Este estudo foi suportado por concessões do Vanderbilt-Ingram Cancer Center de Suporte Grant (P30 CA068485), a Childhood Foundation tumor cerebral e os Institutos Nacionais de Saúde (NS042205).

1. Micro dissecção de Tumor-rolamento Cerebelo e dissociação do tecido tumoral <ol><li> Recuperação do tecido tumoral <ol><li> Ratos doentes são muitas vezes tendo meduloblastoma runted, e exibir hidrocefalia e típicos sintomas neurológicos, incluindo paralisia posterior e insuficiência de recuperar a postura, quando capotou. Para recuperar tecido tumoral, euthanize ratos por inalação de dióxido de carbono. É imporant não realizar deslocamento cervical, um procedimento que gera pressão para a posterior do crânio e pode comprometer a integridade do tecido do tumor. </li><li> Decapitação é realizada imediatamente após a morte usando um par de tesouras, a remoção de pêlos e tecido muscular, tanto quanto possível para uma boa visualização do crânio. Limpe a superfície do crânio com Kimwipe embebido com álcool 95%. </li><li> Use uma tesoura fina para cortar uma abertura ao longo da linha média do crânio, e remover o tecido usando uma pinça fina crânio, altura em que todo o cérebro, incluindo tumor-rolamento cerebelo é exposto. </li><li> Enquanto o cerebelo de adultos saudáveis ​​mostrar bem definidas hemisférios e vermis, o cerebelo de camundongos portadores de tumor são muitas vezes alargada, amorfa com uma superfície lisa e vasos sanguíneos visíveis. Usando técnicas estéreis, recuperar o tumor cerebelar usando uma pinça e coloque em gelada de tampão fosfato salina (PBS) sem Mg 2 + e Ca 2 +. </li></ol></li></ol> Nota: todos os instrumentos são desinfectados em etanol 95% e vapor autoclavado antes da utilização. <ol start="2"><li> Dissociação do tecido tumoral <ol><li> Transferir o tecido tumoral a partir de PBS para Accutase 50% (diluído em PBS), que é cerca de 4 vezes o volume do tecido tumoral, mince o tecido com uma tesoura bem por 3 minutos em temperatura ambiente, seguido de incubação a 37 ° C por 4 minutos , após o qual o tecido passa por pipetagem repetitiva com um Pipetman de 1 ml por mais 3 minutos. Este método deve render uma mistura de células isoladas e pequenos agregados celulares. </li><li> Diluir a suspensão celular 3 vezes com PBS e centrifugar durante 5 minutos a 1000xg para agregar as células. (Os procedimentos acima foram descritos em &quot;enriquecimento de isolamento e manutenção de células-tronco meduloblastoma&quot;, Jove no prelo) </li></ol></li></ol> Ressuspender o pellet celular em recém-preparados meio de células-tronco neurais consistem de médio Neurobasal com glutamina, penicilina-estreptomicina, N2, B27, EGF humano (25 ng / mL) e FGF básico (25 ng / mL). Calcular a densidade de células da solução e as devidas diluições com meio de células-tronco neurais adicionais, que se pode carregar 4 mL de solução com um número adequado de células tumorais dissociada, por exemplo, 5x10 5, para uma seringa com ponta de bisel estéril 10μL. O número exato de células a serem inoculadas devem ser determinados pelo pesquisador de acordo com requisitos específicos experimental. Manter a solução de células em um tubo de microcentrífuga mini-200μL (tubo PCR) no gelo. 2. Procedimentos de anestesia para o destinatário Ratos Para o procedimento cirúrgico, uma área dedicada à cirurgia desinfectados roedores é necessário para a duração do procedimento. Idealmente, criar um banco comprido, com áreas separadas, mas adjacente para a preparação de animais, campo operatório e recuperação animal. Luvas cirúrgicas, luvas não exame, são necessários para a cirurgia. Técnica asséptica é mantido por dar as mãos com luvas acima da cintura e usado apenas para manipular objetos estéril de uma superfície seca e esterilizada. <ol><li> Uma mistura de cetamina (100 mg de cetamina por 1 kg de peso do mouse) e xilazina (10 mg de xilazina por 1 kg de peso mouse) é usado para anestesia. Administrar os anestésicos por via intraperitoneal. </li><li> Ele normalmente leva 3-5 minutos para os anestésicos para tirar pleno efeito. Determinar se um mouse é totalmente anestesiado por tep / cauda pinch. Coloque uma pequena quantidade de pomada oftálmica nos olhos do mouse. Trazer o mouse totalmente anestesiado para a área operacional, monitorando constantemente para se certificar de que o mouse está respirando normalmente. </li></ol> 3. Intracraniana Grafting das células tumorais <ol><li> Raspar a região da cabeça dorsal posterior do mouse, usando um cortador de cabelo, para revelar o crânio posterior acima do mesencéfalo e cerebelo. </li><li> Aplicar a solução sobre o couro cabeludo Betadine expostos usando um cotonete, seguido de limpeza com uma compressa embebida em álcool. Repita essas duas etapas de esterilização duas vezes. </li><li> Faça uma incisão quarto de polegada no couro cabeludo posterior, usando um bisturi esterilizado. Perfurar um orifício de trepanação 0,5 milímetros 2 milímetros para a direita e 2 mm posterior à lambda usando uma broca estéril dental. </li><li> Posicione o mouse em um quadro estereotáxico ligando seus incisivos para o porão frame. Cuidadosamente descer e posição do bisel com ponta de seringa de 10 mL carregado com 4 mL de solução de células tumorais no furo da rebarba. Uma vez que o chanfro do SyriESL agulha está abaixo da superfície do crânio, descendo para outro de 3 mm e, em seguida, subir para 0,5 mm. Injecte lentamente a quantidade desejada de células tumorais, com uma força constante em um período de tempo de 30 segundos, para o cerebelo do destinatário. Deixe a agulha em seu lugar após o término da injeção por mais dois minutos. Em seguida, retire a agulha e seringa. Clipe da ferida usando um autoclip. </li><li> Mantenha o mouse sobre um cobertor aquecimento após a cirurgia para ajudar a manter a sua temperatura corporal. Padrões de mobilidade e respiratórias serão observados de forma contínua. Uma vez que o mouse se recupera da anestesia, coloque-o novamente em uma gaiola de habitação estéril. Monitorar diariamente para consumo de alimentos e água, comportamento, aparência e coloração vermelho dos olhos. Verifique se há sinais de infecção no local da incisão. Remover o autoclips no dia após a cirurgia 7. Manter os ratos para uma média de 2-6 meses e sacrifício daqueles que apresentem sintomas típicos meduloblastoma para estudos posteriores. </li></ol> 4. Resultados representante Meduloblastomas secundário desenvolvido em camundongos altamente destinatário se assemelhavam aos dos tumores primários. O cerebelo portadores de tumores secundários foram ampliadas frequentemente, amorfa com os vasos sanguíneos ectópica aparente quando visto como um todo-montagens. Imunohistoquímica do tecido do tumor secundário revelou que as células do tumor são altamente proliferativas, expressa forte SmoM2-YFP e marcadores de precursores granulares do cerebelo neurônio, como Math1 e Pax6. Quando dissociado e cultivadas através de métodos relatados (&quot;O isolamento de enriquecimento, e manutenção de células-tronco meduloblastoma&quot;, Jove no prelo), as células do tumor secundário meduloblastoma podem ser expandidas rapidamente, marcadores de células-tronco expressar múltiplos, são clonogênica e pode ainda iniciar a formação de tumores quando transplantadas para recipientes adicionais imunocomprometidos. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="Figura 1" /> Figura 1. Típico de todo o monte e vistas de secções de um tecido meduloblastoma secundário Um exemplo típico de tecido meduloblastoma secundário desenvolvido 26 dias após a injeção de 5x10 5 células tumorais primárias. Coloração com hematoxilina revela a morfologia típica celular de meduloblastoma, incluindo relação nuclear / cytoplasmic elevada e polimorfismo nuclear. Estas células tumorais secundárias são altamente proliferativas, como indicado pela expressão Ki67 e também expressar robustamente membranosa SmoM2-YFP. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="Figura 2" /> Figura 2. Meduloblastoma células secundárias podem ser cultivadas e expandidas in vitro Utilizando o protocolo descrito para as células primário do mouse meduloblastoma (&quot;Isolamento de enriquecimento, e manutenção de células-tronco meduloblastoma&quot;, Jove no prelo), essas células meduloblastoma secundário pode ser cultivado e expandido para passagens múltiplas. A imagem mostra a colônia mesmo tumor cultivadas representante de 4 dias e 14 dias. As células tumorais cultivadas são bipolares, alongadas com citoplasma escasso e muitas vezes irradiam a partir de um núcleo denso do agregado celular. Eles não apresentam inibição por contato crescimento. As células pequenas redondas são as células vermelhas do sangue.

<ol>
	<li>Schuller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+granule+neuron+precursor+identity+is+a+critical+determinant+of+progenitor+cell+competence+to+form+Shh-induced+medulloblastoma.">Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Shh-induced medulloblastoma.</a> Cancer Cell. 14, 123-134 (2008).</li><li>Yang, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Kessler, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+alters+the+fate+of+preneoplastic+cells+in+a+mouse+model+of+medulloblastoma.">N-myc alters the fate of preneoplastic cells in a mouse model of medulloblastoma.</a> Genes Dev. 23, 157-170 (2009).</li><li>Flora, A., Klisch, T. J., Schuster, G., Zoghbi, H. Y., Y, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+Atoh1+disrupts+Sonic+Hedgehog+signaling+in+the+developing+cerebellum+and+prevents+medulloblastoma.">Deletion of Atoh1 disrupts Sonic Hedgehog signaling in the developing cerebellum and prevents medulloblastoma.</a> Science. 326, 1424-1427 (2009).</li><li>Salsano, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+neurogenic+basic+helix-loop-helix+transcription+factor+NEUROG1+identifies+a+subgroup+of+medulloblastomas+not+expressing+ATOH1.">Expression of the neurogenic basic helix-loop-helix transcription factor NEUROG1 identifies a subgroup of medulloblastomas not expressing ATOH1.</a> Neuro Oncol. 9, 298-307 (2007).</li></ol>

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Vários fatores críticos para garantir a célula meduloblastoma sucesso allografting que produz a formação de tumores secundários são os seguintes: Primeiro, use apenas Accutase 50% para o tecido dissociação e não mais de digerir o tecido como o tratamento enzimático prolongada leva à redução significativa da viabilidade celular. Também usar apenas o tamanho Pipetman 1 mL como dicas menores também podem gerar dano físico às células dissociadas. É bom ter uma mistura de células individuais e agregados ...

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<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
<td>&nbsp;</td>
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<td>hEGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0311</td>
<td>25ng/ml</td>
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<td>bFGF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>PHG0023</td>
<td>25ng/ml</td>
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<td>N2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17502048</td>
<td>1X</td>
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<td>B27(-RA)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12587010</td>
<td>1X</td>
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<td>Accutase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1110501</td>
<td>50% </td>
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<td>Glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030081</td>
<td>2mM</td>
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<td>Stereotaxic instrument</td>
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<td>Stoelting </td>
<td>51730</td>
<td>&nbsp;</td>
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<td>Foredom flexible shaft drill, hang-up style</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>58650</td>
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<td>Large probe holder</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51633</td>
<td>&nbsp;</td>
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<td>10 &mu;L syringe, 26 ga., bevel tip</td>
<td>&nbsp;</td>
<td>Stoelting </td>
<td>51105</td>
<td>&nbsp;</td>
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<td>Drill bit .75mm</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

Meduloblastoma é o tumor mais comum em idade pediátrica do sistema nervoso. Um grande número de estudos com animais tem se concentrado em precursores granulares do cerebelo neurônio (CGNPs) como a célula de origem para meduloblastoma 1-4. No entanto, as diversas apresentações clínicas de subtipos de meduloblastoma em pacientes humanos (nodular, desmoplásico, clássicos e grandes células / anaplásico), eo fato de que meduloblastoma é encontrado em um subgrupo de pacientes humanos com nenhuma expressão ectópica de CGNP marcador 5, sugerem que o origens celular e molecular de meduloblastoma são mais complexas e longe de ser completamente decifrado. Portanto, é essencial para determinar se existe um tumor meduloblastoma alternativa de células de origem com base em qual tipo de célula-modalidade terapêutica específica pode ser desenvolvida. Para este fim, ortotópico intracraniana allografting de organismos geneticamente marcadas tipos de células do tumor seguido por análises subseqüentes do desenvolvimento do tumor secundário em receptores permitirá a determinação da origem celular do tumor, iniciando células. Aqui nós descrevemos o protocolo experimental para ortotópico intracraniana allografting meduloblastoma de células derivadas do tecido do tumor primário, e esse procedimento também pode ser usado para o transplante de células de linhagens celulares estabelecidas.

Watch this Scientific Journal Video about Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice at JoVE.com

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Este protocolo descreve o isolamento e dissociação do mouse tecido meduloblastoma, e subsequente allografting das células tumorais em camundongos imunocomprometidos destinatário a fim de iniciar meduloblastoma secundário.

Allografting ortotópico intracraniana de Células Meduloblastoma em camundongos imunocomprometidos

Medulloblastoma is the most common pediatric tumor of the nervous system. A large body of animal studies has focused on cerebellar granule neuron ...

intracranial-orthotopic-allografting-medulloblastoma-cells

Research

JoVE Journal

Neuroscience

12.1K visualizações.  Vanderbilt University. Este protocolo descreve o isolamento e dissociação do mouse tecido meduloblastoma, e subsequente allografting das células tumorais em camundongos imunocomprometidos destinatário a fim de iniciar meduloblastoma secundário.

Vídeo: Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Enriquecimento de isolamento, e Manutenção de Células-Tronco Meduloblastoma

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

Neurociência

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Injeção intracraniana da adeno-associado vetores virais

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Farmacoterapia intracraniana e ensaios de dor em roedores

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

A detecção de 5-Hidroximetilcitosina em células-tronco neurais e cérebros de camundongos

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Medição quantitativa de proteínas sintetizadas intrathecally em camundongos

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

Registro eletrocorticográfico de áreas do córtex cerebral manipulado usando um vírus associado ao Adeno visando cofilina em camundongos

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.