Este trabalho foi apoiado por fundos de pesquisa do Conselho Nacional de Saúde e Pesquisa Médica da Austrália, o Instituto de Pesquisa Médica Hunter, e da Universidade de Newcastle.

Todos os procedimentos são realizados com a aprovação da Universidade de Newcastle Animal Care e do Comitê de Ética, bem como de acordo com as diretrizes e regulamentos pertinentes, incluindo a Lei de NSW Pesquisa Animal, o regulamento Animal Research NSW, e do Código australiano de Prática para a cuidado e uso de animais para fins científicos. 1. Dissecção do Tecido Cerebral <ol><li> Sacrifício ratos machos 12 semanas de idade por decapitação. Os ratos podem ser sacrificados por atordoamento e decapitação, ou pode ser primeiro anestesiados com isoflurano (5% de indução, manutenção de 1,5-2%) com 70% de N2 e 30% de O 2, e em seguida decapitados. </li><li> Remover o cérebro do crânio, tão rapidamente quanto possível (de preferência, este deve ser realizado em menos de 2 min). </li><li> Retire o córtex por dissecção à mão livre. A dissecção deve ser realizada no mínimo tempo possível, com o mínimo de danos no cérebro quanto possível, de modo a maintain a integridade das fatias. </li><li> A fim de remover qualquer resíduo de pele, pele, sangue, etc, imergir o cérebro de uma pequena quantidade (3-5 mL) de 37 ° C de Krebs-tampão (118 mM cloreto de sódio, cloreto de potássio 4,7 mM, cloreto de cálcio 1,3 mM, 1,2 mM de dihidrogenofosfato de potássio, sulfato de magnésio 1,2 mM, carbonato de hidrogénio de sódio 25 mM, glicose 11,7 mM, ácido etilenodiaminotetracético dissódico 0,03 (EDTA), equilibrado com 95% de oxigénio / 5% de dióxido de carbono (carbogénio), pH 7,4). </li></ol> 2. Preparação de Microslices <ol><li> Coloque o cérebro no palco de um triturador de Mcllwain em que dois pedaços de papel de filtro foram colocados e humedecido com tampão de Krebs quente. Mantenha o cérebro eo papel de filtro umedecido com tampão Krebs quente, mas não tão molhada que há líquido livre na superfície do papel, que pode causar o cérebro a deslizar. </li><li> Fatie o cérebro em 250 um cortes coronais. Como nutrientes, oxigênio, e resíduos ªa são normalmente trocados com o fornecimento de sangue são trocados com um fluido que rodeia o tecido em tecido neural isolado, o tamanho das fatias não deve ser mais do que 400 um de espessura para permitir a oxigenação e troca de nutrientes adequadas para ocorrer. </li><li> Vire o palco McIlwain 90 °. </li><li> Fatie cérebros em 250 mM fatias sagital. Aqui, estas secções do cérebro (250 um x 250 fim fatias de comprimento variável em função da espessura do tecido) são referidos como microslices. </li><li> Coloque as microslices para um tubo de fundo redondo com 37 ml de 10-15 ° C de tampão de Krebs (a quantidade de tampão de Krebs utilizada vai depender da quantidade de tecido). </li></ol> 3. A equilibração do Microslices <ol><li> Agite suavemente até microslices individuais são suspensas, e, em seguida, permitir-lhes que se contentar com a gravidade. </li><li> Remover o sobrenadante cuidadosamente, que irá ser turvo, devido aos restos de células em suspensão, e adicionar 10 ml fresco 37 ° CKrebs tampão para o tubo. </li><li> Repetir este processo de lavagem, quatro vezes, o que vai resultar na remoção da maioria dos detritos. </li><li> Seguindo este, equilibrar as microslices a 37 ° C com agitação suave / inversão, e mudar o tampão de Krebs a cada 15 min durante 1 hora no total. </li><li> Neste momento, adicionar 2 ml de tampão de 37 ° C de Krebs fresca, e transferir 15-20 microslices (150 ul de suspensão MicroSlice) ao poliestireno de fundo plano, utilizando tubos de 5 ml de um furo ponteira extra largo com arestas suavizadas (que pode ser criada por corte ~ 2 mm da extremidade da ponta de um P1000). Espalhe os microslices uniformemente sobre a base do tubo, em uma única camada, de modo que cada MicroSlice não é mais do que alguns milímetros da atmosfera oxigenada. </li></ol> 4. Estimulação excitotóxico <ol><li> Incubar as microslices a 37 ° C, e passam continuamente CARBOGEN sobre a superfície da suspensão de 150 ul MicroSlice, utilizando uma linha de gás que é a posiçãoed apenas acima da superfície da suspensão MicroSlice (para evitar ruptura mecânica). </li><li> Tratar microslices quer com 150 ul de 5 mM de glutamato + 100 mM de glicina no tampão de Krebs (estímulo excitotóxico: concentração final 2,5 mM de glutamato + 50 pM de glicina) ou 150 ul de tampão de Krebs sozinho (controlo não estimulados). Uma variedade de estímulos pode ser utilizado neste passo (incluindo, mas não limitado a, de AMPA, de NMDA + glicina, KCl despolarização, e privação de oxigénio / glucose). </li><li> Remover a solução de incubação a vários tempos pós-estimulação (0, 30, 60, 90, 120, e 300 seg), e substituí-la com 300 ul de tampão de homogeneização arrefecido em gelo (30 mM Tris, pH 7,4, 1 mM etilenoglicol ácido tetra-acético, 4 mM de EDTA, 100 ^ M de molibdato de amónio, pirofosfato de sódio a 5 mM, fluoreto de sódio 25 mM, ortovanadato de sódio 1 mM, phenylmethanesulfonylfluoride 1 mM, cocktail inibidor de protease completo). </li><li> Homogeneizar microslices no gelo, usando um copo de Teflon Dounce Homogeneizador(20 ciclos; de 700 rpm). </li><li> Armazenar homogeneizados a -80 ° C, e várias moléculas de sinalização, as moléculas de morte, etc, podem ser medidos por western blot. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3H]MK-801+binding+in+immature+and+mature+chicken+forebrain.">3H]MK-801 binding in immature and mature chicken forebrain.</a> Neurosci. Lett. 134 (1), 83-87 (1991).</li><li>Kavanagh, J. M., Bunn, S. J., Boyd, T. L., Rostas, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+glutamate+receptor+stimulated+inositol+phospholipid+metabolism+and+45Ca(2+)-accumulation+in+posthatch+chicken+forebrain.">Developmental changes in glutamate receptor stimulated inositol phospholipid metabolism and 45Ca(2+)-accumulation in posthatch chicken forebrain.</a> Neurosci. Lett. 194 (3), 161-164 (1995).</li><li>Sharps, E. S., McCarl, R. 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Os autores declaram não haver conflito de interesse em relação a qualquer dos trabalhos realizados no âmbito deste manuscrito.

Nisto, uma técnica in vitro em para a geração de microslices que podem ser utilizados para examinar os mecanismos moleculares envolvidos na excitotoxicidade e morte celular mediada por isquemia em tecido cerebral intacto madura é descrito. Esta técnica produz tecido viável (Figuras 1-3), que é metabolicamente semelhante para maiores fatias organotípicas 15. Além disso, este modelo MicroSlice corresponda à resposta observada após excitotoxicity mediada lesões cerebrais <em...

A forma mais comum de AVC é acidente vascular cerebral isquêmico, que ocorre quando um vaso sanguíneo cerebral torna-se obstruída. A isquemia do tecido que resulta da cessação do fluxo sanguíneo provoca a despolarização das membranas generalizada, a libertação de neurotransmissores excitatórios, e a elevação sustentada do cálcio intracelular, que conduz à activação de vias de morte celular 1. Este processo tem sido denominado &quot;excitotoxicidade&quot;, e é uma via comum envolvido na morte neuronal produzido por uma variedade de patologias, incluindo acidente vascular cerebral 2. A inibição das vias de sinalização envolvidas na excitotoxicidade e outras cascatas de morte celular neuronal é uma abordagem atraente para limitar os danos neuronal após o AVC. Identificar os mecanismos moleculares precisos envolvidos na excitotoxicidade e acidente vascular cerebral isquêmico pode ser difícil quando se utiliza sistemas animais inteiros. Como, embrionário primário, e &#39;neuronal-like&#39; (por exemplo, neuroblastoma e adenocarcinoma linhas imortalizados) sistemas de cultura de células são usadas frequentemente. As principais vantagens destes modelos é que eles são fáceis de manipular, relativamente eficaz em termos de custos, e a morte celular pode ser facilmente medido e quantificado. No entanto, as vias de sinalização pode ser alterada por condições de cultura utilizadas de 3,4, e os neurónios imaturos e imortalizado linhas podem expressar diferentes receptores e moléculas de sinalização, quando comparado a amadurecer cérebro 5-8. Além disso, os neurónios cultivados apenas permitir o exame de um tipo de célula (ou dois, se um sistema de co-cultura é utilizado), enquanto que o tecido cerebral intacto é heterogénea, contendo uma variedade de tipos de células que interagem uns com os outros. Sistemas de cultura de explantes (organotípicas de fatias finas de tecido de cérebro), também são utilizados, e estes modelos permite que o estudo de populações heterogéneas de células à medida que são encontrados in vivo. No entanto, apenas uma quantidade limitada de tecido pode ser obtida a partir de cada animal, ao utilizar esta técnica, as fatias podenão ser cultivados durante o tempo que as linhas de células imortalizadas, e meio de cultura de longo prazo pode resultar em alterações nas vias de sinalização e receptores nas fatias. Embora cérebro maduro pode ser usado para gerar fatias organotípicas, fatias de cérebro imaturo são mais propícios para a cultura, e são mais vulgarmente utilizados. Existe, portanto, a necessidade de se desenvolver modelos que imitam ou representam cérebro intacta madura, que são fáceis de usar, em que as vias de sinalização neuronal pode ser examinada. Nisto, uma técnica in vitro, que envolve o tecido nervoso intacto, que pode ser utilizado para elucidar os mecanismos moleculares envolvidos na morte celular na sequência de um insulto excitotóxico ou isquémico é descrito. Esta técnica reduz o número de animais necessários para realizar uma experiência, é reprodutível, e gera tecido viável que se comporta de uma forma semelhante à metabolicamente fatias organotípicas maiores. Além disso, o circuito neuronal, as interacções celulares, e co pós-sinápticampartment permanece parcialmente intacta. O tampão fisiológico utilizado permite que os membranas celulares para &#39;lacrar novamente&#39;, e permite que as células para recuperar sua resistência da membrana 9 original. Este modelo de fatia de cérebro é capaz de imitar fielmente as respostas observadas a seguir excitotoxicity mediada lesões cerebrais 10, e pode ser usado para examinar os mecanismos moleculares envolvidos no acidente vascular cerebral.

<table><tbody><tr><td>Guillotine</td><td></td><td></td><td>Used to decapitate animal</td></tr><tr><td>Surgical equipment</td><td></td><td></td><td>Forceps, scissors, tweezers, &lt;em&gt;etc.&lt;/em&gt;, for brain removal and dissection</td></tr><tr><td>McIlwain chopper</td><td>McIlwain choppers are manufactured/distributed by a range of companies including Mickle Engineering, Harvard Apparatus, Campden Instruments and Ted Pella.</td><td></td><td>Used to generate 100-400 &amp;#181;m brain sections.</td></tr><tr><td>Round bottom plastic tubes</td><td>Greiner</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>For keeping tissue at 37 &amp;deg;C</td></tr><tr><td>Humidifier/aerating apparatus</td><td></td><td></td><td>Used to keep microslices in a humidified, oxygenated environment</td></tr><tr><td>Flat bottomed polystyrene tubes</td><td>Nunc</td><td></td><td></td></tr><tr><td>Dounce Homogenizer</td><td></td><td></td><td></td></tr></tbody></table>

excitotoxic stimulation of brain microslices as an in vitro model of stroke

Examinando os mecanismos moleculares envolvidos em condições neuropatológicas, tais como acidente vascular cerebral isquêmico, pode ser difícil quando se utiliza sistemas animais inteiros. Como são comumente utilizados &#39;neuronal-como&#39; sistemas de cultura de células, tais, primária ou. Enquanto que estes sistemas são relativamente fáceis de trabalhar, e são úteis em sistemas modelo que vários resultados funcionais (tais como a morte celular) pode ser facilmente quantificados, os resultados analisados ​​e vias em neurónios imaturos em cultura (tais como as vias de morte celular mediada por excitotoxicity) não são necessariamente as mesmas que as observadas no cérebro maduro, ou em tecidos intactos. Portanto, existe a necessidade de desenvolver modelos em que os mecanismos celulares de tecido neural maduro podem ser examinados. Desenvolvemos uma técnica in vitro, que pode ser usado para investigar uma variedade de vias moleculares no tecido nervoso intacto. A técnica aqui descrita utiliza tecido cortical de rato, mas esta técnica pode ser adaptado para o uso de tecidoa partir de uma variedade de espécies (tais como o rato, coelho, cobaia, e galinha) ou regiões do cérebro (por exemplo, hipocampo, estriado, etc.) Além disso, uma variedade de estimulações / tratamentos podem ser utilizados (por exemplo, excitotóxica, a administração de inibidores, etc.) Em conclusão, o modelo de fatia cerebral aqui descrito pode ser usado para examinar uma variedade de mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade.

Microslices gerados utilizando este procedimento são viáveis, e uma variedade de espécies (por exemplo, rato, ratinho, galinha e) pode ser usado para produzir microslices. Três medidas independentes de viabilidade foram utilizadas: taxa de respiração (Figura 1), as relações de nucleótidos de adenina (figura 2), e o conteúdo de potássio do tecido (Figura 3). Utilizando estas medidas, foi demonstrado que microslices permanecem viáveis ​​durante pelo menos ...

Watch this Scientific Journal Video about Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke at JoVE.com

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Nós desenvolvemos um modelo de fatia de cérebro que podem ser utilizados para examinar os mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade. Esta técnica gera tecido cerebral maduro viável e reduz o número de animais necessários para a experimentação, mantendo o circuito neuronal, interações celulares e compartimentos pós-sinápticos em parte intacta.

Excitotóxico Estimulação do Microslices cérebro como um<em&gt; In vitro</em&gt; Modelo de AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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Research

JoVE Journal

Medicine

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

8.4K visualizações.  The University of Newcastle.  Nós desenvolvemos um modelo de fatia de cérebro que podem ser utilizados para examinar os mecanismos moleculares envolvidos na lesão cerebral mediada por excitotoxicidade. Esta técnica gera tecido cerebral maduro viável e reduz o número de animais necessários para a experimentação, mantendo o circuito neuronal, interações celulares e compartimentos pós-sinápticos em parte intacta. 

Vídeo: Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombosis is a quick, minimally-invasive technique for inducing small and well-delimited infarction in areas of interest in highly reproducible manner. It is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Isquemia Photothrombotic: Um Modelo de lesão cortical fotoquímica Minimamente Invasiva e reprodutível para mouse Estudos curso

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected ...

Medicina

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Um<em&gt; Ex vivo</em&gt; Modelo de um Ataque de T-Cell dirigiu-Oligodendrocyte em Agudos fatias de cérebro

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Imunologia e Infecção

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Neurociência

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and contributes significantly to the development of vascular dementia. Few models of focal white matter stroke exist and this lack of appropriate models has hampered understanding of the neurobiologic mechanisms involved in injury response and repair after this type of stroke. The main limitation of other subcortical stroke models is that they do not focally restrict the infarct to the white matter or have primarily been validated in non-murine species. This limits the ability to apply the wide variety of murine research tools to study the neurobiology of white matter stroke. Here we present a methodology for the reliable production of a focal stroke in murine white matter using a local injection of an irreversible eNOS inhibitor. We also present several variations on the general protocol including two unique stereotactic variations, retrograde neuronal tracing, as well as fresh tissue labeling and dissection that greatly expand the potential applications of this technique. These variations allow for multiple approaches to analyze the neurobiologic effects of this common and understudied form of stroke.

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

A Stroke Versátil Modelo murino de Subcortical Branco Matéria para o Estudo da axonal Degeneration e branco Matéria Neurobiology

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic fatias Hippocampal culturas como um modelo para estudo neuroproteção e invasão de células tumorais

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Uma abordagem alternativa para estudar eventos primários na neurodegeneração usando fatias de cérebro de rato Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Imagem de dois fotões de atração Microglial processos para ATP ou de serotonina no cérebro aguda fatias

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Um Modelo de Epipetogênese em Culturas de Fatia Organotipica Do Cortex-Hipocampo Rhinal

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Um modelo de cultura celular para estudar o papel das interações neurônio-glia na isquemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

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A Análise de Neurovascular Remodelação em Entorhino-hipocampal culturas Organotípicas Fatia

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Imagem de dois fotões de atração Microglial processos para ATP ou de serotonina no cérebro aguda fatias

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Um modelo de cultura celular para estudar o papel das interações neurônio-glia na isquemia

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model