Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00
Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

모든 프로토콜이 적용 어디에 기관 동물 관리 및 사용위원회의 정책을 준수하여 수행 하였다. 1. 준비 <ol><li> 75 %로 안정화 매체를 준비 둘 베코의 수정 독수리 중간 (DMEM), 25 % 행크스 &#39;균형 소금 솔루션 (HBSS). 사용할 때까지 -20 ° C에서 잘 나누어지는 및 저장을 섞는다. </li><li> 인산염 완충 식염수 (PBS)를 준비하고 고압 증기 멸균에 의해 소독. 실온에서 용액을 저장한다. ?...

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In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

증거의 성장 몸은 혈액 망막 장벽 (BRB) 1-5과 혈액 뇌 장벽 (BBB) ​​6,7과의 유사성의 존재를 지원합니다. BBB를의 타협은 단단히 이러한 정신 착란 (10)로 인과 적 또는 알츠하이머 병 (AD)과 같은 만성 신경 퇴행성 질환에 대한 진단 마커로 8,9 급성 조건을 연결되어 있습니다. 잠재적 인 약물 표적에 대한 이러한 병리 및 발견에 기계적인 통찰력은 일반적으로 뇌의 제한된 액세스 및 네트워크 복잡함에 의해 방해된다. 이러한 생체 내 이미징 (11), 뇌의 Organotypic 문화 (12), 차 세포 배양 13, 14 및 공동 문화 시스템 (15)과 같은 대안이 생성되었습니다. 그러나, 이러한 모델의 대부분은 세포를 식별하는 특수 장비, 긴 실험 기간 또는 여러 개의 마커를 필요로한다. 기능적 및 구조적 및 BBB BRB 유사성뿐만 아니라 DY 간의 상관이 둘의 sfunctions는 16-19을 주장하고있다. 또한, 쉽게 액세스 잘 정의 세포 유형 및 적층 구조는 뇌 윈도우로 잘 특성화 망막 수있다. BBB를하고 BRB의 구조적 및 기능적 아이덴티티 상세히 비교 남아. 그러나, 망막 병증은 특히 BRB 파기는 또한 밀접 당뇨병 18-19 및 AD (21, 22)을 포함하는 다양한 질병의 진행과 연관되어있다. 따라서, BRB는 부전 시스템 메커니즘을 묘사 할뿐만 아니라 잠재적 인 약물 스크리닝뿐만 아니라 확립 관심사이다. 이 보고서에서, 단순 급성 망막 문화를 사용하여 프로토콜 가능 BRB 부전을 개발하고 제공됩니다. 증가 BBB 투과성 및 AD와 같은 병리학 적 변화는 히스타민, 프로 염증 중재자 (12)와 함께 배양 뇌의 Organotypic 문화에 설립되었습니다. 따라서, 표시 시스템에서, 히스타민은 APPL이었다BRB 부전을 유도하기 위해 생체 망막 문화 IED. 이러한 뮤스의 musculus과 보스 황소 같은 여러 종에서 망막은, 테스트되었습니다. 때문에 상업적 가용성 및 인체 조직에 유사에 신선한 돼지의 시선은 여기에보고 된 데이터를 제공하기 위해 사용되었다. 히스타민 및 / 또는 다른 약물과 배양 한 후 망막은 면역 글로불린 G (IgG의) 혈액의 주요 구성 요소 중 하나로서 여러 단백질 (12)에 대한 면역 염색으로 평가 처리 하였다; glial fibrillary 산성 단백질 (GFAP), 아교 활성화를위한 잘 알려진 마커; 및 미세 소관 - 관련 단백질 2 (MAP2), 미세 소관의 조립에 필수적인 신경 세포 특이 적 세포 골격 단백질. 또한, 망막의 적층 구조는 그 폭과 연속성의 변화로 뮐러 세포 및 신경절 세포의 처리를 상세하게 분석 할 수있다. 따라서, 몇 가지 추가 매개 변수의 영향을 평가하기 위해 사용할BRB 위반 초기 단계에서뿐만 아니라 잠재적 인 치료의 반전 효과를 평가하는. 혈관 누출 (이어진), 아교 세포의 활성화 및 신경 세포의 손상 응답이 프로토콜에서, 약물 스크리닝의 전위 반전 효과는 세 가지 관점에서 평가된다. 몇몇 정량화 방법은 예를 들어, 발현이 면역, 프로세스 및 인핸스먼트 필터에 의해 도시 신경 프로세스 연속성 폭 측정 강도에 의해 도시 된 바와 같이, 사용된다. 더 나은 방법을 설명하기 위해 결과를 해석하는 데 도움이, 폭신 (lipoxin) A4 (LXA4), 내생 적 염증 부상에 대한 응답과 내피 기능 장애 (23)를 감쇠에서 합성 된 화합물은 데모 용으로 선택되었다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">1.45s</td>
			<td></td>
		</tr>
	</tbody>
</table>

an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

우리는 히스타민에 의해 유도 된 BRB 유출을 방지 할 수 잠재적 치료를 평가하는 저비용 시간 효율적이고 사용하기 쉬운 시스템을 제시한다. IgG의 제어 망막 (도 1a)의 용기 내에 구속되지만, 모델이 성공적으로 설정되었음을 확인 히스타민 노출 (도 1b)시 혈관 밖으로 유출. LXA4는 제시된 시스템의 BRB ?...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

낮은 - 비용, 사용하기 쉽고 강력한 시스템은 히스타민에 의해 유도 된 혈액 망막 장벽 위반을 개선 할 수 잠재적 인 치료를 평가하기 위해 설립된다. 혈관 누수, 뮐러 세포 활성화 및 신경 프로세스의 연속성 잠재적 약물 폭신 (lipoxin) A4를 갖는 손상 응답 및 역전을 평가하기 위해 이용된다.

치료를 위해 혈액 망막 장벽 위반 및 잠재적 인 마약을 평가하는 급성 망막 모델

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

Research

JoVE Journal

Medicine

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

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There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

연구

의학

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Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

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같은 산소 포도당 박탈과 재산 소화<em&gt; 체외</em&gt; 허혈 - 재관류 손상 모델 공부 뇌 혈관 장벽 부전

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

망막 질환의 평가에 대한 정량적 안저 자기 형광

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Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

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생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

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The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

설치류에 있는 허혈성 망막 질병을 위한 산소 유도한 망막병증 모형

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

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Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...