint changes in Twin Block and Bionator appliance therapy: a magnetic resonance imaging study.</a> Prog Orthod. 15 (57), (2014).</li><li>Dutra, E. H., 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=Cellular+and+Matrix+Response+of+the+Mandibular+Condylar+Cartilage+to+Botulinum+Toxin.">Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.</a> PLoS ONE. 11 (10), 0164599 (2016).</li><li>Benjamin, M., Ralphs, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fibrocartilage+cells.">Biology of fibrocartilage cells.</a> Int Rev Cytol. 233, 1-45 (2004).</li><li>Shen, G., Darendeliler, 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=The+Adaptive+Remodeling+of+Condylar+Cartilage-+A+Transition+from+Chondrogenesis+to+Osteogenesis.">The Adaptive Remodeling of Condylar Cartilage- A Transition from Chondrogenesis to Osteogenesis.</a> J Dent Res. 84 (8), 691-699 (2005).</li><li>Utreja, 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=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Kaul, R., 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=The+Effect+of+Altered+Loading+on+Mandibular+Condylar+Cartilage.">The Effect of Altered Loading on Mandibular Condylar Cartilage.</a> PLoS ONE. 11 (7), 0160121 (2016).</li><li>Dyment, N. 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=High-Throughput,+Multi-Image+Cryohistology+of+Mineralized+Tissues.">High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.</a> J Vis Exp. , e54468 (2016).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch Histol Cytol. 66 (2), 123-143 (2003).</li><li>Hayman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+(TRAP)+and+the+osteoclast/immune+cell+dichotomy.">Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.</a> Autoimmunity. 41 (3), 218-223 (2008).</li></ol>

저자 아무 경쟁 금융 관심사 있다.

이 원고는 형태학 측정 및 세포 분석 murine 악의 condyles mandibles의 방법을 설명합니다. 방사선 형태학 측정 작은 실험 동물에서 다른 뼈 분석을 사용할 수 있습니다. 또한, 세포 분석 (셀 정량화 및 연골 거리 매핑) 설치류 악의 관절 돌기에 국한 되지 않습니다 하지만 수많은 조직의 조직학 섹션 척도를 사용할 수 있습니다.
유전자 변형 마우스 모델 표현 형광 기자는 유전자 발현에 변화를 시각적으로 이해 우수한 도구입니다. Transgenes는 고객 센터의 prehypertrophic 및 hypertrophic 지역에서 그렇게 표현 됩니다 이후 더블 콜라겐 형광 기자 마우스 모델 (Col2a1XCol10a1)이이 보고서에 사용 되는 고객 센터의 연구에 특히 적합 했다 그 콜라겐 배포 평가 될 수 있습니다. 연구원에 형광 기자를 표현 하는 유전자 변형 마우스 모델에 액세스할 수 없는 경우에 조직학 단백질 표정 분석, 면역 형광 검사, 등의 다른 방법은 사용할 수 있습니다.
형태학 측정 캐비닛 방사선 사진 이미지를 복용 하기 전에 중요 한 단계는 mandible 또는 분석 하기 위해 뼈의 모든 부드러운 조직 제거 것입니다. 과도 한 근육 이나 뼈에 연결 하는 부드러운 조직 구조의 실제 측정 마스크 수 있습니다. 그것은 캐비닛 방사선 사진 시스템의 제한 사항에 유의 해야 합니다. 모든 x 선 처럼 그것은 3 차원 구조의 2 차원 표현 하 고 구조 및 유물의 중복을 신중 하 게 해석 되어야 합니다. 또한, 방사선 캐비닛에 뼈의 위치 일치 해야한다.
긍정적인 픽셀의 수를 계산에 의하여 세포 정량화 셀-풍부한 지역, 고객 센터 등에서 조직학 정량화는 효율적인 방법 이지만이 방법의 한계는 그것은 세포의 정확한 수를 제공 하지 않습니다. 또한,만 픽셀 선택 단정, 그것 것이 좋습니다 같은 "샘플링" 픽셀을 사용 하 여 다른 이미지를 분석할 때 다른 농도의 픽셀의 정량화를 피하기 위해의 모든 이미지를 계량 하기.
조직학 단면도 측정 하는 경우 조언의 일반적인 조각 여러 직렬 섹션의 각 샘플을 분석 하는 (이 원고 3 직렬 섹션 분석 되었다 각 관절 돌기에 대 한), 이후 유사 각 샘플 내에서 일반적으로 관찰 된다.
우리의 실험에 언급 된 방법론 간단 하 고 사용 하기 쉬운, 그리고 어떤 기자에 마우스 사용 되는 어떤 광물 화 된 조직 연구에 사용할 수 있습니다. 기존의 방법론으로 셀을 셀의 공동 지역화를 시각화 하 여 트랩 (Col2a1 또는 Col10a1) 또는 듀 Col1a1 (Col2a1)에 대 한 스테인드 시각화 가능 하다.

TMJ craniofacial 지역에 위치한 독특한 로드 베어링 조인트 이며 fibrocartilage의 형성 된다. MCC는 TMJ의 말하기와 masticating, unhindered 턱 운동을 하 여 관절 기능을 위해 필수적 이지만 일반적으로 관절염1를 포함 한 퇴행 성 질환에 의해 영향을 받습니다. TMJ 고객 센터2,3,,45의 구성 요소 구조와 세포 변화를 선도 외부 자극 및 로드 변경에 적응 하는 능력이 있다. MCC의 부하 베어링 속성 그것의 성분, 물, 콜라겐 네트워크를 포함 하 고 밀도가 proteoglycans 포장 사이 상호 작용에 의해 설명 될 수 있다. 고객 센터는 콜라겐과 비 콜라겐 단백질의 종류를 나타내는 4 개의 고유 셀룰러 영역: 1) 표면 또는 관절 영역; 요구; 로드에 응답 하는 2) 증식 영역, 미 분화 간 엽 세포의 구성 성숙한 chondrocytes 표현 콜라겐 유형 2; 3) prehypertrophic 영역 구성 그리고 4)는 hypertrophic 영역, 콜라겐 표현 hypertrophic chondrocytes 10 다를 입력 하는 지역 및 석 회화를 받 다. 비 mineralized 지역 proteoglycans 압축 력을6에 저항을 제공 하는 풍부 하다.
Hypertrophic 영역에서 골 chondrogenesis에서 전환 발생 하면 고객 센터의 지속적인 강화는 악의 관절 돌기7의 마찰의 뼈의 강력한 미네랄 구조를 보장. Unmineralized 및 광물 화 된 지역에서 세포질 변화는 궁극적으로 악의 관절 돌기에 mandible 형태학 및 구조 변화에 리드. 고객 센터의 모든 세포질 영역 및 마찰의 부분의 강화 작용의 항상성 유지, 부하 베어링 용량, 건강과 TMJ의 무결성을 필수적입니다.
여러 콜라겐 유전자 변형 마우스 모델 (Utreja 그 외 여러분에 의해와 같이) 8 모든 transgenes는 MCC에서 표현 됩니다 때문에 콜라겐 식의 변화를 이해를 사용 하 여 훌륭한 도구입니다. 깊이 조직학 평가, 대 한 조직학 얼룩 매트릭스 증 착, 강화 작용, 세포 증식, apoptosis, 뿐만 아니라 고객 센터의 다른 세포 층에 단백질 식 공부 하는 데 사용 됩니다.
이 원고, 조직학 및 형태학 분석 쥐의 악의 관절 돌기의 MCC 및 마찰의 뼈에서 세포 및 구조 변화를 평가 하는 데 사용 됩니다. 또한, 세포 정량화 방법, 형광 조직학 이미지를 분석 하 고 가벼운 현미경 슬라이드를 매핑 설명 되어 있습니다. 고객 센터 및 마찰의 뼈9세포 형태학 변화 원인, 메서드를 로드 압축 정적 TMJ 또한 우리의 방법을 확인 하기 위해 그림입니다.
형태학 및 악의 관절 돌기에 설치류의 mandible 조직학 변화 결정 또는 endochondral 나오고의 다른 지역 및 추가 광물 화 된 직물의 형태 분석 여기에 설명 된 방법은 사용할 수 있습니다.

<table width="896">
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			<td style="width:219px;">Faxitron X-Ray LLC&#160;</td>
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			<td style="width:219px;">MedCalc Software</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">Shandon Cryomatrix embedding resin</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">6769006</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">Manual microscope Axio Imager Z1</td>
			<td style="width:219px;">Carl Zeiss</td>
			<td style="width:119px;">208562</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">yellow fluorescent protein filter&#160; - EYFP</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49003</td>
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			<td height="21" style="width:392px;height:21px;">cyan fluorescent protein filter - ECFP</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49001</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">red fluoresecent protein filter - Cy5</td>
			<td style="width:219px;">Chroma Technology Corp</td>
			<td style="width:119px;">49009</td>
			<td></td>
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			<td height="21" style="width:392px;height:21px;">sodium acetate anhydrous</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">S2889</td>
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			<td height="23" style="width:392px;height:23px;">sodium L-tartrate dibasic dihydrate&#160;</td>
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			<td style="width:119px;">228729</td>
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			<td height="21" style="width:392px;height:21px;">sodium nitrite&#160;</td>
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			<td height="21" style="width:392px;height:21px;">ELF97 substrate</td>
			<td style="width:219px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">E6600</td>
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			<td height="21" style="height:21px;">ClickiT EdU Alexa Fluor 594 HCS kit</td>
			<td style="width:219px;">Life Technologies</td>
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			<td height="21" style="width:392px;height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
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			<td>S3264</td>
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			<td height="25" style="width:392px;height:25px;">Adobe Photoshop&#160;</td>
			<td style="width:219px;">Adobe Systems Incorporated</td>
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			<td style="width:219px;">Research Products International</td>
			<td style="width:119px;">P32080-100T</td>
			<td></td>
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			<td height="21" style="height:21px;">CNA Beta III Nickel-Free Archwire</td>
			<td>Ortho Organizers, Inc.</td>
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			<td height="21" style="height:21px;">GraphPad Prism&#160;</td>
			<td>GraphPad Software, Inc.</td>
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</table>

a morphometric and cellular analysis method for the murine mandibular condyle

턱 관절 (TMJ) 외부 자극에 적응 하는 능력 있고 변경 로드 condyles의 위치 뿐만 아니라 악의 condylar 연골 (MCC)의 구조와 세포 구성 요소에 영향을 미칠 수 있습니다. 이 원고에는 이러한 변경 및 마우스 (즉, 압축 정적 TMJ 로드)에서 TMJ의 로드를 변경 하는 방법을 분석 하는 방법을 설명 합니다. 여기에 그림 구조 평가 Digimizer 소프트웨어를 사용 하 고 작은 뼈의 방사선에서 수행 되는 간단한 형태학 접근 이다. 또한, 휴대의 분석 선도 콜라겐 식, 뼈, 세포 분열, 변경에 변화와 proteoglycan 메일 고객 센터에서 설명 하는. 이러한 변화는 긍정적인 형광 픽셀을 사용 하 여 계산 하 여 조직학 단면도-의 정량화 이미지 소프트웨어 및 측정 거리 매핑 및 스테인드 영역 Digimizer-는 또한 설명 했다. 여기에 표시 된 메서드는 murine TMJ에 국한 되지 않습니다 하지만 추가 뼈 작은 실험 동물의 및 endochondral 나오고의 다른 지역에서 사용 될 수 있습니다.

기술 통계는 형태학 측정 (악의 길이, condylar 길이, condylar 폭) 및 조직학 분석의 분포를 검사를 수행 했다. 결과 로드 그룹 (즉, 마우스 베타 티타늄 스프링 압축 선적을 복종)와 (즉, 일치 하는 모든 프로시저에는 영향을 받지 않은 통제 쥐) 컨트롤 그룹 사이 비교 되었다. 수단 간의 통계적으로 유의 한 차이 짝이 없는 t-검정, 및 p-값에 의해 결정 되었다 < 0.05 통계적으로 중요 한 것으로 간주 되었다.
형태학 측정, 단계 4 및 그림 3에서 설명한 대로 로드의 mandibles 및 제어 그룹에서 수행 했다. 크게 증가 mandible 길이 제시 로드 그룹 (로드: 16.62 ± 0.23 m m 제어 대: 16.21 ± 0.2 m m; p < 0.05) 및 관절 돌기 머리 길이 (로드: 4.6 m m, SD 제어 대 0.08 m m: 4.4 m m; SD 0.06; p < 0.05) 컨트롤 그룹에 비해. 그럼에도 불구 하 고, 그룹 사이 관절 돌기 폭에 큰 차이가 없었다 (로드: 3.06 ± 0.12 m m 제어 대: 2.9 ± 0.11; p = 0.09).
크게 공개 8 단계에서 설명 하는 방법을 사용 하 여 콜라겐 배포의 정량화 Col10a1 컨트롤에 비해 로드 그룹의 condyles MCC에 표현의 증가 (로드: 컨트롤에 비해 21% ± 4.46%: 7.50% ± 2.03%; p < 0.005; 그림 8 A와 D)입니다. 다른 한편으로, 아니 통계적으로 로드 사이 Col2a1 식에 차이 제어 그룹 (로드: 4.85% ± 1.95% 제어 대: 2.92% ± 1.89%; p = 0.13; 그림 8 B와 E)입니다. 또한, 트랩 활동의 분석 로드 된 마우스의 condyles의 마찰의 지역에서 증가 트랩-긍정적인 지역 보였다 (로드: 5.28% ± 1.45% 제어 대: 2.41% ± 1.39%; p < 0.05; 그림 8 C와 F)입니다. 컨트롤 마우스에 비해 로드 그룹의 고객 센터에서 증가 듀 긍정적인 셀에 표시 된 대로 증가 세포 증식, 마찬가지로, 관찰 (로드: 6.23% ± 1.89% 제어 대: 1.90% ± 1.03%; p < 0.05; 그림 9 A와 C)입니다.
로드의 고객 센터에 통제 쥐 Proteoglycan 배포 단계 9 및 그림 7에 설명 된 대로 톨루이 블루 스테인드 지역 및 거리 지도, 평가 하 여 정량 했다. 우리는 컨트롤에 비해 로드 그룹의 condyles의 고객 센터에 있는 크게 증가 거리 매핑 (로드: 컨트롤 대 210.22 µ m ± 4.11 µ m: 187.36 µ m ± 8.64 µ m; p < 0.005; 그림 9 B와 D)입니다. 그러나, proteoglycan 스테인드 영역 이었다는 로드 사이 통계적으로 다른 그룹을 제어 하 고 (로드: 203,897.93 µ m2 ± 10,171.00 µ m2 제어 대: 202,875.09 µ m2 ± 33,419.09 µ m2; p = 0.94; 그림 9 B와 E)
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55998/55998fig8.jpg"> 그림 8 : 대표 결과: (A) condyles 제어 및 로드 된 마우스에서의 고객 센터의 화살 단원의 Col10a1 긍정적인 세포. 증가 숫자 Col10a1 긍정적인 셀 로드 그룹 (D)에 있다. (B) Col2a1-긍정적인 세포 제어 및 로드 쥐에. 그룹 (E)사이 Col2a1-긍정적인 세포에 차이가 있다. ((C)) 트랩 제어 및 로드 된 마우스의 condyles에 얼룩. 제어 (F)에 비해 로드 그룹에서 트랩 활동 증가 히스토그램 (D-F) 대표 n 수단 ± SD = 4 그룹 당. * * 중요 한 차이 제어 및 로드 그룹 (p < 0.005). 눈금 막대 = 200µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig8large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55998/55998fig9.jpg"> 그림 9:대표 결과: (A) 듀 제어 및 로드 된 마우스의 condyles에 얼룩. 로드 된 그룹에 있는 증가 세포 증식으로 증가 듀-긍정적인 표현 (C)세포. (B) 톨루이 블루는 제어 및 로드 된 마우스의 condyles에 얼룩. 증가 거리 매핑 실험 그룹 (D)에서 같이 로드 된 마우스에서 연골 두께 증가. 아무 차이 proteoglycan 스테인드 영역에서 제어 및 로드 그룹 ((E)). 히스토그램 (C-E) 대표 n 수단 ± SD = 4 그룹 당. * * 중요 한 차이 제어 및 로드 그룹 (p < 0.005). 눈금 막대 = 200 µ m. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig9large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

Watch this Scientific Journal Video about A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle at JoVE.com

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

이 원고 형태학 및 설치류의 악의 관절 돌기 내의 세포질 변화를 분석 하기 위한 메서드를 제공 합니다.

형태학 및 Murine 악의 관절 돌기 세포 분석 방법

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

a-morphometric-cellular-analysis-method-for-murine-mandibular

Nanyang Technological University

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JoVE Journal

Biology

8.2K 조회수.  University of Connecticut Health Center. 이 원고 형태학 및 설치류의 악의 관절 돌기 내의 세포질 변화를 분석 하기 위한 메서드를 제공 합니다.

비디오: A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

쥐에서 도보의 질적 분석 방법으로 Rotorod의 사용

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

연구

생물학

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murine 신장 이식 절차

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Neurotoxicity 높은 컨텐츠 분석의 연결 및 Astrocyte 공동 문화 분석

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

세계 S. cerevisiae의에서 organelle 형태를 연구하기 위해 높은 처리량 방법

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

높은 처리량 Zebrafish의 정자 Cryopreservation에 대한 방법과 체외에서 수정

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

인간 사전 mRNA에 매핑 Ribonucleoproteins에 대한 신속한 하이 스루풋 방법 (RNPs)

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

셀룰러 Lysates에서 Multiprotein의 단지의 분석을 위해 블루 기본 Polyacrylamide 젤 전기 영동 (BN - PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

두개골 Mesenchyme의 셀룰러 행동을 평가하기위한 Explant 분석

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

휴대 통신 분석을위한 단일 세포 미세 주입

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...