Os autores gostaria de agradecer os doadores e suas famílias para sua generosa oferta. Obrigado ao Sr. Xiao Xuan Li que gravou o vídeo e ajudou com edição de vídeo; Sra. Hannah Lewis e Sr. Louis Szabo para fornecimento de técnico suporte; e o Professor Jan Provis para rever o vídeo e fornecer dados para o conteúdo de vídeo.

todos os métodos descritos aqui foram aprovados pelo Comitê de ética humana pesquisa da Universidade Nacional da Austrália. Para criar o modelo ventricular, usamos o Klingler fibra dissecação técnica 12 , 14. A técnica de Klingler é um método de dissecação tátil que envolve a remoção de pequenas porções de matéria cinzenta do córtex e descolar-se feixes de fibras nervosas, fornecendo assim um guia passo a passo através das camadas de tecido da superfície para as profundas estruturas do cérebro.
 Nota: O espécime de cérebro usado para demonstrar este protocolo no vídeo que acompanha e imagens cuidadosamente foi removido de um cadáver humano formalina-embalsamado, obtido do programa doador do corpo da faculdade de medicina, australiano Universidade Nacional. O doador não tinha conhecido histórico de doença neuropatológica. Após a remoção da dura-máter, o cérebro foi armazenado em solução 10% de etanol na temperatura de quarto por três anos.
 1. preparação <ol><li>obter um cérebro inteiro de um cadáver humano embalsamado e remover a dura-máter e armazenar o cérebro em 10% de etanol à temperatura ambiente antes da dissecação. 
	Atenção: Use equipamentos de proteção individual em um quarto ventilado de acordo com as diretrizes locais ao manusear. Certifique-se de todos os participantes estão familiarizados com os procedimentos institucionais para a manipulação segura e eliminação de um bisturi e sharp objetos antes de iniciar o protocolo de dissecação.</li>
	<li>Preparar os seguintes instrumentos: tesouras, pinças, lâminas de bisturi (n º 15 e n º 22), sonda de metal e o fim brusco de uma alça de metal bisturi ( Figura 1). Use a extremidade romba do punho bisturi para minimizar danos para as delicadas fibras nervosas e conservar a grande matéria branca fibra intervalos ( Figura 2) 13.</li>
	<li>Posicionar o cérebro para que sua superfície ventral é virada para cima.</li>
</ol> 2. Procedimento de dissecação Nota: A dissecação leva aproximadamente 2 a 3 h para completar <ol><li>remover a aracnoide e a vasculatura associada de ambos os hemisférios cerebrais, usando um par de Pinças atraumáticas (blunt).</li>
	<li>Gentilmente levante o cerebelo e localize o colliculi inferior. Coloque a lâmina de bisturi (n º 15) unida a um punho longo bisturi apenas caudal para o colliculi inferior e corte axialmente através do tronco cerebral. Mantenha a lâmina como perto de horizontal quanto possível para evitar danos no cerebelo. Tome cuidado para preservar o tectum do mesencéfalo.</li>
	<li>Posição do cérebro para ver a fissura lateral direita ou esquerda. Começando com o giro supramarginal, use a extremidade romba do punho bisturi para remover suavemente as camadas corticais superficiais. Movimente suavemente para a frente primeiro acima e, em seguida, abaixo do sulco lateral para revelar os feixes de fibra de associação horizontal executando no parietal, frontal e lobos temporais, respectivamente.</li>
	<li>Siga a direção das fibras arqueamento ao redor da borda posterior da insula conectando o fasciculi longitudinal superior e inferior para revelar o fascículo de arcuate.</li>
	<li>Anteriormente, retire com cuidado as restantes camadas corticais superficiais da média temporal e corticais frontais inferiores para revelar as fibras fascicular uncinadas, que se conectam a temporal e lóbulo frontal</li>
	<li>Identificar os curtos corticais do córtex insular e remova o insula. Em seguida, remova a cápsula extrema e claustrum para revelar a cápsula externa subjacente. Observe a protuberância formada pelo núcleo lentiforme profundo da cápsula. Se movendo em direção a superfície dorsal do córtex, revelar as fibras da Coroa radiada ( Figura 4).</li>
	<li>Remover o restante do córtex e subjacente substância branca na superfície dorsal do cérebro para alcançar o giro cingulado. Continuar a usar o sem corte-extremidade da alça bisturi para remover o córtex cingulado para revelar o cíngulo, o trato de matéria branca conectando a substância perfurada anterior com o giro parahipocampal.</li>
	<li>Usar a mesma técnica para remover o cíngulo de posterior para anterior revelam o corpo caloso, composto por fibras commissural conectando os dois hemisférios cerebrais. O dorso do corpo (tronco) do corpo caloso agora será visível ( Figura 6).</li>
	<li>Repita etapas 2.3 a 2.8 no hemisfério cerebral contralateral.</li>
	<li>Palpate e identificar a extensão do ventrículo lateral em um dos hemisférios. Usando uma sonda, perfure a parede lateral do ventrículo no local do trígono colateral. Usando uma lâmina de tamanho 24 (unida a um punho de bisturi n º 4) entram através do local de punção e corte inferiormente para abrir todo o comprimento do corno inferior do ventrículo lateral de.</li>
	<li>Agora retornam para o trígono colateral ventricular para estender o corte superiormente em direção a splenium do corpo caloso (linha pontilhada na Figura 5).</li>
	<li>Repetir passos 2.10 e 2.11 no outro hemisfério.</li>
	<li>Abrir o corpo do ventrículo lateral, continuando a incisão do trígono rostral usando um corte de aproximadamente 3 cm paralelo ao corpo caloso em ambos os hemisférios (linhas pontilhadas na Figura 6).</li>
	<li>Junte-se a duas incisões paralelas em cada hemisfério rostral ao nível do genu e caudalmente ao nível da splenium do corpo caloso. Usando a pinça, realizada na mão não dominante, levante suavemente o corpo caloso na splenium. Com uma pequena afiada tesoura, realizada na mão dominante, separe a splenium o septo pelúcido subjacente. Depois de ter atingido o fim rostral do corpo, corte do corpo caloso e retire la</li>
	<li>Nestlé longitudinalmente a superfície ventral do cérebro na palma da sua mão não-dominante para estabilizar as áreas temporais e occipitais (parte posterior). Ao mesmo tempo, usar sua mão dominante para firme mas delicadamente segurar a extremidade anterior do cérebro, colocando os dedos contrário e o polegar no núcleo lentiforme de ambos os lados do cérebro.</li>
	<li>Usando suave puxando e torcendo os movimentos, separar fisicamente as partes anteriores e posteriores do cérebro tendo especial cuidado para manter intacto o plexo coroide. É recomendável que um colega estar presente para orientar a separação e suavemente seção qualquer restantes conectando tecidos durante o processo usando um bisturi.</li>
</ol>

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Os autores declaram que não têm nenhum conflito de interesses.

O objetivo deste trabalho foi a elaboração de um guia de dissecação para divulgação para professores e alunos que poderiam ser usados para melhorar o ensino e a aprendizagem do profundo ventricular e estruturas periventriculares do cérebro humano. Nós criaram um guia passo a passo com acompanhamento de imagens, juntamente com um recurso de vídeo, que pode ser usado para ajudar a compreensão da morfologia dos ventrículos e suas estruturas associadas. A técnica de dissecação em si não é nova. Dissecação d...

Muitos alunos se esforçam para compreender os espaços negativos do sistema ventricular, localizado profundamente dentro do cérebro humano1,2. Comumente utilizados recursos disponíveis para os alunos a estudar os ventrículos fornecem representações relativamente brutas das intrincadas relações 3D dessas estruturas cerebrais profundas. Compreender a anatomia 3D do sistema ventricular e estruturas relacionadas é particularmente importante em neurocirurgia, porque o acesso ao sistema ventricular é uma das técnicas mais utilizadas para medir a pressão intracraniana, descomprimir o ventricular sistema e administrar medicamentos3. Além disso, os rápidos avanços em imagens médicas implicaram o desenvolvimento de habilidades na interpretação da Anatomia 3D.
Seções a bidimensional (2D) do cérebro em diferentes planos são normalmente usadas para visualizar as estruturas cerebrais profundas que formam os limites dos espaços negativos ventricular4. No entanto, 2D fatias do cérebro sozinho são insuficientes para permitir que os alunos a compreender toda a extensão da arquitetura 3D dos ventrículos e os detalhes da região como feixes de fibras conectando o córtex e estruturas subcorticais5. Por conseguinte, educadores têm que confiar na capacidade dos alunos para calcular uma concepção 3D compreensível dos ventrículos4. Os estudantes que lutam com consciência espacial encontrá-lo extremamente difícil criar esta imagem 3D. Enquanto modelos de plástico e moldes ventriculares fornecem uma representação 3D do sistema ventricular, falham ao demonstrar as relações abrangentes que formam os limites dos ventrículos. Os alunos muitas vezes negligentemente remover partes do modelo plástico para acessar o sistema ventricular e entender suas interconexões. Neste processo, eles frequentemente negligenciam detalhadas sobre posições relativas de cada estrutura em perdem a compreensão de suas relações (por exemplo, a formação do telhado dos ventrículos laterais pelo corpo caloso).
O desenvolvimento de novas ferramentas de ensino informatizado abordou algumas destas limitações. No entanto, muitos destes modelos são limitados para imagens e texto estático e não em aproveitar a interatividade oferecida por estas novas tecnologias7,8. Enquanto tecnologias interativas permitem ao usuário rodar modelos de computador 3D para estudar múltiplos pontos de vista, isso pode confundir alguns usuários, especialmente os noviços que desafio para orientar estruturas6. Além disso, recursos de computador interativo foram mostrados para ser menos eficaz em ensinar mais complexas estruturas anatômicas6. Assim, um dos desafios no ensino de neuroanatomia é proporcionar aos alunos os recursos que lhes permitam adequadamente Visualizar os ventrículos e apreciar sua estrutura 3D e relações anatômicas, incluindo o delicado associativa, projeção, e os molhos de fibra commissural que formam relações complexas com o de estruturas periventriculares2.
Dissecação tem demonstrada ser um excelente método de ensino para o aprendizado de anatomia7,8. Um estudo recente fornece evidências dos benefícios da dissecação do estudante na aprendizagem de neuroanatomia. Em 2016, Rae et al encontraram melhorada de retenção de curto e longo prazo dos conhecimentos de neuroanatomia em alunos participando de dissecações9. Enquanto os avanços da tecnologia continuarem a melhorar a precisão e a interatividade dos modelos de computador 3D, os conhecimentos adquiridos através de dissecação hands-on não podem ser replicados digitalmente no presente tempo10.
Neste estudo, tivemos como objetivo produzir uma dissecação reprodutível de um cérebro humano. Escolhemos um método de dissecação de fibra, porque isso permite preservação dos pacotes fibra delicado e estruturas de matéria cinzenta periventricular para melhor definem o espaço negativo dos ventrículos.
Aqui nós apresentamos um guia passo a passo completo para a criação de um modelo prosection dos ventrículos e estruturas periventriculares juntamente com um vídeo de treinamento que acompanha para usam em neuroanatomia, ensino e aprendizagem. Estes recursos podem ser usados para ensinar e aprender a neuroanatomia do cérebro por educadores e estudantes.

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Scalpel Blade No 15</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0205</td>
			<td style="width:167px;">Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade No 11</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0203</td>
			<td>Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Blade No 24</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0211</td>
			<td>Scalpel blade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Long Scalpel handle No3L</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0913</td>
			<td>Scalpel handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Short Scalpel handle No4G</td>
			<td style="width:113px;">Swann-Morton</td>
			<td style="width:119px;">0934</td>
			<td>Scalpel handle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Atraumatic Forceps</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Atraumatic forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothed Forceps</td>
			<td style="width:113px;"></td>
			<td style="width:119px;"></td>
			<td>Toothed forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Genelyn Arterial Enhanced</td>
			<td style="width:113px;">GMS Inovations</td>
			<td style="width:119px;">AE-475</td>
			<td>Arterial embalming media</td>
		</tr>
	</tbody>
</table>

exploring deep space - uncovering the anatomy of periventricular structures to reveal the lateral ventricles of the human brain

Estudantes de anatomia são normalmente fornecidos com seções a bidimensional (2D) e imagens quando estudando anatomia ventricular cerebral e estudantes encontrar este desafiador. Porque os ventrículos são espaços negativos localizados profundamente dentro do cérebro, a única maneira de entender sua anatomia é por apreciando seus limites formados por estruturas relacionadas. Olhando para uma representação 2D destes espaços, em qualquer um dos planos cardinais, não permitirá a visualização de todas as estruturas que formam os limites dos ventrículos. Assim, usar seções 2D sozinhos exige que os alunos calcular a sua própria imagem mental dos espaços 3D ventriculares. O objetivo deste estudo foi desenvolver um método reprodutível para dissecar o cérebro humano para criar um recurso educacional para melhorar o estudante compreender as intricadas relações entre os ventrículos e estruturas periventriculares. Para conseguir isso, criamos um recurso de vídeo que apresenta um guia passo a passo usando um método de dissecação de fibra para revelar os ventrículos laterais e terceiros juntamente com as estruturas intimamente relacionadas do sistema límbico e os gânglios basais. Uma das vantagens deste método é que ele permite que a delimitação dos tratos de substância branca que são difíceis de distinguir usando outras técnicas de dissecação. Este vídeo é acompanhado por um protocolo escrito que fornece uma descrição sistemática do processo para ajudar na reprodução de dissecação do cérebro. Este pacote oferece uma anatomia valiosa recurso de ensino para educadores e estudantes iguais. Seguindo estas instruções educadores podem criar recursos de ensino e alunos podem ser guiados para produzir seu próprios dissecação cerebral como uma atividade prática hands-on. Recomendamos que este guia de vídeo seja incorporado na neuroanatomia, ensinando a realçar o aluno compreensão da morfologia e relevância clínica dos ventrículos.

Esse método de dissecação expõe o sistema ventricular, separando o cérebro em uma anterior e uma parte posterior (Figura 7 e Figura 8). A parte posterior oferece uma vista interna para o trígono colateral do qual os cornos posteriores e inferiores podem ser vistos estendendo-se até o occipital e os lobos temporais, respectivamente (Figura 8). No corno inferior e temporal, o h...

Watch this Scientific Journal Video about Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain at JoVE.com

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

Explorando o espaço profundo - descobrindo a anatomia das estruturas periventriculares para revelar os ventrículos laterais do cérebro humano

Este documento demonstra o uso efetivo de um método de dissecação de fibra para revelar os panfletos superficial matéria branca e estruturas periventriculares do cérebro humano, no espaço tridimensional, para ajudar à compreensão do aluno de morfologia ventricular.

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

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Research

JoVE Journal

Neuroscience

16.5K visualizações.  Australian National University. Este documento demonstra o uso efetivo de um método de dissecação de fibra para revelar os panfletos superficial matéria branca e estruturas periventriculares do cérebro humano, no espaço tridimensional, para ajudar à compreensão do aluno de morfologia ventricular.

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

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.

Modelos de mouse de leucomalácia periventricular

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

Neurociência

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Análises morfométricas de secções da retina

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

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.

Drenagem Manual dos Zebrafish ventrículos cerebrais embrionárias

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

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.

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 ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Análise das Alterações Gene expressão no Rat Hippocampus Após Estimulação Cerebral Profunda do Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Investigating the Function of Deep Cortical and Subcortical Structures Using Stereotactic Electroencephalography: Lessons from the Anterior Cingulate Cortex

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure involves the chronic placement of multiple depth electrodes into regions of the brain typically inaccessible via subdural grid electrode placement. SEEG thus provides a unique opportunity to investigate brain function. In this paper we demonstrate how SEEG can be used to investigate the role of the dorsal anterior cingulate cortex (dACC) in cognitive control. We include a description of the SEEG procedure, demonstrating the surgical placement of the electrodes. We describe the components and process required to record local field potential (LFP) data from consenting subjects while they are engaged in a behavioral task. In the example provided, subjects play a cognitive interference task, and we demonstrate how signals are recorded and analyzed from electrodes in the dorsal anterior cingulate cortex, an area intimately involved in decision-making. We conclude with further suggestions of ways in which this method can be used for investigating human cognitive processes.

Stereotactic Electroencephalography (SEEG) is an operative technique used in epilepsy surgery to help localize seizure foci. It also affords a unique opportunity to investigate brain function. Here we describe how SEEG can be used to investigate cognitive processes in human subjects.

Investigar a função do Deep Cortical e estruturas subcorticais Usando estereotáxica Eletroencefalografia: Lições da Cortex cingulado anterior

Stereotactic Electroencephalography (SEEG) is a technique used to localize seizure foci in patients with medically intractable epilepsy. This procedure ...

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.

Imagens ao vivo da ependimária Cilia nas laterais ventrículos do cérebro do rato

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

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

Um de uma etapa simples protocolo de dissecação para Whole mount-Preparação do Adulto<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sublimação da DAN Matrix para a Detecção e Visualização de gangliosídeos no cérebro de um rato do tecido por MALDI imagem Espectrometria de Massa

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Gravação de plasticidade sináptica em fatias Hippocampal agudas, mantidas em um pequeno volume reciclagem - perfusão- e sistema de câmara de submersão-tipo

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...