Cette étude a été soutenue par des subventions du Vanderbilt-Ingram Cancer Center Grant Support (P30 CA068485), la Fondation des tumeurs cérébrales enfance et les National Institutes of Health (NS042205).

1. Micro-dissection des porteuses de tumeur cervelet et la dissociation du tissu tumoral <ol><li> Récupération de tissu tumoral <ol><li> Souris malades portant médulloblastome sont souvent chétifs, et afficher l&#39;hydrocéphalie et typique des symptômes neurologiques, y compris paralysie des membres postérieurs et l&#39;incapacité à retrouver une posture en cas de retournement. Pour récupérer le tissu tumoral, euthanasier les souris par inhalation de dioxyde de carbone. Il est imporant de ne pas effectuer dislocation cervicale, une procédure qui génère une pression sur le crâne postérieur et peut compromettre l&#39;intégrité des tissus tumoraux. </li><li> Décapitation est effectuée immédiatement après la mort en utilisant une paire de ciseaux, enlever les poils et les tissus musculaires, autant que possible pour une bonne visualisation du crâne. Nettoyer la surface du crâne avec Kimwipe imbibé d&#39;éthanol à 95%. </li><li> Utilisez des ciseaux pour couper fines une ouverture le long de la ligne médiane du crâne, et éliminer les tissus du crâne avec une pince fine, à quel point l&#39;ensemble du cerveau, y compris porteurs d&#39;une tumeur du cervelet est exposée. </li><li> Alors que les cervelets des adultes sains d&#39;affichage bien définis hémisphères et du vermis, les cervelets de souris porteuses de tumeurs sont souvent élargie, amorphe avec une surface lisse et les vaisseaux sanguins visibles. En utilisant des techniques stériles, extraire la tumeur du cervelet en utilisant une pince à épiler et le placer dans la glace froide tampon phosphate salin (PBS) sans Mg 2 + et Ca 2 +. </li></ol> Remarque: tous les instruments sont désinfectés dans de l&#39;éthanol à 95% et de vapeur autoclavés avant usage. </li><li> La dissociation du tissu tumoral <ol><li> Transférer le tissu tumoral du PBS pour Accutase 50% (dilué dans du PBS), qui est environ 4 fois le volume du tissu tumoral, émincer les tissus avec des ciseaux fins pour 3 minutes à température ambiante, suivie d&#39;une incubation à 37 ° C pendant 4 minutes , après quoi le tissu subit pipetage répétitifs avec un Pipetman de 1 ml pendant 3 minutes supplémentaires. Cette méthode devrait donner un mélange de cellules individuelles et de petits agrégats cellulaires. </li><li> Diluer la suspension cellulaire 3 fois avec du PBS et centrifuger pendant 5 minutes à 1000xg pour culotter les cellules. (Les procédures ci-dessus ont été décrits dans &quot;L&#39;isolement, l&#39;enrichissement et la maintenance des cellules souches médulloblastome&quot;, Jupiter, sous presse) </li> Reprendre le culot cellulaire dans fraîchement préparés moyennes cellules souches neurales composé de milieu Neurobasal avec de la glutamine, pénicilline-streptomycine, N2, B27, EGF humain (25 ng / ml) et FGF basique (25 ng / ml). Calculer la densité de cellules de la solution appropriée et de faire d&#39;autres dilutions moyennes avec d&#39;autres cellules souches neurales de telle sorte que l&#39;on peut charger 4 pi de solution avec un nombre approprié de cellules tumorales dissociées, par exemple 5x10 5, dans une stérile coniques à pointe seringue 10 ul. Le nombre exact de cellules à inoculer doit être déterminée par le chercheur en fonction des exigences spécifiques d&#39;expérimentation. Conserver la solution de cellules dans un microtube 200 pl mini (tube de PCR) sur la glace. </ol></li></ol> 2. Anesthésies pour les souris receveuses Pour l&#39;intervention chirurgicale, une zone désinfectée dédié à la chirurgie chez les rongeurs est nécessaire pour la durée de la procédure. Idéalement, mis en place un banc de long avec des zones séparées, mais voisines pour la préparation des animaux, champ opératoire et la récupération des animaux. Les gants chirurgicaux, des gants d&#39;examen non, sont nécessaires pour la chirurgie. Une technique aseptique est maintenu en tenant les mains gantées dessus de la taille et utilisées uniquement pour traiter des objets stériles à partir d&#39;une surface sèche stérile. <ol><li> Un mélange de kétamine (100 mg de kétamine par 1 kg de poids de la souris) et de xylazine (10 mg de xylazine par 1 kg de poids de la souris) est utilisée pour l&#39;anesthésie. Administrer par voie intrapéritonéale d&#39;anesthésiques. </li><li> Il prend généralement 3-5 minutes pour les anesthésiques de prendre pleinement effet. Déterminer si une souris est totalement anesthésié par tep / queue de pincement. Placer une petite quantité de pommade ophtalmique dans les yeux de la souris. Apportez de la souris totalement anesthésiés à la zone d&#39;exploitation, la surveillance constante pour s&#39;assurer que la souris est à respirer normalement. </li></ol> 3. Intracrânienne greffe des cellules tumorales <ol><li> Raser la région de la tête dorsale postérieure de la souris, en utilisant une tondeuse à cheveux, pour révéler le crâne au-dessus du mésencéphale postérieur et le cervelet. </li><li> Appliquer la solution Betadine sur le cuir chevelu exposés à l&#39;aide d&#39;un applicateur coton-tige, puis en l&#39;essuyant avec une compresse imbibée d&#39;alcool. Répétez ces deux étapes de stérilisation deux fois. </li><li> Faire une incision un quart de pouce dans le cuir chevelu postérieure à l&#39;aide d&#39;un scalpel stérilisé. Percez un trou de 0,5 mm bavure 2 mm à droite et 2 mm en arrière du lambda en utilisant une perceuse stériles dentaire. </li><li> Placez la souris sur un cadre stéréotaxique en accrochant ses incisives sur la cale de trame. Soigneusement descendre et la position du biseau incliné seringue de 10 uL chargé avec 4 pl de la solution de cellules tumorales dans le trou de trépan. Une fois que le biseau de l&#39;aiguille de la seringue est inférieure à la surface du crâne, descend pour une autre de 3 mm et ensuite monter à 0,5 mm. Injecter lentement la quantité désirée de cellules tumorales, avec une force constante dans un délai de 30 secondes, dans le cervelet du destinataire. Laisser l&#39;aiguille à sa place après la fin de l&#39;injection d&#39;un deux minutes supplémentaires. Ensuite, retirer l&#39;aiguille et la seringue. Clip de la plaie en utilisant un autoclip. </li><li> Gardez la souris sur une couverture chauffante après la chirurgie pour aider à maintenir sa température corporelle. Schémas de mobilité et respiratoires seront observés en permanence. Une fois que la souris se remet de l&#39;anesthésie, le replacer dans une cage de logement stérile. Moniteur quotidienne de nourriture et d&#39;eau, le comportement, le toilettage, et la coloration rouge des yeux. Inspectez les signes d&#39;infection au site d&#39;incision. Retirez le autoclips sur la chirurgie après jour 7. Maintenir la souris pour une moyenne de 2-6 mois et le sacrifice celles affichant les symptômes typiques médulloblastome pour des études ultérieures. </li></ol> 4. Les résultats représentatifs Médulloblastomes secondaire développé en souris receveuses très ressemblaient à celles des tumeurs primaires. Les cervelets portant des tumeurs secondaires ont souvent été élargie, amorphe avec des vaisseaux sanguins apparents ectopique lorsqu&#39;elles sont considérées comme l&#39;ensemble de montures. Analyses immunohistochimiques de tissus tumoraux secondaires ont révélé que les cellules tumorales sont très prolifératives, expriment une forte SmoM2-YFP et des marqueurs de cérébelleuse précurseurs de neurones granulaires, tels que Math1 et Pax6. Lorsque dissociés et cultivés selon des méthodes déclarés («L&#39;isolement, l&#39;enrichissement et la maintenance des cellules souches médulloblastome&quot;, Jupiter, sous presse), les cellules tumorales secondaires médulloblastome peut être développé rapidement, d&#39;exprimer de multiples marqueurs des cellules souches, sont clonogéniques et peut encore initier la formation de tumeur lorsque transplantées dans d&#39;autres destinataires immunodéprimés. <img src="/files/ftp_upload/2153/2153fig1.jpg" alt="Figure 1" /> Figure 1. Typiques entier monture et des vues en coupe d&#39;un tissu médulloblastome secondaires Un exemple typique de tissu médulloblastome secondaires développé 26 jours après l&#39;injection de 5x10 5 cellules tumorales primaires. Coloration hématoxyline révèle la morphologie cellulaire typique de médulloblastome, y compris nucléaires de haute activité / cytoplasmique ratio et le polymorphisme nucléaire. Ces cellules tumorales secondaires sont très proliférante, comme indiqué par l&#39;expression de Ki67 et ils ont aussi robuste expresse membraneuse SmoM2-YFP. <img src="/files/ftp_upload/2153/2153fig2.jpg" alt="Figure 2" /> Figure 2. Cellules de médulloblastome secondaires peuvent être cultivées et développées in vitro En utilisant le protocole décrit pour les cellules primaires médulloblastome de la souris («L&#39;isolement, l&#39;enrichissement et la maintenance des cellules souches médulloblastome&quot;, Jupiter, sous presse), ces cellules de médulloblastome secondaires peuvent être cultivées et élargi pour des passages multiples. La photo montre la colonie même représentant tumorales en culture à 4 jours et 14 jours. Les cellules tumorales en culture sont bipolaires, de forme allongée avec un cytoplasme peu et souvent rayonnent à partir d&#39;un noyau dense d&#39;agrégats cellulaires. Ils ne présentent pas d&#39;inhibition de contact de la croissance. Les petites cellules rondes sont des globules rouges.

<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>

Plusieurs facteurs essentiels pour assurer la cellule médulloblastome succès allogreffe que la formation de tumeurs secondaires sont des rendements comme suit: Premièrement, utilisez uniquement Accutase 50% pour les tissus de dissociation et de ne plus digérer le tissu comme traitement enzymatique prolongée conduit à une réduction significative de la viabilité cellulaire. Aussi utilisez uniquement le Pipetman taille 1 mL comme petits trucs peuvent aussi générer des dommages physiques aux cellules dissociées. ...

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<td>Neurobasal medium</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21103049</td>
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<td>&nbsp;</td>
<td>Invitrogen</td>
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<td>Invitrogen</td>
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<td>Invitrogen</td>
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<td>Invitrogen</td>
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intracranial orthotopic allografting of medulloblastoma cells in immunocompromised mice

Le médulloblastome est la tumeur la plus fréquente en pédiatrie du système nerveux. Un grand nombre d&#39;études ont porté sur des animaux précurseurs de neurones du cervelet granules (CGNPs) que la cellule d&#39;origine pour un médulloblastome 1-4. Cependant, les diverses présentations cliniques des sous-types médulloblastome chez les patients humains (nodulaire, desmoplastique, classique et à grandes cellules / anaplasique), et le fait que le médulloblastome se trouve dans un sous-ensemble de patients humains sans aucune expression ectopique de CGNP marqueurs 5, suggèrent que le origines moléculaires et cellulaires de médulloblastome sont plus complexes et loin d&#39;être entièrement déchiffré. Par conséquent, il est essentiel de déterminer s&#39;il ya une tumeur médulloblastome alternatives de cellules d&#39;origine sur la base duquel la cellule-type spécifique modalité thérapeutique peut être développée. À cette fin, orthotopique intracrânienne allogreffe d&#39;organismes génétiquement marqués types de cellules tumorales suivie par des analyses ultérieures de développement de tumeurs secondaires chez les receveurs permettra de déterminer l&#39;origine cellulaire des tumeurs initier les cellules. Nous décrivons ici le protocole expérimental pour orthotopique intracrânienne allogreffe de cellules provenant de tissus de médulloblastome tumeur primaire, et cette procédure peut également être utilisé pour la transplantation de cellules de lignées cellulaires établies.

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

Ce protocole décrit l&#39;isolement et la dissociation des tissus médulloblastome de la souris, et après allogreffe de cellules tumorales dans des souris immunodéprimées bénéficiaires afin d&#39;initier un médulloblastome secondaire.

Intracrânienne allogreffe orthotopique de cellules dans des souris immunodéprimées Médulloblastome

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

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12.1K vues.  Vanderbilt University. Ce protocole décrit l'isolement et la dissociation des tissus médulloblastome de la souris, et après allogreffe de cellules tumorales dans des souris immunodéprimées bénéficiaires afin d'initier un médulloblastome secondaire.

Vidéo: 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.

L&#39;isolement, l&#39;enrichissement et l&#39;entretien des cellules souches Médulloblastome

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

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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.

Injection intracrânienne des vecteurs adéno-associés virale

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

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

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

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.

Une méthode simple et peu coûteuse pour déterminer Sensibilité au froid et adaptation chez la souris

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.

Pharmacothérapie intracrânienne et dosages de la douleur chez les rongeurs

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.

La détection de 5-Hydroxymethylcytosine dans les cellules souches neurales et le cerveau des souris

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.

Mesure quantitative des protéines synthétisées intrathéquement chez les souris

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 ...

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.

Un modèle évolutif pour étudier les effets des blessures contondantes chez le poisson-zèbre adulte

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 ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Shuttle Box Assay en tant qu’outil d’apprentissage associatif pour l’évaluation cognitive dans les études d’apprentissage et de mémoire à l’aide de poissons-zèbres adultes

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Intracranial Pressure Monitoring In Nontraumatic Intraventricular Hemorrhage Rodent Model

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage animal models is essential. In this study, we sought out ways to measure intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during nontraumatic intraventricular hemorrhage in rats. The experimental design included three Sprague Dawley groups: sham, standard 200 &#181;l intraventricular hemorrhage, and vehicle control groups. By introducing an intraparenchymal fiberoptic pressure sensor, precise intracranial pressure measurements were obtained in all groups. Cerebral perfusion pressures were calculated with the knowledge of intracranial pressure and mean arterial pressure values. As expected, the intraventricular hemorrhage and vehicle control groups both experienced a rise in the intracranial pressure and subsequent decline in cerebral perfusion pressure during intraventricular injection of autologous blood and artificial cerebrospinal fluid, respectively. The addition of an intraparenchymal fiberoptic pressure sensor is beneficial in monitoring precise intracranial pressure changes.

Monitoring intracranial pressure in rodent&#160;models of nontraumatic intraventricular hemorrhage is not common in the current literature. Herein, we demonstrate a technique for measuring intracranial pressure, mean arterial pressure, and cerebral perfusion pressure during intraventricular hemorrhage in a rat&#160;animal model.

Surveillance de la pression intracrânienne dans un modèle de rongeur hémorragique intraventriculaire non traumatique

Survivors of intraventricular hemorrhage are often left with significant long-term memory impairment; thus, research utilizing intraventricular hemorrhage ...