Name: in vivo vascular permeability detection in mouse submandibular gland
Uploaded: 2022-08-04T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

Cette étude a été financée par la Fondation nationale des sciences naturelles de Chine (subventions 31972908, 81991500, 81991502, 81771093 et 81974151) et la Fondation des sciences naturelles de Beijing (subvention 7202082).

Toutes les procédures expérimentales ont été approuvées par le Comité d’éthique de la recherche animale du Centre des sciences de la santé de l’Université de Pékin et étaient conformes au Guide pour le soin et l’utilisation des animaux de laboratoire (publication des NIH n ° 85-23, révisée en 1996). Des souris mâles de type sauvage (WT) âgées de 8 à 10 semaines ont été utilisées pour la présente étude. Les animaux de laboratoire ont été soigneusement traités pour minimiser leur douleur et ...

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	<li>Carpenter, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretion,+components,+and+properties+of+saliva.">The secretion, components, and properties of saliva.</a> Annual Review of Food Science and Technology. 4, 267-276 (2013).</li><li>Garrett, 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=The+proper+role+of+nerves+in+salivary+secretion:+A+review.">The proper role of nerves in salivary secretion: A review.</a> Journal of Dental Research. 66 (2), 387-397 (1987).</li><li>Berndt, P., 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=Tight+junction+proteins+at+the+blood-brain+barrier:+Far+more+than+claudin-5.">Tight junction proteins at the blood-brain barrier: Far more than claudin-5.</a> Cellular and Molecular Life Sciences. 76 (10), 1987-2002 (2019).</li><li>Cong, X., 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=Disruption+of+endothelial+barrier+function+is+linked+with+hyposecretion+and+lymphocytic+infiltration+in+salivary+glands+of+Sj&#246;gren's+syndrome.">Disruption of endothelial barrier function is linked with hyposecretion and lymphocytic infiltration in salivary glands of Sj&#246;gren's syndrome.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (10), 3154-3163 (2018).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nature Methods. 2 (12), 932-940 (2005).</li><li>Zipfel, W. 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S., 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=Functional+dissociation+of+paracellular+permeability+and+transepithelial+electrical+resistance+and+disruption+of+the+apical-basolateral+intramembrane+diffusion+barrier+by+expression+of+a+mutant+tight+junction+membrane+protein.">Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.</a> The Journal of Cell Biology. 134 (4), 1031-1049 (1996).</li><li>Enis, D. 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=Induction,+differentiation,+and+remodeling+of+blood+vessels+after+transplantation+of+Bcl-2-transduced+endothelial+cells.">Induction, differentiation, and remodeling of blood vessels after transplantation of Bcl-2-transduced endothelial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (2), 425-430 (2005).</li><li>Wang, X., 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=Application+of+digital+subtraction+angiography+in+canine+hindlimb+arteriography.">Application of digital subtraction angiography in canine hindlimb arteriography.</a> Vascular. 30 (3), 474-480 (2022).</li><li>Trani, M., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+in+the+control+of+vascular+permeability:+vascular+endothelial-cadherin+and+other+players.">New insights in the control of vascular permeability: vascular endothelial-cadherin and other players.</a> Current Opinion in Hematology. 22 (3), 267-272 (2015).</li><li>Viazzi, F., 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertension Research. 31 (5), 873-879 (2008).</li><li>Scheppke, L., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> The Journal of Clinical Investigation. 118 (6), 2337-2346 (2008).</li><li>Blanchet, M. 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=Loss+of+CD34+leads+to+exacerbated+autoimmune+arthritis+through+increased+vascular+permeability.">Loss of CD34 leads to exacerbated autoimmune arthritis through increased vascular permeability.</a> Journal of Immunology. 184 (3), 1292-1299 (2010).</li><li>Egawa, G., Ono, S., Kabashima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+Imaging+of+vascular+permeability+by+two-photon+microscopy.">Intravital Imaging of vascular permeability by two-photon microscopy.</a> Methods in Molecular Biology. 2223, 151-157 (2021).</li><li>Vestweber, D., Wessel, F., Nottebaum, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarities+and+differences+in+the+regulation+of+leukocyte+extravasation+and+vascular+permeability.">Similarities and differences in the regulation of leukocyte extravasation and vascular permeability.</a> Seminars in Immunopathology. 36 (2), 177-192 (2014).</li><li>Schulte, D., 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=Stabilizing+the+VE-cadherin-catenin+complex+blocks+leukocyte+extravasation+and+vascular+permeability.">Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability.</a> The EMBO Journal. 30 (20), 4157-4170 (2011).</li><li>Uhl, B., 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=A+novel+experimental+approach+for+in+vivo+analyses+of+the+salivary+gland+microvasculature.">A novel experimental approach for in vivo analyses of the salivary gland microvasculature.</a> Frontiers in Immunology. 11, 604470 (2020).</li></ol>

Le maintien et la régulation de la fonction barrière endothéliale sont essentiels à l’homéostasie vasculaire. Les cellules endothéliales et leurs jonctions intercellulaires jouent un rôle essentiel dans le maintien et le contrôle de l’intégrité vasculaire12. La force de cisaillement du flux sanguin, les facteurs de croissance et les facteurs inflammatoires peuvent provoquer des changements dans la perméabilité vasculaire et, ainsi, participer à l’apparition et au développement ...

Diverses glandes salivaires produisent de la salive, qui agit principalement comme première ligne de défense contre les infections et aide à la digestion, jouant ainsi un rôle essentiel dans la santé bucco-dentaire et globale1. L’approvisionnement en sang est crucial pour la sécrétion des glandes salivaires car il fournit constamment de l’eau, des électrolytes et des molécules qui forment la salive primaire. La fonction barrière endothéliale, régulée par le complexe de la jonction serrée (TJ), limite strictement et délicatement la perméation des capillaires, qui sont hautement perméables à l’eau, aux solutés, aux protéines et même aux cellules se déplaçant des vaisseaux sanguins circulants dans les tissus des glandes salivaires 2,3. Nous avons précédemment constaté que l’ouverture des TJ endothéliaux en réponse à un stimulus cholinergique facilite la sécrétion de salive, alors que l’altération de la fonction de barrière endothéliale est liée à l’hyposécrétion et à l’infiltration lymphocytaire dans les glandes sous-maxillaires (SMG) dans le syndrome de Sjögren4. Ces données suggèrent que la contribution de la fonction de barrière endothéliale doit être suffisamment prise en compte en ce qui concerne une variété de maladies des glandes salivaires.
Un microscope à balayage laser à deux photons est un outil puissant pour observer la dynamique des cellules dans les tissus intacts in vivo. L’un des avantages de cette technique est que la lumière proche infrarouge (NIR) a une pénétration tissulaire plus profonde que la lumière visible ou ultraviolette lorsque les échantillons sont excités par le NIR et ne provoque pas de dommages évidents aux tissus par la lumière dans des conditions appropriées 5,6. En effet, les glandes salivaires sont un tissu très homogène et superficiel, dans lequel les cellules acineuses de surface ne sont qu’à environ 30 μm de la surface de la glande 7,8. Il a été démontré que la microscopie confocale intravitale peut étudier la sécrétion exocrine et le cytosquelette d’actine dans les glandes salivaires de souris vivantes à une résolution subcellulaire8. Néanmoins, la microscopie à balayage laser à deux photons présente non seulement l’avantage de la microscopie confocale conventionnelle, mais peut également être utilisée pour détecter plus clairement les tissus et les images plus profonds. Ici, les dextrans marqués par fluorescence, qui sont fréquemment utilisés comme traceurs de perméabilité paracellulaire et ont l’avantage de différentes tailles, peuvent être utilisés pour tester l’ampleur du pore TJ9. Dans la présente étude, une technique de microscopie intravitale à balayage laser à deux photons en temps réel est établie pour l’évaluation in situ de la fonction de barrière endothéliale chez les SMG de souris. Chaque étape de travail pour la détection in vivo de la perméabilité vasculaire chez les SMG de souris est décrite dans le protocole actuel. Voici un exemple de détection de la fonction de barrière endothéliale dans le modèle de ligature du canal SMG de souris.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-photon microscope (TCS-SP8 DIVE)</td>
			<td>Leica, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 kDa FITC-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>46944</td>
			<td></td>
		</tr>
		<tr>
			<td>70 kDa rhodamine B-labeled dextran</td>
			<td>Sigma Aldrich</td>
			<td>R9379</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt tissue separation nickel</td>
			<td>Bejinghuabo Company</td>
			<td>NZW28</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Veet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable sterile syringe</td>
			<td>Zhiyu Company</td>
			<td></td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>0253316</td>
			<td>1 mL</td>
		</tr>
		<tr>
			<td>Leica Application Suite X software</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline 1x</td>
			<td>Servicebio</td>
			<td>G4207-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors</td>
			<td>Bejinghuabo Company</td>
			<td>M286-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tribromoethanol</td>
			<td>JITIAN Bio</td>
			<td>JT0781</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo vascular permeability detection in mouse submandibular gland

La salive joue un rôle important dans la santé bucco-dentaire et globale. La fonction de barrière endothéliale intacte des vaisseaux sanguins permet la sécrétion de salive, tandis que le dysfonctionnement de la barrière endothéliale est lié à de nombreux troubles sécrétoires des glandes salivaires. Le présent protocole décrit une méthode de détection de perméabilité paracellulaire in vivo pour évaluer la fonction des jonctions serrées endothéliales (TJ) dans les glandes sous-maxillaires de souris (SMG). Tout d’abord, des dextranes marqués par fluorescence avec différents poids moléculaires (4 kDa, 40 kDa ou 70 kDa) ont été injectés dans les veines angulaires de souris. Par la suite, le SMG unilatéral a été disséqué et fixé dans le support personnalisé sous un microscope à balayage laser à deux photons, puis des images ont été capturées pour les vaisseaux sanguins, les acini et les canaux. En utilisant cette méthode, la fuite dynamique en temps réel des traceurs de différentes tailles des vaisseaux sanguins dans les côtés basaux de l’acine et même à travers l’épithélium acineux dans les canaux a été surveillée pour évaluer l’altération de la fonction de barrière endothéliale dans des conditions physiologiques ou physiopathologiques.

Conformément au protocole, le SMG unilatéral a été attaché à un support sur mesure, et la glande a été maintenue aussi loin que possible du corps de la souris pour empêcher la respiration de provoquer des artefacts de mouvement. Le flux rapide des globules rouges (points noirs) dans les vaisseaux sanguins a été observé au microscope. Après avoir trouvé le champ tissulaire sous une lentille oculaire, il faut passer à manipuler le logiciel du microscope. Dans le groupe témoin, les deux traceurs existaient d...

Watch this Scientific Journal Video about In Vivo Vascular Permeability Detection in Mouse Submandibular Gland at JoVE.com

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Dans le présent protocole, la fonction de barrière endothéliale de la glande sous-maxillaire (SMG) a été évaluée en injectant différents traceurs fluorescents pondérés moléculairement dans les veines angulaires de modèles animaux testés in vivo sous un microscope à balayage laser à deux photons.

In vivo Détection de perméabilité vasculaire dans la glande sous-maxillaire de souris

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Le présent protocole décrit une mesure de détection de perméabilité paracellulaire in vivo pour évaluer la fonction des jonctions endothéliales serrées dans les glandes sous-maxillaires de souris. Néanmoins, la microscopie à balayage laser à deux photons présente non seulement l’avantage de la microscopie confocale conventionnelle, mais peut également être utilisée pour détecter plus clairement les tissus et les images plus profonds. Cette expérience fournit une bonne mesure méthodologique pour évaluer la perméabilité vasculaire de différents tissus, en particulier les tissus de surface et les organes des animaux.Commencez par choisir des traceurs appropriés tels que la fluorescéine, l’isothiocyanate, le dextrane marqué et la rhodamine B, le dextrane marqué avec des spectres d’émissions d’excitation distincts, afin de minimiser l’interférence entre les signaux de fluorescence pour le test de perméabilité. Diluer les traces dans une solution saline tamponnée au phosphate stérile en 100 milligrammes par millilitre et stocker les aliquotes à l’abri de la lumière à moins 20 degrés Celsius. Après avoir anesthésié la souris mâle de type sauvage âgée de 8 à 10 semaines, tenez doucement la tête et le cou de la souris pour faire légèrement saillir un côté du globe oculaire.Insérez une seringue à insuline contenant 100 microlitres de mélange de solution traceur fluorescente le long du coin de l’œil perpendiculaire à l’œil. Après l’injection, placez rapidement la souris sur le carton en décubitus dorsal, les membres et la tête scotchés. Pour l’isolement de la glande sous-maxillaire ou de l’ASM, au microscope stéréoscopique, coupez l’épiderme du cou avec des ciseaux de tissu généraux pour exposer les deux côtés des SMG.Ensuite, utilisez une aiguille de séparation des tissus émoussés pour séparer doucement la capsule de la surface de la glande sans endommager le tissu glandulaire, les vaisseaux sanguins et la structure glandulaire. Connectez le support personnalisé au dispositif à pression négative et vérifiez si l’effet de pression négative est correctement obtenu à l’avance. Placez doucement le SMG exposé sur le support personnalisé et aspirez le tissu par aspiration à pression négative, aussi loin que possible du corps de la souris, afin de réduire les artefacts de mouvement causés par la respiration et d’autres conditions.Commencez l’imagerie peu de temps après le choix du site de la glande. Acquérir des images séquentielles le long de l’axe Z à deux microns de la surface de la glande jusqu’à 70 à 140 microns dans le tissu pour générer une construction tridimensionnelle. Ensuite, acquérir une imagerie accélérée des vaisseaux pour mesurer la perméabilité vasculaire dans le SMG.Dans la boîte de dialogue Explorer, cliquez sur Ajouter un nouveau dossier et modifiez le nom du fichier. Ensuite, revenez à la colonne d’acquisition. En XY, le format est de 512 par 512 et la vitesse est de 400 hertz.Activez l’option Bidirectionnelle X », définissez le facteur de zoom sur 0,75 et 1,5 pour le zoom. Ensuite, sélectionnez XYT sous Mode d’acquisition » pour acquérir l’imagerie accélérée. Définissez la durée sur 20 secondes, 30 minutes ou plus selon le besoin expérimental.Après avoir défini les conditions, cliquez sur En direct pour observer l’imagerie vasculaire de deux canaux et des canaux qui se chevauchent dans le cadre de formation de photos et choisissez les zones d’intérêt. Cliquez sur Démarrer pour commencer l’imagerie. Essai de perméabilité vasculaire in vivo et images tridimensionnelles des vaisseaux sanguins chez la souris.Les glandes sous-maxillaires sont montrées ici. Dans le groupe témoin, les deux traceurs existaient dans les vaisseaux sanguins du SMG. En raison de son faible poids moléculaire, FD4 a pu s’échapper des vaisseaux sanguins vers les côtés basaux des acini et des canaux, représentant ainsi clairement la forme des acini et des canaux.Le RD70 a été distribué dans des vaisseaux sanguins et des microvaisseaux de grande taille. Dans le groupe ligature des canaux, FD4 et RD70 ont été extravasés vers les côtés basaux des acini, ce qui indique que la ligature des canaux pourrait perturber la fonction de barrière endothéliale et augmenter la perméabilité aux grosses molécules. De plus, les résultats semi-quantitatifs de l’intensité de fluorescence FD4 et RD70 ont confirmé ces observations.Les images tridimensionnelles ont montré une fluorescence beaucoup plus obscure de FD4 et RD70 autour des vaisseaux sanguins dans le groupe ligature par rapport au groupe témoin. Une isolation SMG soigneuse, un placement approprié et le microscope sont des conditions préalables essentielles pour une détection réussie. En suivant les procédures, le débit sanguin peut être calculé en mariant le mouvement des globules rouges et l’infiltration des cellules sanguines marquées par fluorescence peut être chassée.

in-vivo-vascular-permeability-detection-in-mouse-submandibular-gland

Research

JoVE Journal

Biology

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Enregistrements aspiration simple cellule à partir de cônes photorécepteurs de la souris

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Recherche

Biologie

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

L&#39;imagerie in vivo et des traitements thérapeutiques dans un modèle de souris orthotopique de cancer des ovaires

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

L&#39;isolement des cellules souches de la souris salivaires Gland

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

Evaluation de développement des glandes mammaires et fonction dans des modèles murins

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-paramètres de mesure de l&#39;ouverture des pores de transition de perméabilité dans le Cœur de souris mitochondries isolées

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Injection intra-canalaire pour localisée Drug Delivery de la glande mammaire de souris

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

La mesure de la perméabilité à l&#39;eau Coefficient osmotique (P<sub&gt; F</sub&gt;) Des cellules sphériques: protoplastes de plantes isolées comme un exemple

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

In Vivo Détection et analyse des Rb SUMOylation de protéines dans les cellules humaines

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.