NIH (1 K08 DK080221 - 01)와 밴더빌트 기관 개발 기금에서 제공하는 자금.

1 부 : 상해 - 복구 대장염 모델 <ol><li> 기준 무게 DSS 치료를 시작하기 전에 각각의 마우스에 대한 취득해야합니다. </li><li> 3 % (W / V) Dextran의 황산 나트륨 소금물에 솔루션과 0.45 μm의의 셀룰로스 아세테이트 필터와 필터를 확인하십시오. </li><li> 나흘 동안 3 % DSS 솔루션 마우스 케이지에서 식수를 교체합니다. 쥐를 물의 다른 소스 (자동 급수 시스템에 위치 즉, 제외 팁)에 액세스할 수 없습니다. </li><li> 일 4 층에 일부 colonic 상피 복구를 허용, 추가로 3 일 동안 물을 DSS 솔루션을 대체합니다. 생쥐는 대장염의 체계적 결과를 계량하기 위해 일 넷 무게해야합니다. 체중 감소는 심각한 부상 일반적입니다. </li><li> 일 7, 무게 및 생쥐를 희생. 마우스가 isoflurane, 또는 institutionally 상대방의 inhalational 과용으로 euthanized 수 IACUC이 방법을 승인했다. </li></ol> 2 부 : 검시와 콜론의 착취 <ol><li> 복부에 가리지 액세스를 보장하기 위해 마우스 및 보안 다리 복부 측면을 폭로. 70 % 에탄올로 완전히 복부가 젖었어. </li><li> 티슈 포셉와 복부의 정중선을 파악하고 tenting 피부를 따라서 위쪽으로 연장. 따라서 복부 내용을 노출 복부를 절개하는 미세 밀고 가위를 사용합니다. 정중선에서 xyphoid 과정의 끝에으로 절개를 확장하고 양자 늑골 여백의 하부 측면 따라 연장. </li><li> 소장, 맹장 및 결장을 식별합니다. 신중하게 해부하다 / 주변 장간막에서 콜론을 괴롭혀. 말초 콜론과 직장을 무료로 골반 깊은 콜론을 가로로 쪼개다. 근위 대장을 무료로 colonocecal 여백에 콜론을 가로로 쪼개다. 케어는 천공이 발생하는 경우에는 콜론이 문제가 될 청소로 콜론을 손상하지 않도록이 과정에서 촬영해야합니다. </li><li> 20G 먹이를 바늘과 10 ML의 주사기를 사용 intubate 및 eluate가 대변을 완전히 명확 때까지 얼음처럼 차가운 PBS로 콜론을 플러시. </li><li> 해부 현미경으로 매크로 분석 원하는 경우이 시점에서, 콜론은 &quot;소시지&quot;으로 해결할 수 있습니다. histologic 분석은 4 &quot;급성 대장염의 Histological 분석을 위해 스위스 롤스 만들기&quot;부분으로 진행보다 원하는 경우. </li></ol> 3 부 : 전체 대장의 매크로 해석에 대해 &quot;소시지&quot;으로 처리 <ol><li> 그것은 콜론의 올바른 방향을 유지하는 것이 중요하다, 따라서, 운영자에 가장 가까운 콜론의 말초 끝을 유지. 비 흡수성 봉합사 두 조각 (SP116 1.5 통계 꼰 실크), 길이 한 약 1 인치, 다른 2인치 컷. </li><li> 여전히 좋은 도장을 유지하면서 가능한 한 여백에 가까운 말초 끝을 묶을 봉합의 2 인치 조각을 사용합니다. </li><li> 10 % 5 ML을 포함 20G 먹이 바늘은 결장의 근위 끝에 포르말린 인산염을 버퍼 플레이스. 대충 먹이 바늘의 전구 즉시 근위 봉합의 나머지 부분을 묶어. 먹이 바늘의 전구에 단단히 콜론을 파악하고 콜론이 확장 때까지 포르말린 달이다. 당신은 따라서 &quot;소시지&quot;으로 주입 확장 콜론을두고, 먹이를 바늘을 철회로 봉합에 매듭을 조인다. </li><li> 10 % 포르말린 인산염을 버퍼와 튜브에 콜론을 장소와 15 ML 원뿔 튜브를 입력합니다. 24 시간 동안 수정. </li><li> 포르말린을 붓고 및 추가 24에 대한 70 % EtOH로 바꿉니다. (콜론은 상온에서 무한정 70 % EtOH에 저장할 수 있습니다.) </li><li> 원뿔 관에서 콜론을 제거하고 대장을 손상하지 않도록주의하고, 메스 각 끝에있는 문자열을 잘라. 말초 끝이 문자열의 가장 긴 부분을했다는 것을 기억 하여라. 그것은 긴 시트를 형성 있도록 결장의 근위 끝에 말초에서 길이 방향 컷. 이 시점에서 콜론은 해부 현미경으로 볼 수 있습니다. </li></ol> 4 부 : 급성 대장염의 Histological 분석을 위해 스위스 롤스 만들기 <ol><li> 그것은 콜론의 올바른 방향을 유지하는 것이 중요하다, 따라서, 운영자에 가장 가까운 콜론의 말초 끝을 유지. </li><li> 콜론의 길이를 측정합니다. 이 통계는 부상의 심각도의 또 다른 지표이다. 대장염은 부종을 증가 시키며, 전체 대장의 길이를 단축. </li><li> 세부적으로 밀고 가위를 사용하여 콜론의 근위 끝에 말초에서 길이 방향 절개. 따라서 평면 시트로 콜론을 표시 절개 중 가장자리를 파악하고 distally -&gt; proximally 길을 작업을 옆으로 열어 좋은 밀고 집게를 사용하십시오. </li><li> 콜론 롤링 것은 포셉 한 켤레, 두 손으로 기술을 필요로합니다. 포셉와 말초 여백 중 하나 가장자리를 파악. 순차적으로 근위 여백을 진행 포셉를 회전하고 공개하여 콜론을 롤로 진행합니다. 따라서 3 차원 또는 &quot;스위스 롤&quot;로 나선형을 생성.압연 양식을 유지하려면, 집게로 단단히 롤을 파악하고 27G1 / 2 바늘로 그것을 가로로 쪼개다. 그것이 출구 롤로 바늘을 절곡하여 바늘을 고정합니다. </li><li> 10 % 병에 적절하게 표시 조직 카세트과 장소에 롤 장소는 조직 고정을 위해 24 시간을위한 상온에서 포르말린 인산염을 버퍼. </li><li> 포르말린을 붓고 및 추가 24에 대한 70 % EtOH로 바꿉니다. (콜론은 상온에서 무한정 70 % EtOH에 저장할 수 있습니다.) </li><li> 샘플가 해결되면, 그것은 파라핀 - 임베디드, sectioned 및 histologic 분석을위한 슬라이드에 장착할 수 있습니다. </li></ol> 제 5 부 : 대표 결과 급성 대장염에 대한 DSS 모델은 연구자가 얻을 수정, 그 모델 급성 대장염 콜론을 분석할 수 있습니다. (그림 1) 정상적으로 압연하는 경우 스위스 롤을 절단하고 장착하면, 그것은 전체 결장의 대표 슬라이스를 형성한다. 장착 롤은 말초 (내부) 끝에서 근위 (외부) 끝 (그림 2), 콜론 손상의 범위를 결정하기 위해 H &amp; E 물들일 수 있습니다. Immunohistochemistry 또한 염증성 infiltrates를 파악하고 계량하는 스위스 롤을 수행할 수 있습니다. 소시지 방법이 올바르게 수행되고있다면, 고정 콜론이 확대되고 전체 점막 표면이 쉽게 해부 현미경 (그림 3)에서 조작하고 볼 수 있습니다. <a href="https://www.jove.com/files/ftp_upload/1652/1652fig1.jpg"><img src="/files/ftp_upload/1652/1652fig1tmb.jpg" alt="그림 1" border="0" /></a> 그림 1. 적절한 실행 &quot;스위스 롤&quot;. (A) 콜론은 근위 끝에 말초에서 굴러 바늘로 가로와 바늘의 끝을 굽힘에 의해 보안됩니다. 그것은 다음 고정을위한 조직 카세트에 배치됩니다. (B) H &amp; E는 DSS (D = 말초 콜론 P = 근위 대장)를 취급 마우스 콜론 만든 스위스 롤 5 μm의 부분을 얼룩. <a href="https://www.jove.com/files/ftp_upload/1652/1652fig2.jpg"><img src="/files/ftp_upload/1652/1652fig2tmb.jpg" alt="그림 2" border="0" /></a> 그림 2. DSS 취급 콜론은 급성 대장염의 흔적을 보여줍니다. 염증과 토굴 손상이 물 처리 제어에 비해 DSS - 처리 대장에 분명 있습니다. <a href="https://www.jove.com/files/ftp_upload/1652/1652fig3.jpg"><img src="/files/ftp_upload/1652/1652fig3tmb.jpg" alt="그림 3" border="0" /></a> 그림 3. &quot;소시지&quot;의 예. 소시지는 포르말린과 함께 주입하고 완전하게 확대했습니다. 약간의 각도는 결장의 자연 굴절률이 존재 보조 것입니다. 열린 소시지가 평평 거짓말을한다.

<ol>
	<li>Okayasu, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Martinez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Mtgr1+sensitizes+the+colonic+epithelium+to+dextran+sodium+sulfate-induced+colitis.">Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis.</a> Gastroenterology. 131 (2), 579-588 (2006).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Mahler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+susceptibility+of+inbred+mouse+strains+to+dextran+sulfate+sodium-induced+colitis.">Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis.</a> Am J Physiol. 274, G544-551 (1998).</li><li>Aharoni, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+therapeutic+effect+of+glatiramer+acetate+on+several+murine+models+of+inflammatory+bowel+disease.">Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease.</a> J Pharmacol Exp Ther. 318 (1), 68-78 (2006).</li><li>Hahm, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+transforming+growth+factor+beta+signalling+in+the+intestine+contributes+to+tissue+injury+in+inflammatory+bowel+disease.">Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease.</a> Gut. 49 (2), 190-198 (2001).</li><li>Green, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+modulation+of+Escherichia+coli+O157:H7+adherence+to+the+colonic+mucosa.">Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.</a> Am J Physiol Gastrointest Liver Physiol. 287 (6), G1238-1246 (2004).</li><li>Dieleman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+experimental+colitis+induced+by+dextran+sulphate+sodium+(DSS)+is+characterized+by+Th1+and+Th2+cytokines.">Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines.</a> Clin Exp Immunol. 114 (3), 385-391 (1998).</li></ol>

동물 실험은 밴더빌트 IACUC에 의해 명시된 지침과 규정에 따라 수행되었다.

이 프로토콜은 급성 부상, 부상 - 수리, 또는 만성 colonic 부상 프로세스를 모델로 수정할 수 있습니다. 급성 상해 변경 : 즉각적인 희생 뒤에 4~6일에 대한 DSS 처리 광고 lib과 상해 - 수리 수정 : 3-4일과 희생의 복구 기간 (위의 프로토콜에서 설명) 다음 DSS 치료 4-6일로 부상. 만성 대장염의 변경 : </p...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dextran Sulfate Sodium Salt</td>
<td>&nbsp;</td>
<td>USB Corp</td>
<td>9011-18-1</td>
<td>mol wt 40,000-50,000</td>
</tr>
<tr>
<td>10% Buffered Formalin Phosphate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>SF100-4</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane, USP</td>
<td>&nbsp;</td>
<td>Phoenix Pharmaceuticals</td>
<td>NDC 57319-474-06</td>
<td>&nbsp;</td>
<tr>
<td>TISSUE PATH Macrosette Cassettes</td>
<td>&nbsp;</td>
<td>Fisher Healthcare</td>
<td>15182706</td>
<td>&nbsp;</td>
<tr>
<td>BD 10 ml Syringe</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>309604</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl Alcohol</td>
<td>&nbsp;</td>
<td>Pharmco-Aaper</td>
<td>E190</td>
<td>Dilute to 70% with distilled water</td>
</tr>
<tr>
<td>20G Straight Feeding Needle</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>20068-612</td>
<td>&nbsp;</td>
<tr>
<td>27G1/2 PrecisionGlide Needle</td>
<td>&nbsp;</td>
<td>B-D</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Scissors Sharp/Blunt Tip</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-588</td>
<td>&nbsp;</td>
<tr>
<td>Waugh Forceps</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>82027-428</td>
<td>&nbsp;</td>
<tr>
<td>Non-absorbable Suture </td>
<td>&nbsp;</td>
<td>LOOK Surgical Specialties</td>
<td>SP116</td>
<td>&nbsp;</td>
<tr>
<td>Whatman Blotting Paper</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>28298-020</td>
<td>Cut as needed</td>
</tr>
<tr>
<td>Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter</td>
<td>&nbsp;</td>
<td>Cole-Parmer</td>
<td>EW-06731-2</td>
<td>&nbsp;</td>
<tr>
<td>Carbon fiber composites digital caliper</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-007-958</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

murine colitis modeling using dextran sulfate sodium (dss)

- Crohn의 대장염 궤양성 대장염 특히 및 Crohn의 질병의 colonic 변종, 대장염은 바이러스 또는 박테리아 감염, 허혈성 모욕, 또는자가 면역 질환에서 발생할 수 있습니다. 급성 대장염은 복부 통증과 팽창, 흡수 장애, 설사, hematochezia 및 배설물에 점액과 함께 제공됩니다. 우리는 환경, 유전 및 대장염의 염증성 장 질환과 동물 모델에서 상피 장벽 부전이 질병의 우리의 이해를 발전에 필수되었습니다 사이의 복잡한 상호 작용을 이해하기 시작하고 있습니다. 하나의 인기있는 모델은 몇 일 지속 상피 손상과 콜론의 강력한 염증 반응의 결과로, 낮은 분자량 Dextran 나트륨 황산 (DSS)와 생쥐의 식수를 보충 포함<sup&gt; 1</sup이 방법의&gt;. 유사 급성 부상, 수리 뒤에 급성 부상 및 복구 모델링 만성 염증성 질환과 interspersed DSS의 반복주기를 모델로 사용할 수 있습니다<sup&gt; 2</sup&gt;. 식수의 3 % DSS의 단일 4 일간 치료 후, 마우스는 체중 감소, 피가 stools, 그리고 설사를 포함한 급성 대장염의 흔적을 보여줍니다. 생쥐는 치료 과정의 결론에 euthanized하고 검시 해부 콜론에서 처리하고 &#39;스위스&#39;압연 수 있습니다<sup&gt; 3</sup&gt; 전체 대장의 미세 분석을 허용하거나 매크로 분석을 허용하는 &quot;소시지&quot;로 포르말린과 함께 들어갈 수 있습니다. 조직은 다음 sectioned 파라핀에 내장하고, histologic 검토를 위해 얼룩이다.

Watch this Scientific Journal Video about Murine Colitis Modeling using Dextran Sulfate Sodium DSS at JoVE.com

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

식수에서 실시 Dextran 황산 나트륨 (DSS)은 급성 대장염의 설립 murine 염증성 손상 모델입니다. 이 프로토콜은 DSS 치료와 조직의 준비를 위해 방법을 설명합니다.

Dextran의 황산 나트륨 (DSS)를 사용 Murine 대장염 모델링

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

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Research

JoVE Journal

Biology

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

49.7K 조회수.  Vanderbilt University. 식수에서 실시 Dextran 황산 나트륨 (DSS)은 급성 대장염의 설립 murine 염증성 손상 모델입니다. 이 프로토콜은 DSS 치료와 조직의 준비를 위해 방법을 설명합니다.

비디오: Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

수학 모델링을 사용 인구 교체 전략의 효과를 예측

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

연구

생물학

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

레이저 Uncagable 플루오레신 Dextran과 Zebrafish 세포의 리니지 라벨

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

IBD의 DSS 유발 모델에서 창자 염증을 조사

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

압력 Myography를 사용 쥐과 저항 동맥의 기능을 평가

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

Danio rerio를 사용하여 병적 인 인간 게놈의 생체 모델링에

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

이미징 FITC-dextran 규제 Exocytosis 위한 기자로 서

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These processes are characterized by the gradual appearance of essential structures in the megakaryocyte cytoplasm such as a polyploid and polylobulated nucleus, an internal membrane network called demarcation membrane system (DMS) and the dense and alpha granules that will be found in circulating platelets. In this article, we describe a standardized protocol for the in situ ultrastructural study of murine megakaryocytes using transmission electron microscopy (TEM), allowing for the identification of key characteristics defining their maturation stage and cellular density in the bone marrow. Bone marrows are flushed, fixed, dehydrated in ethanol, embedded in plastic resin, and mounted for generating cross-sections. Semi-thin and thin sections are prepared for histological and TEM observations, respectively. This method can be used for any bone marrow cell, in any EM facility and has the advantage of using small sample sizes allowing for the combination of several imaging approaches on the same mouse.

In Situ Exploration of Murine Megakaryopoiesis using Transmission Electron Microscopy

Here, we present a protocol to analyze ultrastructure of the megakaryocytes in situ using transmission electron microscopy (TEM). Murine bone marrows are collected, fixed, embedded in epoxy resin and cut in ultrathin sections. After contrast staining, the bone marrow is observed under a TEM microscope at 120 kV.

시투에서 전염 전자 현미경 검사를 사용하여 뮤린 메가 카요포이스의 탐사

Differentiation and maturation of megakaryocytes occur in close association with the cellular and extracellular components of the bone marrow. These ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Fluorescein-Isothiocyanate-labeled dextran을 사용한 생쥐의 장 장벽 무결성 평가

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Molecular Dynamics를 사용한 Lipid Bilayers 시뮬레이션 및 분석

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

저자 스포트라이트: Advancing Cell Membrane Biophysics - Exploring Interactions and Challenges Through Experimental and Computational Approaches(세포막 생물물리학의 발전 - 실험 및 계산적 접근법을 통한 상호 작용 및 과제 탐색) 

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

시뮬레이션 연구에서 복잡한 지질 혼합물을 사용한 사실적인 막 모델링

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...