A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

1. 계란 부화 <ol><li> 팔일 된 무균 유정란을 얻습니다. </li><li> 계란 트레이, 위쪽으로 향하게 스탬프 끝을 회전에 알을 놓고 계란 인큐베이터 내부의 회전 트레이를 배치합니다. 36 ° C에서 48 시간 50 %의 습도 알을 품어. </li></ol> 2. CAM을 삭제하고 계란 열기 <ol><li> 인큐베이터에서 계란을 수집합니다. 달걀 랙에 넣어 달걀, 최대 측면을 각인하고, 층류 후드에 가져다. </li><li> 룸과 후드 등을 끕니다. </li><li> CAM의 혈관뿐만 아니라 공기 주머니를 노출 계란 캔 들러 가볍게 계란의 스탬프 끝을 잡고. 참고 : 계란 캔 들러가 개발 배아 및 관련 공기 주머니와 혈관을 시각화하는 데 사용되는 광원이다. </li><li> 두 개의 주요 혈관 사이에 연필 표시를합니다. </li><li> 불을 켜고 PE에서 공기 주머니 (스탬프 끝) 위의 계란의 팁과 하나의 구멍에 하나의 구멍을 만들기 위해 멸균 고정 핀을 사용ncil 마크. 푸시 핀을 통해 모든 방법을 밀어하지 마십시오 3mm 주위에 구멍이 깊은 일반적으로 충분합니다. </li><li> 불을 끄십시오. 공기 주머니 위의 구멍에 꽉 열린 끝을 눌러 안전 전구를 짠다. CAM은 연필 마크에서 멀리 쉘에서 드롭하는 원인, 연필 마크의 구멍으로 공기를 끌어 흡입을 적용합니다. </li><li> 공기 주머니가 캔 들러를 사용하여 연필로 표시된 구멍에 계란의 스탬프 끝에서 이동 한 것을 확인합니다. 그렇지 않은 경우, 안전 전구를 사용하여 흡인을 다시 적용한 후 두 구멍 약간 깊게 만들어하는 푸시 핀을 사용한다. </li><li> 불을 켜다. 부착 된 15/16 인치 컷 - 오프 휠 드레 멜 로터리 툴을 켜고 쉘을 통해 잘라 그냥 깊은만큼 두 가로 삭감을하지만 충분히 깊은하지가 우울에 이르기까지 모든 방법을 잘라, 한 손에 계란을 잡고 CAM. 서로 떨어져 길이 2cm과 1cm 주위의 상처를 확인합니다. 직경 1cm 실리콘 링을 통과 할 수 있도록 충분히 넓은 상처를 확인표준 스카치 테이프의 폭보다하지만 얇은. </li><li> 다음, 가볍게 쉘 컷 - 오프 휠을 만져 사이에 두 가로 인하의 끝에서 1 종 컷을합니다. </li><li> 쉘 쉘 조각 및 병렬에서 멸균 집게를 밀어 넣습니다. 집게로 껍질 조각을 잡고 깨끗하게 전체 조각을 제거합니다. </li><li> 제거의 용이성을 위해 자체에 한쪽 끝을 통해 접어 확인하고, 새로 열린 창을 통해 스카치 테이프를 놓습니다. </li><li> 다시 인큐베이터 (NOT 회전 달걀 랙에서) 계란 트레이에 접종 때까지 장소 달걀. </li></ol> 3. 예방 접종 <ol><li> 이러한 매트 리겔과 같이, 지하 세포막 단백질 혼합물을 얼음에 보관 후드 (중합 반응을 방지하기 위해) 및 장소. </li><li> 5 분 동안 조직 문화 인큐베이터에서 - (T75 플라스크 당 5 ㎖의 4) 장소 플라스크 세포를 분리하는 2 mM의 EDTA를 사용합니다. </li><li> 미디어에서 분리 된 세포를 재현 탁하고 혈구와 트리 판 블루를 사용하여 계산합니다. 500,000t 사이에 사용캠에 접목 세포주에 따라 2,000,000 세포 오. 예비 실험에서 적절한 수를 최적화 할 수 있습니다. </li><li> 5 분 동안 500 XG에 적​​절한 휴대폰 번호와 원심 분리기에 필요한 측정 볼륨. </li><li> 뜨는을 취소하고 다음 PBS의 40 μ L ++ 및 기저막 혼합물의 20 μ L에서 세포 펠렛을 재현 탁. 참고 : 셀 / 기저막 혼합물 현탁액에 재현 탁하여 실험에 필요에 따라이 시점에서, 세포는 약물 또는 다른 테스트 제로 처리 될 수있다. 또한, 에이전트는 이식 후 세포 / 지하 막 혼합물 서스펜션에 분주 할 수 있습니다. </li><li> 층류 후드의 보육과 장소에서 계란 계란 랙을 검색합니다. </li><li> 껍질 테이프 창 떨어져, 멸균 집게를 사용하여 극저온 유리 병의 뚜껑에서 1mm 실리콘 링을 검색하고, 창을 통해 반지를 놓습니다. </li><li> 피펫 PBS의 60 μ L 셀 솔루션 ++ 및 기저막 mixtur직접 CAM에 휴식 실리콘 링의 중심으로 전자. </li><li> 다시 테이프 창을하는 인큐베이터에 다시 계란 랙을 이동합니다. 쉽게 치환 될 수있는 링과 세포 현탁액으로 계란을 이동할 때이 시점에서주의하십시오. </li><li> 종양이 5-7일에 대한 성장하도록 허용합니다. </li></ol> 4. 수확 종양 <ol><li> 후드 아래에 패드를 놓습니다. 조직학에 필요한 35 X 10mm 판과 파라 포름 알데히드 용기를 준비합니다. 각 35 X 10mm 판에 1 ml의 PBS ++를 넣습니다. </li><li> 후드에서 생물 학적 가방을 놓습니다. 멸균 해부 가위를 가지고 스탬프 끝 부분 구멍에 계란에 뾰족한 끝을 삽입합니다. </li><li> 길이 방향 전체 계란 주위에 잘라. 참고 : 링과 캠과 종양이 창 쉘의 상단 절반에 충실해야한다. </li><li> 해부 가위와 집게를 사용하여, 멀리 CAM에서 종양을 해부하고, 35 X 10mm 판에서 종양을 배치합니다. 측정하고 종양의 무게. paraformaldehy에 고정 종양드 후 조직 학적 및 면역 조직 화학 실험에 사용할 수 있도록 파라핀-포함. </li></ol> 5. (선택 사항) : 종양 세포 해리 <ol><li> 35 X 10mm 판에서 종양을 말하다 해부 가위를 사용합니다. 15 ㎖의 원심 분리기 튜브에 다진 종양을 붓고 피펫을 통해 가능한 ++ 많은 PBS를 제거합니다. </li><li> (PBS에 1 ㎎ / ㎖ ++) 콜라게나 제 2 mL를 넣어 여러 번에 걸쳐 튜브를 켜고 (몇 시간까지) 적어도 30 분 동안 37 ° C에서 튜브를 품어. </li><li> 각각의 튜브에 5 ml의 미디어를 추가하고 아래로 (20)로 pipetting에 의해 철저하게 재현 탁 - 40 배를. 철저한 피펫은 완전한 세포 분리 중요하다. </li><li> 침전물이 정착 할 수 있도록 허용하고, 새로운 튜브에 뜨는을 전송합니다. 5 분 500 XG에 원심 분리기. 해리 세포 펠렛을 남겨두고, 상층 액을 제거합니다. 주 :이 시점에서, 세포 펠릿을 각종 실험에 사용될 수있다 (유동 세포 계측법, 웨스턴 블 롯팅, RNA 분리 등의 용도 용해). 전체 종양 세포 수는 혈구 및 트립 판 블루 염색을 이용하여 얻어 질 수있다. 작은 타원형 셀 닭 세포이며 CAM으로부터 종양 세포로 간주되어서는 안된다는 참고. 종양이 조심스럽게 CAM에서 멀리 해부 경우 비 종양 (CAM) 세포를 오염 최소한이 있어야한다. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

이 프로토콜의 몇 가지 주요 단계는 대부분 개선 된 배아의 생존뿐만​​ 아니라 종양 성장의 신뢰성 증가를 차지하고있다. 공기 주머니에 흡입을 적용하여 멀리 쉘에서 CAM을 삭제하면 (계란, 무딘 절개 등으로부터 알부민을 제거하기 위해 바늘을 사용) 기존의 방법보다 덜 침습적입니다. 이 방법에 필요한 두 개의 작은 구멍을 만들기 위해 멸균 푸시 핀을 사용하는 프로토콜의 가장 어려운 부분?...

간세포 암종 (HCC)은 세계에서 1 암 사망률 제 3 주요 원인이다. 현재 간암 환자의 30 %가 잠재적으로 치료 수술 치료 (2)을받을 수 있으며, 전신 항암 화학 요법은 3 효과가 없습니다. 따라서, 새로운 간암 치료 용 임상 가압 충족해야하고, 새로운 에이전트의 효율적인 시험에 적합한 모델 시스템의 개발이있다. 병아리 융모 막 (CAM) 시험은 생체 내에서 잠재적 인 항암 약물을 시험의 재현성, 비용 효율적이고 빠른 중간 처리 방법을 제공한다. CAM 혈관 형성 검정은 4를 연구하기 위해 광범위하게 사용되고있다. 또한 성공적 5 아교 모세포종, 췌장암 6 흑색 7-9, 10-11 및 골육종을 포함한 암의 종양 이종 이식 모델로 개발되었다. 모두 비켜 (12)과 전 비켜에서13 기술은 프로토콜과 프로토콜에 다양한 세부 사항, 문헌에 이용되어왔다. 50 % 11 ~ 14 - 캠 이종 이식 모델에 하나의 주요 과제는 25에 이르기까지 출판 된 병아리 배아 사망률, 계란의 조작 후 배아 사망의 상대적으로 높은 발병률이다. 이 글에서, 우리는 확실하게 조직 학적 분화 간세포 암과 유사한 입체, 혈관 종양의 성장을 생산 간세포 암의 이종 이식 모델 비켜에서의 개발을 설명합니다. 우리는 먼저 Ossowski 등. (14)에 의해 기술 된 프로토콜을 적용하고 매우 높은 종양 생착으로 최대 93 %의 병아리 배아 생존율을 달성했다.

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			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
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			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

병아리 융모 막 (CAM)는 CAM이 종양 이식을위한 이상적인 시스템을 만들고, 자연 면역 결핍 높은 혈관입니다 수정 후 7 일째에 의해 개발을 시작하여 하루 (12)에 의해 성숙. 또한, CAM은 그것을 종양 침윤과 전이를 연구하는 매력적인 모델을 만드는 등 피브로넥틴, 라미닌, 콜라겐, 인테그린 알파 (V) 베타 3, 및 MMP-2와 같은 세포 외 기질 단백질을 포함하고 있습니다. 과학자들은 긴 혈관 신생의 모델로서 사용하여 CAM의 생리 학적 장점을 가지고있다. 최근 CAM 검정 기본 체외 일 더 복잡한 동물 암 모델을 절충 각종 암 이종 이식 생체 내 모델 시스템으로 동작하도록 변형되었다. CAM 분석은 비용과 시간이 효과적이다 중요한 생물학적 시스템에서 종양 성장, 항 종양 치료 및 종양 친 분자 경로의 연구 허용한다. 여기서, 우리는 CAM의 젠의 개발을 기술최대 93 %의 배아 생존율하고 신뢰할 수있는 종양과 간세포 암 (HCC)의 ograft 모델은 세 가지 차원, 혈관 종양의 성장을 선도 걸릴.

프로토콜의 주요 단계의 대표 사진이 여기에 표시됩니다. 그림 1A는 배아, 공기 주머니와 CAM의 혈관을 시각화하기 위해 캔 들러의 사용을 보여줍니다. 그림 1B-1c는 만들어 CAM을 삭제하는 과정을 보여 두 개의 구멍 후 안전 전구를 사용하여 부압을인가하고,도 1d는 성공적 연필 마크 구멍 하에서 큰 기포로 CAM 떨어 나타낸다.도 1E 및도 1F는 필요한 ?...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

<em&gt; 비켜에서</em간세포 암의 효율적인 이종 이식 모델로&gt; 병아리 융모 막 (CAM) 분석

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

34.9K 조회수.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

비디오: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

구강 편평 세포 암종의 Orthotopic 마우스 모델에서 종양 세포 침략의 다중 광자 이미징

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

연구

의학

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

역 형성 갑상선 암의 동소 마우스 모델

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; OVO에서</em육종을위한 Xenograftmodel으로&gt; CAM-분석

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

면역 결핍 마우스의 피하 및 간내 인간의 간세포 암 이종 이식의 생성

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

같은 뇌 Microslices의 흥분 독성 자극<em&gt; 체외</em&gt; 스트로크의 모델

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

치과 복합 재료의 Cytocompatibility 평가하기위한 효율적인 방법으로 공 촛점 시간 경과 영상

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

&quot;환자와 같은&quot;동소 이식 동계 마우스 모델 간세포 암의 전이

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

내부 접근을 사용 하 여 상 악 재건을 위한 디자인 CAD/CAM 수술 가이드

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

기계 학습을 사용 하 여 이미지 기반 요법 치료 반응 예측: 간세포 암 종의 트랜스 동맥 치료 예

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...