Les auteurs tiennent à remercier m. David Rowe pour aimablement les souris transgéniques et Li Chen pour l’assistance histologique.
La recherche rapportée dans cette publication a été financée par le National Institute of Dental & Craniofacial Research de la National Institutes of Health, sous attribution numéro K08DE025914 et par l’Association américaine de Foundation orthodontique à Sumit Yadav.

<ol>
	<li>LeResche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Temporomandibular+Disorders:+Implications+for+the+Investigation+of+Etiologic+Factors.">Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors.</a> Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+functional+loading+causes+differential+effects+in+the+subchondral+bone+and+condylar+cartilage+in+the+temporomandibular+joint+from+young+mice.">Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice.</a> Osteoarthr Cartil. 17 (3), 354-361 (2009).</li><li>Pirttiniemi, P., Kantomaa, T., Sorsa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decreased+loading+on+the+metabolic+activity+of+the+mandibular+condylar+cartilage+in+the+rat.">Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat.</a> Eur J Orthod. 26 (1), 1-5 (2004).</li><li>Chavan, S. J., Bhad, W. A., Doshi, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+temporomandibular+joint+changes+in+Twin+Block+and+Bionator+appliance+therapy:+a+magnetic+resonance+imaging+study.">Comparison of temporomandibular joint changes in Twin Block and Bionator appliance therapy: a magnetic resonance imaging study.</a> Prog Orthod. 15 (57), (2014).</li><li>Dutra, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+Matrix+Response+of+the+Mandibular+Condylar+Cartilage+to+Botulinum+Toxin.">Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.</a> PLoS ONE. 11 (10), 0164599 (2016).</li><li>Benjamin, M., Ralphs, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fibrocartilage+cells.">Biology of fibrocartilage cells.</a> Int Rev Cytol. 233, 1-45 (2004).</li><li>Shen, G., Darendeliler, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Adaptive+Remodeling+of+Condylar+Cartilage-+A+Transition+from+Chondrogenesis+to+Osteogenesis.">The Adaptive Remodeling of Condylar Cartilage- A Transition from Chondrogenesis to Osteogenesis.</a> J Dent Res. 84 (8), 691-699 (2005).</li><li>Utreja, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Kaul, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Altered+Loading+on+Mandibular+Condylar+Cartilage.">The Effect of Altered Loading on Mandibular Condylar Cartilage.</a> PLoS ONE. 11 (7), 0160121 (2016).</li><li>Dyment, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput,+Multi-Image+Cryohistology+of+Mineralized+Tissues.">High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.</a> J Vis Exp. , e54468 (2016).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch Histol Cytol. 66 (2), 123-143 (2003).</li><li>Hayman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+(TRAP)+and+the+osteoclast/immune+cell+dichotomy.">Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.</a> Autoimmunity. 41 (3), 218-223 (2008).</li></ol>

Les auteurs n’ont aucun intérêt financier concurrentes.

Ce manuscrit décrit les méthodes de mesure morphométriques et analyse cellulaire des condyles mandibulaires murins et mandibules. Les mesures radiographiques morphométriques permet également d’analyser d’autres ossements de petits animaux de laboratoire. En outre, l’analyse cellulaire (quantification de la cellule et la cartographie de distance du cartilage) ne se limitent pas à du condyle mandibulaire rongeur, mais peut être utilisé pour quantifier les coupes histologiques de nombreux tissus.
Des modèles de souris transgéniques exprimant des reporters fluorescents sont d’excellents outils à comprendre visuellement les changements dans l’expression des gènes. Le modèle de souris double-collagène journaliste fluorescent (Col2a1XCol10a1) utilisé dans le présent rapport a été particulièrement adapté pour l’étude de la MCC, puisque les transgènes sont exprimés dans la région prehypertrophic et hypertrophique de la MCC ainsi que distribution de collagène pu être évaluée. Si le chercheur n’a pas accès à un modèle de souris transgénique exprimant reporters fluorescents, les autres méthodes d’analyse de l’expression protéique histologiques, tels que l’immunofluorescence, utilisable.
Pour les mesures morphométriques, l’étape essentielle avant de prendre des images de radiographie du cabinet est d’enlever tous les tissus mous de la mandibule ou des os à analyser. Musculaire excessive ou des tissus mous attachés aux os pourraient masquer les mesures réelles des structures. Il est important d’être conscient des limitations du système de radiographie du cabinet. Comme chaque rayon x, c’est une représentation de dimension 2 d’une structure en 3 dimensions et chevauchement des structures et des objets doit être interprétée avec soin. En outre, le positionnement des os dans le cabinet radiographique doit être cohérent.
La quantification cellulaire au moyen de compter le nombre de pixels positifs est une méthode efficace pour la quantification histologique dans une région riche en cellules tels que le MCC, mais la limitation de cette approche est qu’elle ne fournit pas le nombre exact de cellules. En outre, puisqu’il quantifie uniquement les pixels sélectionnés, il est recommandé de chiffrer toutes les images d’intérêt en utilisant le même pixel « échantillonnés » pour éviter la quantification des pixels des intensités différentes lors de l’analyse des images différentes.
Un Conseil lors de la quantification des coupes histologiques générales est d’analyser plusieurs coupes sériées de chaque échantillon (dans ce manuscrit, trois coupes sériées ont été analysés pour chaque condyle), étant donné que les variations au sein de chaque échantillon sont généralement observées.
La méthodologie mentionnée dans nos expériences est simple, facile à utiliser et peut être utilisée dans toute étude de tissus minéralisés dans lequel reporter souris sont utilisés. Avec la méthodologie existante, il est possible de visualiser les cellules qui sont colorées pour piège (Col2a1 ou Col10a1) ou EdU (Col1a1 ou Col2a1) en visualisant la co-localisation des cellules.

L’ATM est une articulation portante unique située dans la région cranio-faciale et est formé de fibrocartilage. Le CMC de l’ATM est essentiel pour la fonction articulaire, y compris le mouvement de mâchoire sans entrave tout en parlant et pétrissage, mais il est souvent affecté par des maladies dégénératives, notamment l’arthrose1. L’ATM a la capacité de s’adapter aux stimuli externes et des altérations de chargement, conduisant à des changements structurels et cellulaires aux composants du MCC2,3,4,5. Les propriétés porteuse du CMC peuvent s’expliquer par les interactions entre ses composants, y compris l’eau, le réseau de collagène et dense de protéoglycanes. Le MCC a quatre zones cellulaires distinctes qui expriment les différents types de protéines de collagène et non-collagène : 1) la zone superficielle ou articulaire ; 2) la zone proliférative, composée de cellules mésenchymateuses indifférenciées et qui répond au chargement des demandes ; 3) la zone de prehypertrophic, composé des chondrocytes matures exprimant le collagène de type 2 ; et 4) subissent une calcification et de zone, la région où les chondrocytes hypertrophiques exprimant collagène type die 10 le hypertrophique. La région non minéralisée est riche en protéoglycanes qui offrent une résistance aux forces de compression6.
Il y a la minéralisation continue à la zone hypertrophique de la MCC, où se produit la transition entre chondrogenèse ostéogenèse, garantissant la robuste structure minérale de l’os sous-chondral du condyle mandibulaire7. Changements cellulaires dans les régions non-minéralisée et minéralisées aboutir à des changements morphologiques et structurelles dans le condyle mandibulaire et de la mandibule. Maintien de l’homéostasie de toutes les régions cellulaires de la MCC et la minéralisation de la portion sous-chondral sont essentiels à la santé, capacité portante et l’intégrité de l’ATM.
Le modèle de souris transgénique de collagène multiples (comme décrit par Utreja et al.) 8 est un excellent outil à utiliser pour comprendre les changements dans l’expression de collagène car tous les transgènes sont exprimés dans le tableau. Pour une évaluation histologique approfondie, taches histologiques sont utilisés pour étudier les dépôts de matrice, minéralisation, prolifération cellulaire et l’apoptose, ainsi qu’expression de la protéine au niveau des couches cellulaires de la MCC.
Dans ce manuscrit, histologique et analyses morphométriques sont utilisés pour évaluer les changements structurels et cellulaires dans l’OS MCC et sous-chondrale du condyle mandibulaire de souris. En outre, une méthode de quantification de cellules, pour analyser des images histologiques fluorescents et de mappage des lames de microscope photonique, est décrite. L’ATM public static compression chargement méthode, ce qui provoque des modifications cellulaires et morphologiques à la MCC et sous-chondral OS9, est également illustrée pour valider nos méthodes.
Les méthodes décrites ici peuvent servir pour déterminer les caractéristiques morphométriques et histologiques dans le condyle mandibulaire et maxillaire inférieur des rongeurs ou d’analyser d’autres régions d’ossification endochondrale et la morphologie des tissus minéralisés supplémentaires.

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			<td height="21" style="height:21px;">ClickiT EdU Alexa Fluor 594 HCS kit</td>
			<td style="width:219px;">Life Technologies</td>
			<td style="width:119px;">C10339&#160;</td>
			<td>includes EdU (5-ethynyl-2&#39;-deoxyuridine)&#160;</td>
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			<td height="21" style="width:392px;height:21px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
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			<td height="21" style="width:392px;height:21px;">Sodium phosphate dibasic&#160;</td>
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			<td>S3264</td>
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			<td height="21" style="width:392px;height:21px;">Sodium phosphate monobasic&#160;</td>
			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">71505</td>
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			<td height="21" style="width:392px;height:21px;">Toluidine Blue O&#160;</td>
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			<td height="25" style="width:392px;height:25px;">Adobe Photoshop&#160;</td>
			<td style="width:219px;">Adobe Systems Incorporated</td>
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			<td height="24" style="width:392px;height:24px;">Phosphate buffered saline tablets (PBS)</td>
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			<td>Ortho Organizers, Inc.</td>
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			<td>GraphPad Software, Inc.</td>
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a morphometric and cellular analysis method for the murine mandibular condyle

L’articulation temporo-mandibulaire (ATM) a la capacité de s’adapter aux stimuli externes et chargement des changements peut affecter la position des condyles, ainsi que les composants structurels et cellulaires du cartilage condylien mandibulaire (MCC). Ce manuscrit décrit des méthodes pour l’analyse de ces changements et un procédé pour modifier le chargement de l’ATM chez la souris (c.-à-d., compression ATM statique de chargement). L’évaluation structurale illustrée ici est une approche morphométrique simple qui utilise le logiciel Digimizer et est réalisée dans les radiographies de petits os. En outre, l’analyse de systèmes cellulaires change conduisant à des altérations dans l’expression du collagène, remodelage osseux, la division cellulaire, et distribution de protéoglycanes dans le MCC est décrite. La quantification de ces changements dans les coupes histologiques - en comptant les pixels fluorescents positives à l’aide d’image logiciel et la cartographie de la distance de mesure et colorées de zone avec Digimizer - est également démontrée. Les méthodes indiquées ici ne se limitent pas à l’ATM murine, mais pourraient être utilisés sur les autres OS de petits animaux de laboratoire et dans d’autres régions de l’ossification endochondrale.

Statistiques descriptives ont été effectuées pour examiner la distribution des mesures morphométriques (longueur mandibulaire, longueur condylienne, largeur condylienne) et des analyses histologiques. Résultats ont été comparés entre le groupe chargé (c.-à-d., souris soumises à une charge de compression avec le ressort en titane bêta) et le groupe témoin (c.-à-d., correspondant à des souris témoins n’ayant pas reçu de toute procédure). Des différences statistiquement significatives entre les moyennes ont été déterminées par les célibataires t-essai et la p-valeur < 0,05 a été jugée statistiquement significative.
Mesures morphométriques, tel que décrit à l’étape 4 et Figure 3, on a effectué les mandibules de la charge et les groupes témoins. Le groupe chargé présenté avec la longueur de la mandibule une augmentation significative (chargé : 16,62 ± 0,23 mm / contrôle : 16.21 ± 0,2 mm ; p < 0,05) et la longueur de la tête condylienne (chargé : 4,6 mm, SD 0,08 mm / contrôle : 4,4 mm ; SD 0,06 ; p < 0,05) comparativement au groupe témoin. Néanmoins, il n’y avait aucune différence significative entre les groupes largeur de condyle (chargé : 3,06 ± 0,12 mm / contrôle : 2,9 ± 0,11 ; p = 0,09).
Quantification de la distribution de collagène à l’aide de la méthode décrite à l’étape 8 a révélé significativement a augmenté l’expression de Col10a1 dans le tableau des condyles du groupe chargé par rapport à la commande (chargé : 21 ± 4,46 % par opposition à contrôle : 7,50 % ± 2.03 % ; p < 0,005 ; Figure 8 A et D). En revanche, il n’y avait pas statistiquement différence d’expression Col2a1 entre la charge et le groupe témoin (chargé : 4,85 % ± 1,95 % par opposition à contrôle : 2,92 % ± 1,89 % ; p = 0,13 ; Figure 8 B et E). En outre, l’analyse de l’activité de piège a montré accrue des régions séropositifs au piège dans la région sous-chondrale des condyles de souris chargés (chargé : 5,28 % ± 1,45 % versus contrôle : 2,41 ± 1,39 % ; p < 0,05 ; Figure 8 C et F). De même, nous avons observé la prolifération cellulaire accrue, comme l’attestent des cellules positives EdU accrues dans le CMC du groupe chargé par rapport aux souris témoins (chargé : 6,23 % ± 1,89 % par opposition à contrôle : 1.90 % ± 1,03 % ; p < 0,05 ; Figure 9 A et C).
Distribution de protéoglycanes dans le MCC de chargés et les souris témoins a été quantifiée en évaluant la zone teinté bleu de toluidine et le plan de la distance, tel que décrit à l’étape 9 et Figure 7. Nous avons trouvé cartographie distance considérablement accrue dans le CMC des condyles du groupe chargé par le rapport de contrôle (chargé : 210.22 µm ± 4.11 µm / contrôle : 187.36 µm ± 8,64 µm ; p < 0,005 ; Figure 9 B et D). Toutefois, la zone tachée protéoglycane n’était pas statistiquement différente entre le chargement et le groupe témoin (chargé : 203,897.93 µm2 ± 10,171.00 µm2 / contrôle : 202,875.09 µm2 ± 33,419.09 µm2; p = 0,94 ; Figure 9 B et E)
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55998/55998fig8.jpg"> Figure 8 : Résultats représentatifs : (A) des cellules Col10a1 positives dans les coupes sagittales de la MCC des condyles de souris chargés et de contrôle. Il y a une augmentation du nombre des cellules positives Col10a1 dans le groupe chargé (D). (B) les cellules de Col2a1 positives en contrôle et chargé de souris. Il n’y a aucune différence dans des cellules positives Col2a1 entre groupes (E). (C) retiennent la coloration chez les condyles de contrôle et de la souris chargés. Augmentation de l’activité TRAP dans le groupe chargé par rapport à la commande (F). Les histogrammes (D-F) représentent le moyen ± SD pour n = 4 par groupe. ** Différence significative entre les témoins et les groupes chargés (p < 0,005). Echelle = 200µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig8large.jpg" target="_blank">s’il vous plaît cliquez ici pour visionner une version agrandie de cette figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55998/55998fig9.jpg"> Figure 9:résultats représentatifs : (A) EdU coloration chez condyles de contrôle et de la souris chargés. La prolifération cellulaire accrue dans le groupe chargé, représentée par EdU-positive accrue cellules (C). (B) le bleu de Toluidine coloration chez les condyles souris chargés du contrôle. A augmenté d’épaisseur de cartilage chez les souris chargés, comme en témoigne la cartographie distance accrue dans le groupe expérimental (D). Aucune différence dans la zone tachée protéoglycane entre les témoins et les groupes de chargé (E). Histogrammes (C-E) représentent le moyen ± SD pour n = 4 par groupe. ** Différence significative entre les témoins et les groupes chargés (p < 0,005). Echelle = 200 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig9large.jpg" target="_blank">s’il vous plaît cliquez ici pour visionner une version agrandie de cette figure.</a>

Watch this Scientific Journal Video about A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle at JoVE.com

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Ce manuscrit présente des méthodes d’analyse morphométrique et changements cellulaires dans le condyle mandibulaire des rongeurs.

Une méthode d’analyse cellulaire pour le Condyle mandibulaire murin et morphométriques

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

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Vidéo: A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

L&#39;utilisation de Rotorod comme une méthode pour l&#39;analyse qualitative de la marche chez le rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

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Biologie

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murin procédure transplantation rénale

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Une méthode à haut débit pour étudier la morphologie globalement organite chez S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

Une méthode à haut débit pour la cryoconservation du sperme de poisson zèbre et de In Vitro La fertilisation

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Un rapide à haut débit Méthode de cartographie ribonucléoprotéines (RNP) sur l&#39;homme pré-ARNm

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

Bleu d&#39;électrophorèse sur gel de polyacrylamide natif (BN-PAGE) pour l&#39;analyse des complexes multiprotéiques de lysats cellulaires

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un test pour évaluer le comportement des explants cellulaires du mésenchyme crânienne

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Microinjection unique cellule pour l&#39;analyse cellulaire de communication

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Isolement des cellules endothéliales murines Valve

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...