이 연구는 신경 장애의 국립 연구소에서 부여 R01NS062842과 건강 (VVD)의 스트로크, 국립 연구소에 의해 및 신경성 질환의 국립 연구소와 ARRA를 통해 건강의 치기, 국립 연구소 (VVD)과 R21에서 보조금 R21 NS064403에 의해 지원되었다 바이오 메디컬 이미징 및 생물의 EB006301 국립 연구소, 건강의 국립 연구소 (VVD).

방법은 포름 알데히드 고정 된 파라핀 조직 절편에 적합하다. 이 기술은 쥐 실험 스트로크 여기의 응용 프로그램에서 보여줍니다. Tyramide 신호 증폭 (TSA) 시스템 1. 준비 구성 요소 TSA 기반 향상이 반응을 준비하고, 라벨의 마지막 단계에 사용되지만 이상의 시간이 걸릴 수 있습니다. 그 때문에, 표시를 시작하기 전에 TSA 시약을 만드는 것이 편리하다. <ol><li> 형광의 tyramide 원액을 확인합니다. TSA 라벨 키트 개별 튜브에 공급되는 형광의 tyramide 시약을 사용합니다. tyramide 시약 유리 병에 300 μL의 DMSO를 추가합니다. 4 ℃에서 저장 될 때 형광의 tyramide 원액은 3 개월 이상 안정 </li><li> 포함 된 TNT의 세척 버퍼 확인 : 0.1 M 트리스 - 염산, pH를 7.5; 0.15 M 염화나트륨; 0.05 % 트윈 -20. 필요한 0.3 % 트리톤 X-100 0.05 % 트윈 -20을 대체 할 수있는 경우. alternat의ively PBS는 세척 완충액으로 사용할 수있다. </li><li> 확인 TNB 포함하는 버퍼 차단 : 0.1 M 트리스 - 염산, pH를 7.5; 0.15 M 염화나트륨; 0.5 % 차단 시약 (키트와 함께 제공). 교반하면서 완전히 차단 시약을 용해, 천천히 버퍼에 조금씩 추가합니다. 점차적으로 연속 교반하면서 55 °의 C에이 솔루션을 가열한다. 이는 30 ~ 60 분 걸릴 수 있습니다. 이 솔루션은 우유를 나타납니다. 사용하기 전에 실온으로 오십시오. 장기 사용을 위해 -20 ° C에서 나누어지는 및 저장. </li></ol> 섹션 2. 준비 포르말린 고정 조직을 잘라 장착 5 μm의 두께 섹션으로 슬라이드를 사용합니다. <ol><li> 15 분 자일 렌 섹션을 처리합니다. </li><li> 다음 섹션을 재수 : 5 분마다 96 % EtOH로 2 배에 담가; 5 분 동안 80 %의 EtOH에 담가; 5 분마다 물을 2 배로 씻어. </li><li> PBS에서 50 ㎍ / ㎖의에 테 K 원액을 희석하여 테 K 작업 솔루션을합니다. </li><li> 테 추가K는 섹션에 솔루션을 작업. 23 ℃에서 15 분 알을 품다 용액의 부피 및 소화의 시간은 섹션 및 조직 유형의 크기에 따라 조정할 필요가있다. 작은 (직경 5 ㎜ 이하) 섹션 테 솔루션의 50 μL은 충분하다. 평균 크기 (~ 직경 10 ㎜) 섹션, 테 K 용액 100 μl를 추가합니다. 매우 큰 부분에 대한 솔루션의 볼륨을 확장 할 수 있습니다. 모든 경우에 신호의 손실을 완료로 연결 조직의 형태를 파괴 섹션 overdigestion을 방지하기 위해주의를 기울입니다. 배양 시간이 25 분을 초과하는 경우에 이러한 상황이 자주 발생합니다. </li><li> 5 분마다 증류수의 2 배에 섹션을 씻으십시오. 섹션은 물에있는 동안 액티브 프로브를 포함하는 라벨 믹스를 준비합니다. </li></ol> 3. VACC TOPO 기반 염색 <ol><li> 다음을 조합하여 액티브 프로브 용액을 준비한다 : 물 - 11.75 μL 1 M 트리스 - 염산, pH를 7.4-1.25 μ엘 12베이스 오버행 자​​살 올리고 뉴클레오티드의 100 pmol의 - 1 μL 어댑터 올리고 뉴클레오티드의 100 pmol의 - 1 μL 50 pmol의의 VACC의 TOPO - 10 μL </li><li> 피펫으로 가볍게 섞는다. 프로브 활성화 할 수 있도록 15 분 동안 실온 (23 ℃)에서 배양한다. </li><li> 세척 항아리에서 절을하고 남은 물을 대기음. 그런 다음 3.1 단계 (~ 10 × 10 밀리 섹션 당 25 μL)에서 제조 된 액티브 프로브 솔루션을 적용 할 수 있습니다. </li><li> 섹션을 커버 슬립, 23 ℃에서 15 분 동안 품어 딥 부드럽게 커버 슬립을 제거하기 위해 물에 슬라이드. 5 분마다 물을 배에 씻으십시오. </li></ol> 4. TSA 신호 향상 <ol><li> TNB 이전에 준비하고 실온 (23 ℃)에서 30 분 동안 가습 실에서 슬라이드를 품어 차단 버퍼와 조직 섹션을 포함한다. </li><li> TNB 버퍼를 차단하는 스트렙 타비 딘 - 고추 냉이 과산화 효소 복합체 (SA-HRP)의 1:100 희석을 준비합니다. 기음 TNBSA-HRP 용액을 버퍼링하고 적용 블로킹. 실온에서 30 분 동안 SA-HRP와 슬라이드를 품어. 조직 섹션, 섹션 당, 100 μl를 충당하기 위해 적절한 시약의 볼륨을 사용합니다. </li><li> 세척은 실온에서 TNT의 세척 버퍼 (또는 PBS)에 5 분마다 배를 슬라이드. </li><li> 키트에서 1X 증폭 희석제에 형광의 tyramide 주식의 1:50 희석을 준비합니다. 각각의 슬라이드에 형광의 tyramide 작업 솔루션을 피펫. 완전히 조직 섹션, 섹션 당 일반적으로 100 μL를 커버하기에 충분한 작업 솔루션을 사용합니다. 바로 각 라벨 전에 형광의 tyramide 작업 솔루션을합니다. 용액을 재사용하므로 라벨링 나서의 미사용 부분을 무시 할 수 없다. </li><li> 3 내지 10 분 동안 실온에서 슬라이드를 배양한다. 구조의 제 힌트를 볼 수있을 때까지 현미경 형광 증가를 모니터링한다. 신속하게 단계를 씻어로 진행합니다. </li><li> 워시 룸 t에서 TNT의 세척 버퍼 (또는 PBS)에 5 분마다 배를 슬라이드교반 온도계 수. </li><li> DAPI와 coverslip에있는 antifading 솔루션을 추가합니다. 형광 현미경 하에서 관찰한다. DAPI 및 FITC 형광이 대역 통과 필터 세트를 사용하여 시각화 : FITC 여기 D490/40 발광 10분의 520; DAPI 여기 D360/40, 배출 20분의 460, 또는 유사한. </li></ol>

<ol>
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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

이 비디오에서 우리는 초점 뇌 허혈에있는 투명 세포 죽음을 활성화 식세포 레이블을하는 방법, 조직 섹션 형식으로 보여줍니다. 제시된 기술은 구체적으로 침몰하고 적극적으로 세포 시체를 소화들만 폐기물 관리 셀 라벨 첫 번째 방법입니다. 이것은이 기능이 없습니다 식세포의 라벨에 대한 기존의 방법에 대한 유익합니다. 두 번 좌초 침몰 핵 소화하는 동안 폐기물 관리 ...

허혈성 뇌졸중은 영향을받는 뇌 조직에 큰 변화를 유도한다. 그것은 미세 아교 기원 주민 식세포의 급속한 활성화로 연결하는 대규모 세포 죽음을 시작합니다. 또한 호중구, 대 식세포, 수지상 및 비만 세포 1,2 등의 혈액에서 파생 된 전문 식세포의 여러 유형에 의해 허혈성 뇌의 침투를 설정합니다. 그것은 여전히​​ 허혈 손상이 반응이 긍정적 또는 부정적인 역할을할지 여부를 논의한다. 그것은 죽은 세포를 지우고 염증을 억제하기 때문에 뇌졸중 다음 식균 작용이 도움이 될 수 있지만, 그것은 또한 신경 세포의 생존에 영향을 미치는 조직의 손상 1-5 악화 유해 활성 산소 종을 생성합니다. 식세포의 여러 종류가 허혈성 뇌에 침투하는 동안, 그들 모두는 세포 시체를 덮어 회생 과정 1 ~ 3을위한 방법을 취소하여 폐기물 관리에 참여한다. 이 CR뇌졸중 세포 죽음의 식세포를 실행할 것을 폐기물 관리 세포의 선택적 식별​​을위한 요구 사항을 eates. 능동 폐기물 관리 셀을 묘화 할 때, 그들은 잠겼 셀 시체 저하 얼마나 효율적의 질문에 응답하는 것도 중요하다. 그것은 자기 면역 병리학 핵 물질 (6)의 방출을 방지하기 때문에 식균 작용에 죽어가는 세포의 DNA의 유효하고 완전 분해가 필수적이다. 여기에서 우리는 활성 식세포의 마커로 특정 DNA 나누기를 사용하여 새로운 프로브를 제시한다. 이 서명 나누기 독점적 기능 폐기물 관리 세포 내부에 가득 채우고 핵의 분해시 생성된다. 따라서 프로브는 선택적으로 침몰 만 식세포 레이블을 적극적으로 세포의 시체를 소화. 그들은 또한 말림 후 강도와 DNA 파괴의 완료를 관찰 허용합니다. 프로브는 강도와 effici의 평가에 도움이됩니다식세포 정리의 ency. 새로운 프로브는 비 단백질 기반의 마커에 의존하기 때문에 광범위한 허혈성 손상은 세포의 형태를 중단 시키거나, 특히 핵심 허혈성 영역 안에, 단백질 기반의 마커를 고갈시킬 뇌졸중의 연구에 특히 유용 할 수 있습니다. 기술의 원리는 그림 1에 제시되어있다. 그림은 효소 우두 토포 이소 머라 제 (VACC TOPO)로 결찰 머리핀 모양의 올리고 뉴클레오티드 프로브를 보여줍니다 5&#39;OH DNA에 DNA 소화 7시 리소좀 DNA 분해 효소 II에 의해 생성 끝납니다.

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:312px;">Name of the reagent or material</td>
			<td style="width:99px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:129px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>JT Baker</td>
			<td>9490-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS Buffer</td>
			<td>Invitrogen</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vaccinia Topoisomerase I</td>
			<td colspan="2">Vivid Technologies</td>
			<td>5 pmol/&#956;L stock&#160;</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-Base Overhang Suicide Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-AAG GGA CCT GCB GCA GGT CCC TTG ATA CGA TTC TA -3&#8217; ; B &#8211; Biotin-dT; 100 pmol/&#956;L stock in ddH2O&#160;&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Adapter Oligonucleotide</td>
			<td>IDT DNA</td>
			<td></td>
			<td>5&#8217;-TAG AAT CGT ATC-3&#8217;; 100 pmol/&#956;L stock in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M Tris-HCl, PH 7.4</td>
			<td>Lonza</td>
			<td>51237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TSA, Fluorescein System&#160;</td>
			<td>PerkinElmer&#160;</td>
			<td>NEL701A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Coverslips</td>
			<td></td>
			<td></td>
			<td style="width:129px;">22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">Rat Brain Sections</td>
			<td></td>
			<td></td>
			<td style="width:129px;">Rat brain taken 48 hours after reproduction of ischemic stroke as described in 11. 5-6&#956;m-thick sections cut from paraformaldehyde-fixed, paraffin-embedded tissue blocks. Use charged and precleaned slides.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td style="width:129px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Video camera and software</td>
			<td>Micromax</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td>Metamorph</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes

우리는 초점 뇌 허혈에 식세포 정리의 시각화를위한 새로운 조직 화학적 방법을 설명합니다. 접근 방식은 모든 세포 계보의 폐기물 관리 식세포에 의한 뇌졸중의 죽은 세포의 제거에 대한 연구를 허용합니다. 식균 작용의 능력이 서로 다른 기원의 수많은 세포가 허혈성 뇌에 존재하지만, 그 중 일부는 적극적으로 침몰 세포 시체를 소화. 선택적 시각화, 정량화 및 능동 식세포 폐기물 관리의 분석은 허혈 뇌의 반응을 평가하는데 도움이됩니다. 그것은 이후의 재생 공정을위한 길을 열어 효율적인 세포 죽음의 간극은, 허혈성 뇌 손상에서 복구를 위해 중요합니다. 시체를 청소하는 고장이 아닌 성능이 저하 된 DNA와 단백질에 의한 유해 반응이 발생할 것입니다. 설명 된 절차는 선택적으로 말림 동안 모든 식세포에서 생산 특성 DNA 나누기에 바이러스 토포 이소 머라 제에 의해 결찰 형광 프로브를 사용세포의 소화 비가 역적 허혈 손상. 이 방법은 허혈 손상에 대한 뇌 반응의 조사를위한 새로운 도구입니다.

활성 폐기물 관리 식세포 레이블을 지정하는 기술의 원리는 그림 1에 제시되어있다. 회로도 보여줍니다 세 단계에서 검출 진행하는. 첫 번째 단계는 프로브 활성화 (도 1a)를 포함한다; 두 번째 단계에서 활성화 된 프로브는 조직 절편 (도 1C)에있는 특정한 DNA 나누기로 결찰된다; 세 번째 단계는 형광 신호 증폭 (도 1D)을 포함한다. 다음 상세한 설?...

Watch this Scientific Journal Video about Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes at JoVE.com

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

우리는 활성화 된 대 식세포의 현장 라벨의 선택을위한 새로운 형광 기술을 제시하는 명확한 행정의 세포 시체 끕니다. 허혈성 뇌에 존재하는 식세포의 단지 작은 비율이 세포 죽음의 정리에 참여하기 때문에 방법은 국소 빈혈에 대한 뇌의 반응을 평가하는 것이 중요합니다.

Ligatable 형광 프로브에 의한 실험 스트로크에서 세포 죽음의 식세포 정리 평가

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

assessing-phagocytic-clearance-cell-death-experimental-stroke

Research

JoVE Journal

Medicine

8.1K 조회수.  Baylor College of Medicine.  우리는 활성화 된 대 식세포의 현장 라벨의 선택을위한 새로운 형광 기술을 제시하는 명확한 행정의 세포 시체 끕니다. 허혈성 뇌에 존재하는 식세포의 단지 작은 비율이 세포 죽음의 정리에 참여하기 때문에 방법은 국소 빈혈에 대한 뇌의 반응을 평가하는 것이 중요합니다. 

비디오: Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

마우스의 모델링 스트로크 - 필라멘트 모델 중간 대뇌 동맥 폐색

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

연구

의학

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

에서 간세포 이식 체외에서 시뮬레이션 국소 빈혈 / Reperfusion 모델

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

태아 알코올 노출의 Zebrafish 모델에서 Teratogenic 변경 사항 평가

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

체외 Mesothelial 정리 분석에서는 그것이 난소 암 전이의 초기 단계 모델

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

실험 인간 폐렴의 운송

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

비정상적인 유사 분열과 관련된 세포의 죽음은 라이브 암 세포의 공 촛점 이미징에 의해 관찰

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

척수 손상의 실험 Contusive 모델에서 신경 줄기 세포 이식

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

실험 녹내장은 안구하는 미소 자석을 투여 한

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

마우스의 담관 결찰 : 폐쇄성 담즙에 의한 염증성 간 손상과 섬유화의 유도

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

공 초점 광학 이미징에 의해 로즈 벵갈 Photothrombosis<em&gt; 생체</em&gt; : 단일 선박 스트로크의 모델

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...