An NINDS Grant NS05033 (JCC) supported this work. The University of Connecticut RAC, SURF and OUR programs provided additional support.

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The authors have nothing to disclose.

우리는 도구와 쥐 및 인간에서 뇌의 심실 시스템의 무결성을 평가할 수있다 프로토콜을 제시한다. 이러한 도구는, 그러나, 또한, 14,21,22 또는 노화 과정에서 손상, 질환으로 인한 변화를 겪는 다른 뇌 구조 또는 기관 시스템에 적용될 수있다. 전략의 단면과 길이 MRI 시퀀스들의 정렬이 특정 영역 또는 관심있는 구조의 3 차원 볼륨 표현을 생성 할 수있는 소프트웨어의 인출 이점을 제시 하였다. 종 MRI 시퀀스 수 시간에 걸쳐 발생하는 총 뇌 체적비로 뇌실 총 뇌 부피, 및 / 또는 기타 뇌 구조 (예 subthalamic 핵 23 또는 뇌량 백색질을 포함하도록 확장 될 수있는 3 차원 볼륨 변경 컴파일 ) 확산 가중 텐서 영상을 사용하여. 함께, 뇌의 구조적인 변화의 포괄적 인 분석이 수행 될 수있다. 궁극적으로,조영 기술 컬렉션 뇌 구조 노화 관련 질병 - 관련 변화를 평가하기 위해 뇌의 건강 상태 (24)의 가장 정확한 그림을 제공하기 위해 필요하다. 함께 주제 주제 변동과 과목에 걸쳐 변화의 범위와 문서 및 중요한 구조적 데이터의 편집은, 이상적으로 건강 또는 질병에있는 조직의 최고 가이드 임상 진단에 초고 필드 MRI를지도 책을 생성하는 데 사용할 수 있습니다. 몇 가지 중요한 단계 사이와 대상 데이터 세트에서 수집 및 처리 변동성을 감소하기 위해주의하는 것이 중요하다. 이상적으로 유사한 데이터 시퀀스를 획득하기 위해, 동일한 MRI 스캐너와 서열 유형 연구 전반에 걸쳐 사용한다. 사후 뇌 조직은 종종 다양한 품질이고; 이러한 정착액에서 사망 원인, 사후 간격, 관류의 품질 및 시간의 길이가 중요한 요소 모두 염색 효능의 필요성에 영향을 미칠 수있다항원 검색 프로토콜. 또한, 인간의 뇌의 크기는 관심 영역을 포함하는 조직을 얻기 위해 추가 조작을 필요로하는 각각의 슬라이스를 다중 슬라이스 단면을 필요로한다. 따라서, 각 구역의 위치 및 방향이 명확하게 표시되어 기록하는 것이 중요하다. 궁극적으로, 사후 조직 분석 주요 문제는 정확하게 그 - 그것은 사후 - 그리고 따라서 수명이 조직의 스냅 뷰를 제공한다. 향상된 조영 기술들은 뇌 세포의 구조 및 해상도 모토 - 작용 정보에 대한 변경의 실시간 분석을 위해 요구된다. 마우스 연구는 변수에 의해 인간의 조건 변수의 심문을 허용하고 인체 조직 작업을 할 때 발견 된 문제의 많은 존재하지 않습니다. 인과 구조 역학의 분석을 통해 인간의 질병이나 상해를 모델링하는데 사용 마우스 연구는 질병 모델 발을위한 향상된 기능을 제공 할 수 있습니다전문화는. 그러나, 종 특이 차이에 유의해야한다. 측뇌실의 분석에서, 마우스로, 측뇌실의 측벽을 따라 활성 줄기 세포 틈새를 유지하고 라이닝 적당한 뇌실막 세포 손실의 경우에 발생하는 심실의 세포 매개 회생 수리 줄기 것을 주목하는 것이 중요하다 세 쥐에서 발견 또는 제한 뇌실막 세포 삭박이 ependyma의 노출을 통해 발생했을 때 19 뉴 라미니다 아제 할 수 있습니다. 어떤, 회생 가능성이있는 경우 반면에, 인간은, 측면 뇌실 벽 25-27 작은 함께 강력한 줄기 세포 틈새를 유지하지 않습니다. 세 마우스와 대조적으로, 세 인간 심실 확장 (6)와 관련된 심실 표면에서 광범위한 astrogliosis를 나타낸다. 발가 벗김과 심실 라이닝의 &#39;상처&#39;의이 레벨은 뇌실막 세포 6,28의 뇌실 안감을 발가 벗기다하는 뉴 라미니다 아제를 사용하여 마우스를 모델링 할 수 있습니다. 또한, 차를 인식하는 것이 중요하다동식물 사육장 - 유지 마우스에서 가장 인체에서 많은 다른 병력을 제시, 감염, 질병이나 외상 발생하지 않습니다. 또한, 마우스는 일반적으로 연령 관련 퇴행성 신경 질환, 뇌실 또는 뇌실 주위 백질 신경교 증을 표시하지 않습니다. 따라서, 그들은 모델링 할 필요가 이러한 표현형을 관찰합니다. 결국, 어떤 동물 모델은 완전히 개체 발생, 병태 생리, 인간 질병의 증상 복잡성을 요점을 되풀이 할 수 없으며 이러한 한계는 인정하고 적절하게 전달 될 필요가있다. 요약하면, 우리는 기술과 프로토콜이 마우스와 인간의 측면 뇌실 볼륨을 평가하기 위해 제시한다. 심실 3D로 렌더링 할 수 및 종 분석은 시공간 볼륨 변경의 편집을 할 수 있습니다. 또한, 한 쌍의 뇌 조직을 이용하여 우리는 심실 부피 변화에 링크 될 수있는 방법을 보여주는 세포 기능. 최근 결과 periventricu의 분야에서 아쿠아 포린-4 발현의 수준을 증가 시연LAR의 신경교 증은 심실 표면을 따라 부종을 제안한다. 조직의 조직 학적으로 FLAIR-MRI 페어링 따라서 미래 연구는 CSF 및 간질 액 교환에 대한 우리의 이해를 향상시키고, 심실 시스템 상태 및 방법의 열화가 다양한 뇌 기능에 영향을 미치는에 대한 중요한 정보를 제공 할 것입니다.

뇌실막 세포 단층 선 뇌척수액 (CSF) 및 격자 간 유체 (ISF) 1-3 간의 양방향 장벽 및 전송 기능을 제공하는 뇌의 심실 시스템. 이러한 기능은 뇌가 독성이없는 생리적 균형 2,3에서 유지하는 데 도움이됩니다. 부상 또는 질병을 통해이 안감의 일부의 인간 손실에서 다른 상피 라이닝에서 발견 회생 교체에 결과에 표시되지 않습니다; 오히려 뇌실막 셀 커버리지의 손실은 심실 표면에서 뇌실막 세포의 무 결함 영역을 커버하는 성상 세포의 메쉬 작업과 뇌실 주위 백질 as​​trogliosis 될 것으로 보인다. 중요한 CSF / ISF 교환 및 통관 메커니즘에 심각한 영향이 상피 층 1,2,4-7의 손실이 발생할 것으로 예상된다. 인간의 노화의 일반적인 기능은 측면 심실 (뇌실) 및 observ 등 관련 뇌실 주위 부종을 확대MRI 및 유체 감쇠 반전 회복 자기 공명 영상 (MRI / FLAIR) 8-14에 의해 에드. 뇌실과 심실 라이닝의 세포 조직 사이의 관계를 조사하기 위해, 사후 인간의 자기 공명 영상 시퀀스는 측면 뇌실 뇌실 주위 조직의 조직 학적 제제와 일치했다. 뇌실 가지 경우에서, 신경교 증의 실질적인 영역은 측면 뇌실 벽을 따라 뇌실막 세포 커버리지를 교체했다. 뇌실 확장이 자기 공명 영상 기반 볼륨 분석에 의해 감지되지 않은 경우, 뇌실막 세포 안감은 그대로이고 신경교 증은 심실 라이닝 (6)에 따라 검출되지 않았다. 이 조합 방식은 부분의 wholemount 준비 또는 전체 측면 뇌실 벽과 심실 볼륨 (6)의 3D 모델링을 사용하여 측면 뇌실 내벽의 세포 무결성의 첫 번째 포괄적 인 문서 자세히 변경을 나타냅니다. 여러 질환 (알츠하이머 병, 정신 분열증)과 부상 (외상성 뇌 손상)초기 신경 병리학 적 기능으로 뇌실을 보여줍니다. 이에 뇌실막 세포 라이닝의 지역의 삭박 정상 뇌실막 세포 기능을 방해하고 CSF / ISF 유체 및 용질 교환 사이의 항상성 균형을 손상 할 것으로 예상된다. 따라서, 기본 또는 이웃 뇌 구조에 심실 시스템, 그것의 세포 구성하고, 결과에 대한 변경 사항에 대해 더욱 철저한 검사는 궁극적으로 뇌실의 확대와 관련된 신경 병리학에 대한 자세한 내용을 공개하기 시작합니다. 함께 학적 조직 샘플에 대응하는 액세스가 제한된 조영 데이터의 부족, 특히 길이 데이터 시퀀스에서, 인간의 뇌 병리 분석 어렵게 만든다. 인간의 노화 또는 질병에있는 모델링 표현형은 종종 마우스 모델을 달성 할 수있는 동물 모델은 인간의 질병 개시 및 진행에 대한 질문을 탐구하기 위해 최선의 방법 중 하나가. 의 여러 연구건강한 젊은 마우스는 좌우 심실 벽의 cytoarchitecture 및 기본 줄기 세포 틈새 4,7-15을 설명했다. 이들 연구는 -6,15- 노화 통해 좌심실 벽의 3D 모델링 및 세포 분석을 포함하도록 확장되어왔다. 마우스는 비교적 강력한 subventicular 영역 (SVZ) -6,15- 라이닝 그대로 뇌실막 세포에 세포 틈새 바로 아래 줄기 표시 오히려 뇌실 주위 백질 신경교 증이나 뇌실 어느 쪽도, 세 생쥐에서 관찰된다. 따라서, 타격 종 특이 차이 -6,15- 노화 과정에서 일반적인 관리 및 뇌실 라이닝의 무결성에 모두 존재한다. 따라서, 인간에서 발견 조건을 심문하는 것이 가장 사용 마우스로 두 종 사이의 차이가 특징으로 적절 어떤 모델링 패러다임에서 고려 될 필요가있다. 여기, 우리는 모두 인간과 M의 측면 심실에 길이 변화와 관련된 뇌실 주위 조직을 평가하는 절차를 제시여러개. 우리의 절차는 세포의 조직과 구조 모두의 특성을 3D 렌더링 및 마우스와 인간의 심실 모두의 용적 측정, 및 뇌실 주위 조직의 전체 마운트 준비의 면역 조직 화학 분석의 사용을 포함한다. 이러한 절차는 함께 심실 시스템의 변화와 연관된 뇌실 조직을 특성화하는 수단을 제공한다.

<table border="0" cellpadding="0" cellspacing="0" width="881">
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:227px;">Name of the Materal/Equipment</td>
			<td style="width:363px;">Company</td>
			<td style="width:131px;">Catalog Number</td>
			<td style="width:160px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Use at 4% in PBS, 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Horse Serum</td>
			<td>Life Technologies</td>
			<td>16050</td>
			<td>10% in PBS-TX (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Life Technologies</td>
			<td>16210</td>
			<td>10% in PBS-TX (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100 (TX)</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>0.1% in PBS (v/v)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Imager.M2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA R2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Stage Controller</td>
			<td>Ludl Electronic Products</td>
			<td>MAC 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereology software</td>
			<td>MBF Bioscience</td>
			<td colspan="2">Stereo Investigator 11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereology software</td>
			<td>ImageJ/NIH</td>
			<td>NIH freeware</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D Reconstruction software</td>
			<td>MBF Bioscience</td>
			<td colspan="2">Neurolucida Explorer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Leica</td>
			<td>TCS SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MRI Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freesurfer</td>
			<td colspan="2">https://surfer.nmr.mgh.harvard.edu/fswiki/DownloadAndInstall</td>
			<td>Segmentation and Volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ITK-Snap</td>
			<td>http://www.itksnap.org/pmwiki/pmwiki.php</td>
			<td></td>
			<td>Segmentation and Volume</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-image Analysis GUI (Mango)</td>
			<td>http://ric.uthscsa.edu/mango/</td>
			<td></td>
			<td>Longitudinal overlay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Mount Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22.5&#176; microsurgical straight stab knife</td>
			<td>Fisher Scientific</td>
			<td>NC9854830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">parafilm</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">wax bottom dissecting dish&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aquapolymount</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Microscope</td>
			<td>Leica</td>
			<td>MZ95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Whole Mount Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse anti-b-catenin</td>
			<td>BD Bioschiences, San Jose, CA, USA</td>
			<td></td>
			<td>1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-GFAP</td>
			<td>Santa Cruz Biotechnology</td>
			<td></td>
			<td>1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-AQP4 (aquaporin-4)</td>
			<td>&#160;Sigma-Aldrich</td>
			<td></td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coronal Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-S100&#946; antibody</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Life Technologies</td>
			<td>D-1306</td>
			<td>10 &#181;g/mL in PBS</td>
		</tr>
	</tbody>
</table>

3d modeling of the lateral ventricles and histological characterization of periventricular tissue in humans and mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

면역 염색 50 μm의 관상 섹션과 3D 재구성 (그림 3)에 따라 마우스 측면 심실의 윤곽 추적은 볼륨 데이터가 질병이나 부상에 대한 모델 시스템으로 마우스를 사용하여 다른 실험 패러다임에서 수집 할 수 있습니다. 이 절차에 긴급 뇌실 벽이 서로 접착 영역을 배제한다. 심실의 영역 subsegmenting 각 영역 (도 3C)에 대해 다른 색을 지정함으로써, 인접한 절편을 준수 할 수 있고, 지역 및 총 부피는 컴파일 서브 세그먼트로부터 계산 될 수있다. 비슷한 연구는 3D 렌더링에 대한 측뇌실 반자동 세그멘테이션 (ITK-SNAP)와 함께 뇌 MRI 스캔을 사용하여 수행 될 수있다. 뇌실 볼륨의 직접 페어링과 뇌실 주위 조직 분석, 사전 또는 사후의 경우 자기 공명 영상 검사는 분할 및 (3D 복원을 만들 정렬 <stronG&gt; 그림 4). 종 MRI 스캔을 사용할 수있는 경우, 3D 재구성 정렬 및 시간이 지남에 확장의 분석 (그림 5)에 대한 중첩 될 수 있습니다. 세로 분석은 미래의 면역 조직 화학 분석을위한 특별한 관심 지역에 대한 정보를 제공합니다. 대응 조직은 심실 표면 (그림 6A)에 그대로 뇌실막 세포 단층 대 astrogliosis의 영역을 나타 내기 위해 면역 조직 화학적 분석된다. 조직의 이미지를 캡처 한 후 심실 표면 (그림 6B)의 넓은 지역을 커버 montaged있다. 컴파일 된 몽타주는 만화 표현으로 변환 한 후 뇌실 주위 세포 무결성 (그림 6C)에서 지역의 변화를 보여주기 위해 자기 공명 영상 기반의 2D 모델에 매핑됩니다. 심실 벽의 전체 마운트 준비는 심실 표면의 큰 넓은 구역의 탁 트인 전망을 허용, 또는 우리는 전체 측면 심실 표면 6 증명으로. 증가 L부종을 암시 표면 신경교 증의 영역에서 아쿠아 포린 -4- 발현 evels은 심실 라이닝 (6)의 무결성을 평가할 수있다. <img alt="그림 1" fo:content-width="6in" src="/files/ftp_upload/52328/52328fig1highres.jpg" width="600" /> 그림 1 : ITK / 스냅 뱀 투자 수익 (ROI) 도구 ()를 사용하여 측면 뇌실 분할 공연은 &#39;거품&#39;는 측면 심실에 추가됩니다. (B) &#39;거품&#39;활성 윤곽 진화하는 동안 확장합니다. 전체 측면 뇌실가 작성 될 때 (C) 분할이 완료됩니다. (케어는 제 3 뇌실 않도록주의한다.) <img alt="그림 2" fo:content-width="6in" src="/files/ftp_upload/52328/52328fig2highres.jpg" width="600" /> 그림 2 : 인간의 측면 뇌실 벽 해부 ( <str옹&gt; AD) 본래 반구 1.5 cm 두께의 관상 슬라브로 절단된다. 심실의 고립 2D 표현은 (D)에 도시되어있다. (E) 심실 벽의 부분은 밖으로 해부와 IHC에 대한 처리됩니다. 조직 세분 크기와 곡률에 따라 요구 될 수있다. 배향을 유지하기 위해, 조직은 심실 표면의 반대측 (녹색)의 상단에서 노치된다. 핀 (검은 점) 조직을 확보하고 최종 해부을 안내하는 데 사용됩니다 (붉은 점선). 최종 전체 마운트 제제는 심실 표면 (녹색)을 위로 향하게하여 슬라이드에 장착된다. <img alt="그림 3" src="/files/ftp_upload/52328/52328fig3highres.jpg" /> 그림 3 : 추적 및 S100β 면역 뇌실막 세포에 의해 설명 된 마우스 측면 심실 (측면 심실 포함 코로나 마우스 뇌 섹션의 3 차원 복원 (*, 측면 심실, 브래킷, adhE 유전자의 지역.<html> 시온; 스케일 바, 500 μm의). (B)는 마우스 측뇌실 체적 분석을 방해 뇌실 접착 영역을 제외한 &#39;등고선&#39;로 추적 및 하위 섹션으로 분할 배치되어있다. 측면 뇌실 윤곽의 (C) 3D 재구성. 측뇌실 함유 관심 영역에 걸친 전체 뇌 용적의 황색, 컨투어. <img alt="그림 4" fo:content-width="6in" src="/files/ftp_upload/52328/52328fig4highres.jpg" width="600" /> 그림 4 : 자기 공명 영상 기반 측면 뇌실 분할 (A) 자기 공명 영상이 조립된다. (B) 뇌실는 ROI (적색)으로서 정의된다. (C) 3 차원 영상 재구성은 체적 정량 및 측면 뇌실의 질적 시각화 할 수 있습니다. 광고 / 52328 / 52328fig5highres.jpg &quot;/&gt; 그림 5 : 종 뇌실 확장 세로 자기 공명 영상의 평가 여러 지점에서 정렬 및 시각화하고 시간이 지남에 따라 과목 내에서 뇌실의 부피 팽창을 정량화하는 오버레이 할 수 있습니다. <img alt="그림 6" src="/files/ftp_upload/52328/52328fig6highres.jpg" /> 그림 6 : 면역 조직 화학적 평가와 β-catenin의 염색으로 설명 인간의 측면 뇌실면 (A) 그대로 ependyma 세포 분야의 지역 매핑, 조약돌 모양 (별표)를 표시하고 심실 표면에 astrogliosis 지역에서 점선으로 경계가된다 (GFAP + 염색). (B)의 직렬 공 초점 이미지는 지역의 몽타주를 생성하는 중첩과 심실 표면에서 세포 조직의 만화 표현 어도비 포토샵을 사용하여 추적됩니다. 의 (C) 만화이미지 몽타주는 자기 공명 영상 기반 3 차원 심실 재구성 대응에 심실 표면에 매핑됩니다. (스케일 바 (A), 40 μm의, 스케일 바 (B), 1mm)

Watch this Scientific Journal Video about 3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse at JoVE.com

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

인간과 마우스의 뇌실 주위 조직의 측면 심실 및 조직 학적 특성의 3D 모델링

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

3d-modeling-lateral-ventricles-histological-characterization

Nanyang Technological University

Research

JoVE Journal

Neuroscience

13.9K 조회수.  University of Connecticut. Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain’s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice. 

비디오: 3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Periventricular Leukomalacia의 마우스 모델

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

연구

신경과학

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

피부와 사지 본즈 Innervating 마우스 감각 신경 섬유의 조직 준비 및 Immunostaining

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

Zebrafish 배아 뇌 심실의 수동 배수

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

Intact Histological Characterization of Brain-implanted Microdevices and Surrounding Tissue

Research into the design and utilization of brain-implanted microdevices, such as microelectrode arrays, aims to produce clinically relevant devices that interface chronically with surrounding brain tissue. Tissue surrounding these implants is thought to react to the presence of the devices over time, which includes the formation of an insulating "glial scar" around the devices. However, histological analysis of these tissue changes is typically performed after explanting the device, in a process that can disrupt the morphology of the tissue of interest.
Here we demonstrate a protocol in which cortical-implanted devices are collected intact in surrounding rodent brain tissue. We describe how, once perfused with fixative, brains are removed and sliced in such a way as to avoid explanting devices. We outline fluorescent antibody labeling and optical clearing methods useful for producing an informative, yet thick tissue section. Finally, we demonstrate the mounting and imaging of these tissue sections in order to investigate the biological interface around brain-implanted devices.

Here we present a histological method for capturing, labeling, optically clearing, and imaging the intact brain tissue interface around chronically implanted microdevices in rodent brain tissue. Results from the techniques comprising this method are useful for understanding the impact of various penetrating brain-implants on their surrounding tissue.

뇌 이식 Microdevices과 주변 조직의 손상 조직 학적 특성

Research  into the design and utilization of brain-implanted microdevices, such as  microelectrode arrays, aims to produce clinically relevant devices ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

마우스 뇌의 좌우 심실의 뇌실막 섬모의 라이브 영상

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

코어 텍스, Hippocampus-, Thalamus-, Hypothalamus-, 횡 중격 Nucleus-과 줄무늬 별<em&gt; 자궁 내</em&gt; C57BL / 6 마​​우스에서 Electroporation에

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

신경 죽음 및 마우스 기본 소 뇌과 립 신경에 변성을 모델링

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

깊은 공간-인간 두뇌의 측면 심 공개 뇌 구조 해부학 적 탐구

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has revealed microglia to be dynamic, capable of assuming both pro-inflammatory and anti-inflammatory phenotypes. Both M1 (pro-inflammatory) and M2 (pro-reparative) phenotypes play an important role in neuroinflammatory conditions such as perinatal brain injury, and exhibit differing functions in response to certain environmental stimuli. The modulation of microglial activation has been noted to confer neuroprotection thus suggesting microglia may have therapeutic potential in brain injury. However, more research is required to better understand the role of microglia in disease, and this protocol facilitates that. The protocol described below combines a density gradient centrifugation process to reduce cellular debris, with magnetic separation, producing a highly pure sample of primary microglial cells that can be used for in vitro experimentation, without the need for 2-3 weeks culturing. Additionally, the characterization steps yield robust functional data about microglia, aiding studies to better our understanding of the polarization and priming of these cells, which has strong implications in the field of regenerative medicine.

A protocol for the isolation of primary microglia from murine brains is presented. This technique aids in furthering the current understanding of neurological conditions. Density gradient centrifugation and magnetic separation are combined to produce sufficient yield of a highly pure sample. Furthermore, we outline the steps for characterization of microglia.

특성화 및 조밀도 기온 변화도 원심 분리 하 여 마우스 기본 Microglia의 절연

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

시냅스 가소성을 가진 수지상 척추의 3D 모델링

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...