이 작품 "피코 Neuromed"에 의해 지원 되었고 건강 V.M. 게 "검색 전류"의 이탈리아 정부에 의해 자금

IRCCS Neuromed 동물 관리 검토 위원회에 의해 그리고 동물에 대 한 모든 절차 승인 &#34; Istituto 레 디 Sanità &#34; (허가 번호: 1163/2015-홍보) EU 지시문 2010의 지침에 따라 했다 / 63/EU 동물 실험에 대 한.
 1. 경 정 맥에 주입 FITC 민 솔루션의 준비 참고: 모든 실험 했다 매니페스트 (11 주 된) HD R6/2 마우스에 고 나이에 날 라와 일치 하는 성별 야생-타입 (WT) 제어 littermates.
<ol><li>디졸브 FITC-알 부 민 분말 (재료의 표 참조) 버퍼링 하는 인산 염 (PBS)에 10 mg/ml에서. 
	참고: 최적의 결과 얻으려면이 준비 되어야 한다 신선한 각 라벨 반응.</li>
</ol> 2. 수술에 동안 사용 장비의 준비 <ol><li>이전 압력가 수술 도구 (재료의 표 참조) 때 려 눕.</li>
	<li>알코올 (70% 에탄올 솔루션) 수술 벤치 청소. 외과 살 균 일회용 수건 드 레이프와 외과 영역 (45 x 45cm)를 준비 하 고 운영 stereomicroscope 설정.</li>
</ol> 3. 경 정 맥에 FITC 알 부 민 주입에 대 한 수술 <ol><li>마 취에 대 한 마우스 몸 무게 기록.</li>
	<li>케 타 민 (100mg/kg)-xylazine (10 mg/kg) 솔루션의 적절 한 복용량의 복 주사 (i.p)와 Anesthetize 마우스.</li>
	Walantus 외. 에서처럼 부드러운 발가락 핀치에 의해 <li>모니터 동물 진정 반사를 철회 11 , 12.</li> <li>보여지는 부정사 위치에 마 취 마우스를 놓고 접착 테이프로 수술 작업 대에 4 개의 다리와 꼬리를 수정.</li>
	<li>마우스 확보 후 신중 하 게 면도 전기 면도기와 함께 목에 적절 한 수술 여백 (재료의 표 참조) 및 povidone-요오드 솔루션 및 70% 에탄올으로 소독.</li>
	<li>면봉와 노출 된 면도 표면 건조.</li>
	<li>부드럽게 면도 피부를 당겨 하 고 1.5 cm 긴 세로 절 개를, 메스는 midclavicular에 의해 흉부에 중간 시작 하 고 바로 목 아래에 확장 라인.</li>
	<li>신중 하 게 외부 경 정 맥 노출 현미경 관찰 (5 배 확대)에서 집게와 떨어져 결합 조직 스트레칭.</li>
	<li>FITC 민 솔루션의 주입에 대 한 30½ G 바늘의 삽입을 촉진 하기 위하여 경 정 맥의 왼쪽 및 오른쪽에 충분 한 공간을 확인 하십시오.</li>
	<li>동물에 정 맥 주입 (오른쪽 경 정 맥) 천천히 (3 분 이상) 100 µ L 10 mL/kg FITC 알 부 민으로 30½ G 바늘을 사용 하 여.</li>
	<li>15 분 후, 잘린 여 마우스를 안락사, 급속 하 게 앞에서 설명한 13, 두개골에서 뇌를 제거 하 고 미리 냉장된 isopentane의 적어도 10 배 뇌 자체의 볼륨에서에서 15 분 담가.</li>
	<li>미리 냉장된 관으로 뇌를 냉동 isopentane 이동한 조직학 단면까지-80 ° C 냉동 고에 저장.</li>
</ol> 4. 조직학 단면의 뇌 주입 FITC 민 <ol><li>포함 복합 최적의 절삭 온도 (10 월)에 뇌를 냉동 (재료의 표 참조) 이전 cryostat 단면에.</li>
	<li>직렬는 cryostat와 20 µ m 두꺼운 부분, 뇌를 잘라 (재료의 표 참조), 현미경 유리 슬라이드에 탑재.</li>
	<li>는 FITC-알 부 민/laminin 공동 얼룩 특정 사용 하 여 수행 안티 laminin 항 체 (재료의 표 참조)으로 설명한 6.
	<ol><li>짧게, 밤에 주 항 체 안티 laminin (pAb 1:1000) 섹션을 품 어와 Cy3에 활용 된 적절 한 이차 항 체 진행.</li>
		<li>Coverslip 슬라이드 4를 포함 하는 설치 매체와 함께 &#39;, 6-diamidino-2-phenylindole (DAPI) (흡수 피크 = 360 nm; 방출 피크 = 460 nm (재료의 표 참조).</li>
		<li>슬라이드 어둠 속에서 4 ° C에서 건조 하룻밤 떠나.</li>
		<li>형광 현미경으로 얼룩이 지기 형광 관찰 (재료의 표 참조) 두 FITC를 갖춘 20 배 확대에 (흡수 피크 = 495 nm; 방출 피크 = 525 nm) 및 Cy3 필터 (흡수 피크 = 550 nm; 방출 최대 = 570 nm).
		<ol><li>샘플은 얼룩 직후, 매니큐어를 사용 하 여 모서리에 coverslip 보안을 보고 (4.3.3) 이상으로 현미경 검사.</li>
		</ol></li></ol></li></ol> 5. 형광 현미경을 설정 하 고 컬러 이미지의 수집 <ol><li>형광 현미경을 다음과 같이 설정 합니다.
	<ol><li>차례 형광 램프에 사용 하기 전에 약 10 분. 현미경, 연결 된 컴퓨터와 디지털 카메라를 켭니다. 이미지 분석 소프트웨어 실행 (재료의 표 참조).</li>
		<li>최적의 신호 수집 및 공간 해상도 대 한 적절 한 목표를 사용 하 고 적절 한 필터를 선택 합니다.</li>
	</ol></li> <li>수집의 컬러 이미지 <ol><li>이미지 분석 소프트웨어에 [라이브] 명령을 선택 (테이블의 자료를 참조) 각 필터에 대 한 최적의 조명 매개 변수를 설정 하 고.</li>
		<li>단일 채널 컬러 이미지를 눈금 막대 추가 [고정] 명령을 선택 (&#39; 편집 &#62; 굽기 규모 &#39;)</li>
		<li>각 이미지에 저장 된 &#39;.tiff &#39; 형식.</li>
		<li>단일 이미지를 선택 하 여 채널 병합 &#39; 파일 &#62; 병합 채널 &#39;에서 그것을 저장 하 고는</li>
		<li>tiff &#39; 형식.</li>
		<li>취득 4 24 비트 컬러의 최소 뇌 조각 (6 섹션 슬라이드 당) 당 (2200 µ m x 3400 µ m)을 사진.</li>
	</ol></li></ol> 6. FITC 민 넘쳐 흐름의 평가 <ol><li>자유롭게 사용할 수 ImageJ 14 , 15를 사용 하 여 각 채널에 대 한 형광 강도 분석.</li>
	<li>FITC-알 부 민 각 이미지 마다 총 CY3 Laminin 형광 강도 의해 총 형광 신호 강도 정상화 하 고 녹색 형광 강도로 혈관 밖에 서 extravasated 알 부 민을 보고.</li>
</ol>

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저자는 공개 충돌의 관심을가지고

우리는 여기에 설명 하는 기술 두뇌 질병 조건 하에서 BBB 누설을 탐지 하는 데 주로 유용 합니다. BBB 역 기능은 다양 한 신경학 적 장애4의 일반적인 기능으로 관심을 얻고 있다. 우리는 이전 HD6같은 희귀 신경 질환의 마우스 모델에서 BBB 무결성의 초기 혼란을 설명 하기 위해이 방법을 사용.
이 방법은 정확 하 고 신뢰할 수 있는 결과 제공 ?...

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author&#39;s names was corrected from:
Maglione Vittorio
to:
Vittorio Maglione

An erratum was issued for: <a href="https://www.jove.com/video/56389/assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc">Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease</a>. One of the author's names was corrected.

Erratum: Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

중앙 신경 조직 (CNS) 내의 항상성 적절 한 커뮤니케이션 및 신경 세포의 기능에 대 한 전제 조건입니다. CNS 실질은 단단히 봉쇄 주변에서는 내 피 혈액-뇌 장벽 (BBB), 주변 혈 류와 뇌 간의 인터페이스와이 두 지구1 사이 잡담에 중추적인 역할을 하 여 ,2. Bbb 등급은 복잡 하 고 동적 3 차원 구조는 주로 전문 마이크로-혈관 내 피 세포 (ECs) 세포 접합 단지-단단한 접속점 (TJs)-를 통해 서로 연결 하 고 pericytes에 의해 포위의 구성 신경 엔딩과 사이토 발 프로세스1,2.
생리 적인 조건 하에서 그대로 BBB의 매우 낮은 침투성 그리고 두뇌, 영양소와 다른 분자의 수송의 엄격한 규제를 보장 하 고 CNS에서 발생 하는 변화에서 독특한 보호를 제공 한다는 구성 및 가능성 주변에 대 한 신경 활동에 영향을 줄 수 있는 혈액의 모욕1,2,3.
그러나 BBB 무결성 및 그것의 향상 된 침투성의 많은 신경에 대 한 핵심 기능을 구성 하는 것 오랫동안 알려져 있다 및 신경 장애4 헌팅턴의 질병 (HD)5,6를 포함 하 여 이러한 장애 원인이 현상 여부 질병 과정에서 propagative 이벤트 아직 명확 하지 않습니다. 그러나 BBB 붕괴의 타이밍은 또한 애매 남아,, 우리의 그룹 및 다른 사람에 의해 증거 신흥 방해 BBB 무결성 늦은 이벤트를 질병의 진행, 하지만 오히려 초기 단계6에서,7 을 대표 하지 않는다 , 8, 장기 결과가 있을 수 있습니다.
이 염두에서에 두고, 그것은 성공적으로 대체 하 고 더 많은 타겟 개입의 수를 개발 하 고 질병의 진행 및 뇌 손상 예측에 유용한 전략을 개발 하기 위하여 조 neurodegeneration BBB 붕괴를 공개 하는 것이 중요 이러한 장애의 임상 결과 완화 BBB 장애의 신뢰할 수 있는 이미징, 그러므로, 실험 연구 및 뇌 질환의 임상 관리에 중요 한 중요성의 이다.
이 문서에서 우리는 높은 분자량 fluorescein isothiocyanate 표시-알 부 민 (FITC-알 부 민)을 사용 하 여 HD 마우스 모델에서 BBB 침투성의 평가 대 한 성공적이 고 간단한 절차를 설명 합니다. FITC-민, 일반적으로 뇌 실질에는 장벽을 교차 수 없습니다의 넘쳐 흐름 BBB 누설의 지표로 측정 되었다. 이 기술은 쉽게 적응할 수 있는 쥐 및 다른 병리학 조건 cerebrovasculature 장애9,10특징 이다.

<table>
	<tbody>
		<tr>
			<td>Albumin-fluorescin isothiocyanate conjiugate</td>
			<td>SIGMA</td>
			<td>A9771-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>pAb anti-Laminin</td>
			<td>Novus Biologicals</td>
			<td>NB300-144</td>
			<td></td>
		</tr>
		<tr>
			<td>CY3 anti-rabbit made in goat</td>
			<td>MILLIPORE</td>
			<td>AP132</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS</td>
			<td>Thermo Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Slips 24 X 50 mm</td>
			<td>Thermo Scientific (DIAPATH)</td>
			<td>61050</td>
			<td></td>
		</tr>
		<tr>
			<td>Kilik Optimal Cutting Temperature (OCT) compound</td>
			<td>Bio Optica</td>
			<td>05-9801</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Mounting Media</td>
			<td>VECTOR</td>
			<td>H-1500</td>
			<td>Mounting media with DAPI</td>
		</tr>
		<tr>
			<td>iNSu/Light Insulin Disposible Syringe</td>
			<td>RAYS Health &#38;Safety</td>
			<td>INS1ML26G13</td>
			<td></td>
		</tr>
		<tr>
			<td>30G 1/2&#34;</td>
			<td>BD Microlance</td>
			<td>304000</td>
			<td>Needle for Insulin disposible Syringe</td>
		</tr>
		<tr>
			<td>Scalpel Handle</td>
			<td>F.S.T.</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>#22 Disposable Scalpel blads</td>
			<td>F.S.T.</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris scissors 10.5 cm</td>
			<td>F.S.T.</td>
			<td>14094-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5745 45&#176; 0.10 x 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe Forceps 10 cm</td>
			<td>F.S.T.</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Forceps #5 0.1 X 0.06 mm</td>
			<td>F.S.T.</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical patch</td>
			<td>Medicalis</td>
			<td>34788</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable towel drape</td>
			<td>Dispotech</td>
			<td>TVO50VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopic Microscope</td>
			<td>NIKON</td>
			<td>SMZ 745 T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic Illuminator LED light (C-FLED2)</td>
			<td>NIKON</td>
			<td>1003167</td>
			<td>Optic Illuminator for Stereoscopic Micrscope</td>
		</tr>
		<tr>
			<td>Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>932162</td>
			<td>Epifluorescence Microscope</td>
		</tr>
		<tr>
			<td>Microscope digital Camera</td>
			<td>Nikon</td>
			<td>DS-Ri2</td>
			<td>Microscope camera</td>
		</tr>
		<tr>
			<td>Intenslight</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Microscope lamp</td>
		</tr>
		<tr>
			<td>NIS-Elements 64 bit</td>
			<td>Nikon</td>
			<td>AR 4.40.00</td>
			<td>Analysis Software</td>
		</tr>
		<tr>
			<td>Electric Razor</td>
			<td>Gemei</td>
			<td>GM-3007</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of blood-brain barrier permeability by intravenous infusion of fitc-labeled albumin in a mouse model of neurodegenerative disease

혈액-뇌 장벽 (BBB) 무결성의 중단은 다른 신경 및 신경 퇴행 성 질환에 대 한 일반적인 기능입니다. 교란된 BBB 항상성 및 뇌 질환의 병 인 사이 상호 작용 추가 조사 필요, 비록 개발 및 BBB 변경 정확 하 게 감지 하는 신뢰할 수 있는 프로시저의 유효성 검사 중요 한 그리고 유용한 도구를 대표 잠재적으로 질병을 예측에 대 한 진행 및 개발 대상 치료 전략.
여기, 우리는 그런 결함 BBB의 침투성에는 천재에 명확 하 게 감지할 수 있는 헌팅턴 질병의 전 임상 마우스 모델에서 발생 하는 신경 상태에서 BBB 누설을 평가 하기 위한 간단 하 고 효율적인 절차를 제시 질병입니다. 특히,는 높은 분자량 fluorescein isothiocyanate 표시 (FITC)-알 부 민, 장애인은 경우에 BBB를 교차 수 있는 마우스 경 정 맥 혈관 이나 parenchymal 지구에 그것의 배급에 심하게 융합은 다음 형광 현미경 검사 법에 의해 결정 됩니다.
뇌 실질에 녹색 형광 민의 축적 탈 BBB 침투성의 색인 기능 그리고 때 quantitated J 이미지 프로세싱 소프트웨어를 사용 하 여 그린 형광 강도로 보고 됩니다.

적절 한 주입 FITC 민의 BBB는 두뇌 parenchymawhen으로 혈 류에서 녹색 형광 추적 프로그램의 넘쳐 흐름에 경 정 맥 결과6손상. 생리 적인 조건 하에서 생긴된 형광 민의 뇌 혈관의 내부에 제한 된 이며 주변 혈관 주위 지역 및/또는 뇌 실질에 신호 감지 (그림 1A, 현미경에는 탑)입니다. 반대로, BBB 신경 병 적인 상황에서 손상 될 때 FITC 민 ?...

Watch this Scientific Journal Video about Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease at JoVE.com

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

이 연구에서 우리는 신경 퇴행 성 조건 하에서 혈액-뇌 장벽 장애를 평가 하기 위한 간단 하 고 효율적인 절차를 제시. 우리의 목표를 달성 하기 위해 우리는 높은 분자량 fluorescein isothiocyanate 표시 (FITC) 주입-마우스 경으로 알 부 민 정 맥 및 형광 현미경 검사 법에 의해 뇌 실질에 그것의 누설을 평가.

FITC 표시 된 알 부 민 신경 질환의 마우스 모델에서의 정 맥 주입에 의해 혈액-뇌 장벽 침투성의 평가

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

assessment-blood-brain-barrier-permeability-intravenous-infusion-fitc

Research

JoVE Journal

Neuroscience

9.8K 조회수.  IRCCS Neuromed. 이 연구에서 우리는 신경 퇴행 성 조건 하에서 혈액-뇌 장벽 장애를 평가 하기 위한 간단 하 고 효율적인 절차를 제시. 우리의 목표를 달성 하기 위해 우리는 높은 분자량 fluorescein isothiocyanate 표시 (FITC) 주입-마우스 경으로 알 부 민 정 맥 및 형광 현미경 검사 법에 의해 뇌 실질에 그것의 누설을 평가.

비디오: Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

마우스 모델에서 정맥 주사 자기 관리를위한 방법

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

연구

신경과학

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

로 불후의 손쥐 뇌 Microvascular 내피 세포 라인의 생성<em&gt; 체외에서</em&gt; 혈액 뇌 장벽 모델

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

초음파 혈액 - 뇌 장벽 파괴 및 망간 향상된 MRI를 사용하여 기능성 Neuroimaging

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process and may be a useful behavioral marker for early detection. Earlier detection in neurodegenerative disease is a major goal in the field because this is when neuroprotective therapies have the best potential to be effective. Therefore, in preclinical studies testing novel neuroprotective strategies in rodent models of neurodegenerative disease, olfactory assessment could be highly useful in determining therapeutic potential of compounds and translation to the clinic. In the present study we describe a battery of olfactory assays that are useful in measuring olfactory function in mice. The tests presented in this study were chosen because they measure olfaction abilities in mice related to food odors, social odors, and non-social odors. These tests have proven useful in characterizing novel genetic mouse models of Parkinson&#8217;s disease as well as in testing potential disease-modifying therapies.

Impairment in olfactory function is a common feature in many neurodegenerative disorders including Parkinson, Alzheimer, and Huntington diseases. In the present article, we describe a set of tests for assessing olfaction discrimination and detection in mice that can be used to measure olfactory abilities in mouse models of neurodegenerative diseases.

신경 퇴행성 질환의 마우스 모델에 대한 후각 분석 실험

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

영구와 광범위한 신경 세포의 형질 도입에 대한 신생아 마우스 뇌의 뇌실 바이러스 감염

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

퇴행성 신경 질환의 초파리 모델에서 등반 결함의 정량 분석

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

마우스 해마에 올리고머 아밀로이드 - 베타의 정위 주입

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Vivo에서 혈액-뇌 장벽 침투성 분석 결과 사용 하 여 마우스에 붙일 추적기 표시

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

허 혈 성 뇌졸중의 쥐 모델에서 뇌혈관 장애의 비보에 평가

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

병원 체 또는 의약품 및 두뇌와 그들의 상호 작용에 의해 장벽 횡단 연구를 인간의 혈액-뇌 인터페이스 모델

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...