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.

Nós apresentamos ferramentas e protocolos que podem ser utilizados para avaliar a integridade de sistema ventricular do cérebro em ratos e em seres humanos. Estas ferramentas, no entanto, pode também ser aplicada a outras estruturas cerebrais ou sistemas de órgãos que sofrem alterações devido a lesões, doença, ou durante o processo de envelhecimento 14,21,22. As estratégias apresentadas tirar proveito do software que permite o alinhamento de seqüências de RM transversais e longitudinais para gerar...

Um ependimais linhas monocamada de células do sistema ventricular do cérebro fornecendo funções de barreira e de transporte bi-direccionais entre o cerebral fluido espinhal (CSF) e fluido intersticial (ISF) 1-3. Estas funções ajudam a manter o cérebro e no equilíbrio fisiológico 2,3 livre de substâncias tóxicas. Nos seres humanos a perda de porções do revestimento esta, devido a lesão ou doença não parece resultar na substituição regenerativa como encontrado em outros revestimentos epiteliais; em vez de perda de cobertura de célula ependimária parece resultar em astrogliose periventricular com uma malha de astrócitos que abrangem regiões desnudados de células ependimais na superfície do ventrículo. As repercussões graves para mecanismos importantes CSF / troca ISF e de apuramento seria previsível como resultado da perda desta camada epitelial 1,2,4-7. Uma característica comum de envelhecimento humano é ampliado ventrículos laterais (ventriculomegalia) e edema periventricular associadas como observed por ressonância magnética e-fluido recuperação inversa da atenuação de ressonância magnética (MRI / DOM) 8-14. Para investigar a relação entre ventriculomegalia e a organização celular do revestimento do ventrículo, seqüências de RM humanos post-mortem foram combinados com preparações histológicas de tecido periventricular ventrículo lateral. Nos casos de ventriculomegalia, áreas substanciais de gliosis tinha substituído cobertura celular ependymal ao longo da parede do ventrículo lateral. Quando a expansão ventrículo não foi detectado por análise de volume com base em ressonância magnética, o revestimento celular ependimária estava intacto e gliose não foi detectada ao longo do revestimento do ventrículo 6. Esta abordagem combinatória representa a primeira documentação detalhando mudanças abrangentes na integridade celular do revestimento do ventrículo lateral usando preparações wholemount de partes ou toda a parede ventrículo lateral e modelagem 3D dos volumes do ventrículo 6. Várias doenças (doença de Alzheimer, esquizofrenia) e lesões (traumatismo crânio-encefálico)ventriculomegalia mostrar como um recurso neuropatológico cedo. Desnudação de áreas do revestimento celular ependymal assim seria previsto para interferir com a função das células ependymal normal e comprometer o equilíbrio homeostático entre CSF / fluido ISF eo intercâmbio soluto. Assim, um exame mais profundo de mudanças no sistema ventricular, a sua composição celular, ea consequência para estruturas cerebrais subjacentes ou vizinhos acabará por começar a revelar mais sobre a neuropatologia associada com aumento do ventrículo. A falta de dados de imagens multimodais, nomeadamente sequências de dados longitudinais, juntamente com acesso limitado a amostras de tecido histológico correspondente torna a análise de patologias cerebrais humanas difícil. Modelando fenótipos encontrados em envelhecimento humano ou doença muitas vezes pode ser conseguido com modelos do rato e modelos animais tornar-se um dos nossos melhores meios para explorar questões sobre iniciação doença humana e progressão. Vários estudos incamundongos jovens saudáveis ​​têm descrito a citoarquitetura das paredes do ventrículo lateral e o nicho de células-tronco subjacente 4,7-15. Esses estudos foram alargados para incluir a modelagem 3D e análise celular das paredes do ventrículo através de envelhecimento 6,15. Nem gliosis periventricular nem ventriculomegalia são observados em ratos envelhecidos, em vez ratos exibir uma zona subventicular relativamente robusta (SVZ) tronco nicho de células subjacente a uma célula ependymal intacto forro 6,15. Assim, as diferenças específicas de espécies de batimento existir tanto na manutenção geral e integridade do revestimento ventrículo lateral durante o processo de envelhecimento 6,15. Portanto, para melhores ratinhos uso para interrogar condições encontradas em seres humanos, as diferenças entre as duas espécies têm de ser caracterizada apropriadamente e considerado em qualquer paradigma de modelagem. Aqui, apresentamos os procedimentos para avaliar as mudanças longitudinais para os ventrículos laterais e do tecido periventricular associadas em seres humanos e mouse. Nossos procedimentos incluem renderização em 3D e volumetria do mouse e ventrículos humanos e uso de análise imuno-histoquímica das preparações inteiras de montagem de tecido periventricular para caracterizar tanto organização e estrutura celular. Em conjunto, estes procedimentos proporcionam um meio para caracterizar as alterações no sistema ventricular e tecido periventricular associado.

<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.

Traçado do contorno dos ventrículos laterais do rato com base em imunomarcadas 50 mm cortes coronais e reconstruções em 3D (Figura 3) permite que os dados de volume a ser coletado em diferentes paradigmas experimentais utilizando o mouse como um sistema modelo para a doença ou lesão. Crítico para este procedimento é a exclusão de regiões onde as paredes ventriculares laterais aderir uns aos outros. Por subsegmenting regiões dos ventrículos e designa uma cor diferente para cada região <stron...

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.

Modelagem 3D do lateral ventrículos e histológica Caracterização do Tecido Periventricular em humanos e mouse

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

Research

JoVE Journal

Neuroscience

13.9K visualizações.  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. 