Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

NOTE: Ce protocole a été examiné et approuvé par l&#39;Université de Pittsburgh institutionnel de protection des animaux et l&#39;utilisation Comité. 1. Préparation de la solution de Krebs modifiée et Drug Test aliquotes <ol><li> Ajouter 6 L d&#39;une solution de Krebs modifiée qui contient (en mM): 117,9 NaCl, 4,7 KCl, 25 NaHCO 3, 1,3 NaH 2 PO 4, 1,2 MgSO 4, 2,5 CaCl2, 11,1 D-glucose, le butyrate de 2 sodium, l&#39;acétate 20 de sodium , 0,004 nifédipine (pour bloquer les contractions musculaires spontanées), et 0,003 indométacine (pour bloquer la synthèse des prostaglandines endogènes). Utiliser des solutions glace froides et chaudes Krebs pour la dissection des tissus et l&#39;enregistrement monofibre, respectivement. </li><li> Préparer des solutions chimiques (par exemple, IS, la capsaïcine, ATP) en aliquotes à des concentrations désirées. </li></ol> 2. Dissection du tissu colorectal nerf <ol><li> Anesthésier et euthanasier les souris mâles (6 - 8 semaines, de 20 à 30 g) dansune chambre de CO 2 à un débit d&#39;écoulement qui déplace 10 - 30% du volume de la chambre par minute jusqu&#39;à ce que la respiration se arrête complètement les souris, comme indiqué par l&#39;absence de mouvement de la poitrine. </li><li> Immédiatement après l&#39;euthanasie, exsangue en coupant ouvert la chambre thoracique, perforation de l&#39;oreillette droite, et en l&#39;immergeant la carcasse de la souris dans un volume suffisant (~ 500 ml) de glace-froid Krebs (4 ° C) solution barboter avec carbogène (95% O 2 , 5% CO 2). </li><li> Retirez soigneusement tous les viscères, mais le côlon et les organes pelviens. Transect la souris dans la moitié à travers le segment rachidien T12 légèrement au-dessus du diaphragme thoracique et transférer la moitié caudale à une chambre de dissection contenant glacée, barboter solution de Krebs. </li><li> Sous un stéréomicroscope, retirez la vessie et les organes reproducteurs par sectionnant à leurs jonctions à l&#39;urètre, et enlever la descente / aorte abdominale jusqu&#39;à ce qu&#39;elle bifurque dans les artères iliaques communes. Libérer le PN ou la LSN de leur surrounding tissus par dissection et suivre le nerf de l&#39;extérieur de la crête iliaque jusqu&#39;à son point d&#39;entrée dans le L6 ventrale et S1 colonne vertébrale (pour PN) ou T13 et L1 colonne vertébrale (pour LSN). </li><li> Couper la symphyse pubienne et à droite et les articulations du cotyle gauche, et enlever l&#39;os iliaque. Sans précaution soit le PN ou LSN du muscle attenant et du tissu conjonctif de près de la rectum jusqu&#39;à où le nerf pénètre dans la colonne vertébrale. </li><li> Réséquer soigneusement l&#39;os iliaque pour exposer le cancer colorectal distal. Disséquer le cancer colorectal distal avec le PN-joint ou LSN en continuum. </li><li> Transférer le rectum avec le nerf fixé à la chambre de bain de la chambre de tissu. Retirer le tissu conjonctif excessive par la poursuite de la dissection, et ouvrez le rectum longitudinalement le long de la frontière anti-mésentérique. </li><li> Avec côté de la muqueuse vers le haut, la broche le bord mésentérique du côlon et du rectum adjacent au compartiment d&#39;enregistrement dans la base de silicone de la chamber et fixer la longueur du côlon et du rectum antimésentérique à un râteau de crochets reliés à un actionneur de force (illustré sur la figure 1 et photographié à la figure 2A). </li><li> Elargir le PN LSN ou dans le compartiment d&#39;enregistrement, qui est relié au compartiment de bain par un trou de souris et la porte. Posez délicatement le tronc de nerf sur un petit miroir de verre dans le compartiment d&#39;enregistrement, qui fournit une surface hydrophile pour le nerf à respecter. Superfuse le compartiment de salle de bain avec chaud (30-32 ° C), une solution de Krebs oxygénée et remplir le compartiment d&#39;enregistrement avec de l&#39;huile minérale. </li></ol> Enregistrement 3. monofibre et la localisation du champ récepteur <ol><li> Retirer délicatement revenir l&#39;épinèvre (gaine des nerfs) du PN ou LSN sous la loupe binoculaire à fort grossissement (50 - 60X). En utilisant des pinces fines, taquiner le tronc nerveux dans 5-8 faisceaux nerveux de ~ 100 um d&#39;épaisseur. </li><li> Placez la référence de platine-iridiumélectrode en contact avec la solution de Krebs dans la chambre de tissu. Placer séquentiellement les faisceaux de nerfs individuels sur l&#39;électrode d&#39;enregistrement constituée de la même matière. </li><li> Utilisez un pinceau souple pour évoquer les points d&#39;accès des afférences colorectal en caressant doucement monter et descendre la surface colorectal. Localisez le pack (s) de nerf qui innervent le côlon par détectable AP (potentiel d&#39;action) des enregistrements. NOTE: Le PN et LSN innervent aussi la vessie et d&#39;autres organes pelviens. </li><li> Utilisez une paire de 30 g de pointes d&#39;aiguilles de diviser davantage le faisceau nerveux en filaments de fascicule fines de ~ 10 um d&#39;épaisseur et placer un filament individuel sur l&#39;électrode d&#39;enregistrement. </li><li> Placer l&#39;électrode concentrique à bout rond perpendiculaire à la surface de la muqueuse pour exciter électriquement des terminaisons afférentes à supraliminaire intensité de stimulation (10 mA d&#39;amplitude, durée 0,5 ms @ 0,3 Hz), qui produit un rayon de ~ 2 mm de propagation courant. Déplacez l&#39;électrode systématiquement (~ 1,5 mm étapes) Le long de la longueur et la largeur du côlon et du rectum aplatie pour localiser les terminaisons réceptives. </li><li> Lorsque une fin afférente est excité, ajuster la position de l&#39;électrode d&#39;identifier le site d&#39;activation (champ récepteur, RF) qui nécessite l&#39;intensité du stimulus minimum (seuil de stimulation). Jeter terminaisons avec un seuil de stimulation&gt; 3 mA 19. </li><li> Calculer la vitesse de conduction (CV) à partir de 1) la distance entre l&#39;électrode de stimulation au champ récepteur (RF) et le site d&#39;enregistrement et 2) le retard de conduction (par exemple, figure 2B) entre l&#39;artefact du stimulus et le début de potentiel d&#39;action . CV (m / s) = distance (mm) / conduction retard (ms). </li></ol> 4. Classification fonctionnelle des mécanosensibles colorectal Afférences <ol><li> Après avoir localisé un RF par la stimulation électrique, appliquer les trois stimuli mécaniques suivantes à la RF: <ol><li> Effectuer le stimulus de sondage en appuyant sur la pointe d&#39;un Calibramonofilament Ted von Frey-comme le nylon (0,4 et 1 g vigueur) perpendiculairement vers la RF sur le cancer colorectal aplatie. </li><li> Effectuer le stimulus caressant en caressant doucement le muqueuse colorectale avec un brin de filament en nylon fine (10 mg de force) pour générer une petite contrainte de cisaillement de surface à la RF. </li><li> Effectuer le tronçon périphérique en utilisant une force actionneur contrôlé par ordinateur, qui fournit une force d&#39;étirement rampe (0-170 mN à 5 mn / sec) en direction circonférentielle le long du bord anti-mésentérique du côlon et du rectum par le râteau de crochets décrites à l&#39;étape 2.8 . </li></ol></li><li> Classer afférences que séreuse (ne répondre qu&#39;aux émousser sondage), la muqueuse (répondre aux muqueuses caressant et émousser le sondage), musculaire (répondre à tronçon périphérique et émousser le sondage) musculaire / muqueuse (répondre à tronçon périphérique, muqueuse caresser et émousser le sondage), ou MIA (non sensible à l&#39;un des trois stimuli mécaniques). </li><li> Pour afférences mésentériques (uniquement enl&#39;innervation LSN) qui sont difficiles à activer sélectivement par la stimulation électrique, localiser leurs terminaisons réceptives en caressant mécanique / sondage du mésentère. </li><li> Pour afférences stretch-sensibles (musculaire et musculo-muqueux), déterminer le seuil de réponse, qui est définie comme la force qui évoque la première AP cours tronçon rampe. </li><li> Pour afférences séreuses, enregistrer leurs réponses à monter les niveaux de sonder ponctuée du champ récepteur entraîné par la force actionneur contrôlé par ordinateur. </li></ol> 5. Chemical Application / Modulation de Endings réceptives <ol><li> Enregistrer une réponse de base à un stimulus mécanique (ce est à dire, la réponse à l&#39;étirement rampe, ponctuée de sondage, ou muqueuse caresser). </li><li> Enduire le bord inférieur d&#39;un morceau de tube (laiton ou en acier inoxydable, 10 mm de haut et 4 x 4 mm 2 carré ou 4 - 5 mm de diamètre) avec de la vaseline et le placer sur le champ récepteur sur le cancer colorectal. </li><li> Supprimerla solution de Krebs à l&#39;intérieur du tube, et exposer la fin réceptif pour les 3 - 5 minutes à 170 ul de la solution contenant le produit chimique (s) à tester. </li><li> Surveiller la réponse du afférent pendant l&#39;application chimique (certains afférences sont chimiosensible). </li><li> Retirer la solution chimique et la tubulure de mettre fin à l&#39;action de la substance chimique. De 4 à 6 min, tester la réponse afférente au même stimulus mécanique comme dans la réponse de référence. </li><li> Réappliquer nouveau le stimulus mécanique après la période suffisante de wash-out (&gt; 15 min). </li></ol> 6. Enregistrement et pointes Discriminating AP <ol><li> Numériser les signaux électriques enregistrés à partir des axones à 20 kHz et enregistrer les données dans un ordinateur. Surveiller le signal en ligne par un moniteur audio. </li><li> Analyser les pointes AP off-line et de discriminer sur la base de simples unités analyse en composantes principales de formes d&#39;onde de pic individuels 25. NOTE: Un enregistrement doit pas contenir plus eun deux unités actives facilement discriminables. </li></ol>

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The authors have nothing to disclose.

Le vitro préparation colorectum-nerf décrit ici se est avérée être une approche puissante pour étudier les fonctions de codage neural de afférences colorectal individuels, qui complète bien d&#39;autres approches non-fonctionnels (par exemple, des études cellulaires, moléculaires et histologiques) sur les neurones sensoriels viscérales ( lire l&#39;article 27 pour plus de détails). Mécanismes neuronaux qui contribuent à la nociception et colorectal hypersensibilité à...

La douleur et hypersensibilité sont les plaintes prédominantes de patients souffrant de troubles gastro-intestinaux fonctionnels, y compris le syndrome du côlon irritable (IBS), qui existent en l&#39;absence de la cause pathobiologique apparente ou dommages aux tissus. Par exemple, les patients IBS présentent une hypersensibilité, y compris les réponses accrues à rectale distension du ballon et une sensibilité accrue au cours de la fonction intestinale normale, ainsi que l&#39;hypersensibilité de la saisine somatiques (ce est à dire, sensibilité à la palpation de la région abdominale) 1. Parce que le ciblage afférences colorectal se est révélé être efficace pour soulager la douleur et l&#39;hypersensibilité chez les patients IBS (par exemple, l&#39;instillation intra-rectale de 2,3 anesthésiques locaux; ingestion orale de la guanylate cyclase C-agoniste linaclotide 4-6), une meilleure compréhension de la innervation afférente du côlon et du rectum est important. Afférences viscérales, y compris les afférences colorectal, Sont capables de répondre aux modalités chimiques / en nutriments et thermiques (par exemple, 7-9). Cependant, les afférences viscérales répondant à des stimuli mécaniques (c.-à-afférences mécanosensibles) ont été le plus étudié à fond parce stimuli mécaniques (par exemple, distension luminale, extensibles) sont celles qui donnent généralement lieu à des sensations conscientes, y compris inconfort et la douleur 10-16. En outre, les viscères sont également innervés par les afférences mécaniquement insensibles (SIAM) de, communément appelés nocicepteurs silencieux ou de dormir 17. Dans des conditions physiologiques normales, MIAs ne répondent pas à la stimulation mécanique ou avoir de très haute réponse seuils 18, mais peuvent devenir actifs et se dotent mécanosensibilité dans des conditions physiopathologiques et contribuer à une hypersensibilité. Utilisation de la préparation et le protocole in vitro décrit ici, nous avons développé et emploie une stratégie de stimulation électrique à la mer<html> RCH pour terminaisons réceptives, permettant l&#39;identification objective des deux terminaisons mécanosensibles et Mia dans le rectum 19. L&#39;innervation colorectal est dérivé de lombaire splanchnique (LSN) et nerf pelvien (PN) les parcours, et comprend afférences colorectal qui peuvent être classés en cinq catégories (mécanosensibles séreuse, muqueuse, musculaire, musculaire-muqueuse, mésentériques) et une classe MIA 20. L&#39;utilisation de cette préparation in vitro, nous avons constaté que MIAs colorectal mécanosensibilité acquise (sensibiliser) après une brève exposition de leurs champs réceptifs à une soupe inflammatoire (IS), qui a sensibilisé 71% des soldats portés disparus dans la voie PN et 23% des soldats portés disparus dans la voie LSN 19. Nous avons également documenté une sensibilisation à long terme (jusqu&#39;à 28 jours) des soldats portés disparus dans le cadre de l&#39;hypersensibilité viscérale comportementale longue durée (ce est à dire, chez les souris recevant des traitements intracoliques avec zymosan 21 ou de l&#39;acide 2,4,6-trinitrobenzène (TNBS) 22) . jove_content &quot;&gt; Parmi afférences mécanosensibles, afférences musculaires et musculo-muqueuses sont les seules classes qui codent tonique tronçon périphérique du côlon et du rectum (ce est à dire, sont tronçon sensible) et subserve l&#39;encodage des nocive distension colorectale 23,24. L&#39;utilisation d&#39;un contrôlé par ordinateur vigueur actionneur, nous avons appliqué une norme, homogène et reproductible tronçon rampe dans la direction circonférentielle du tissu colorectal aplatie et subdivisés afférences extensibles sensible à seuil bas et seuil haut 23. En outre, le cours du temps de la sensibilisation d&#39;étirement afférences -sensibles après zymosan intracolique 21 ou 22 TNBS traitement correspond à l&#39;apparition, la persistance et / ou le rétablissement de l&#39;hypersensibilité viscérale comportement, suggérant un rôle des afférences colorectal extensibles sensible dans la douleur et l&#39;hypersensibilité viscérale.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Name of Material/ Equipment</td>
			<td style="width:291px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:372px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica MZ16 stereo microscope&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica IC D camera</td>
			<td>Leica Microsystems Inc.</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Amplifier</td>
			<td>World Precision instruments, Inc.</td>
			<td>SYS-DAM80&#160;</td>
			<td>Low-noise differential amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Two-compartment tissue chamber</td>
			<td style="width:291px;">Custom made</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Power1401</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;">Power1401</td>
			<td>Data acquisition, analog signal input/out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Spike2 v5.02</td>
			<td>Cambridge Electronic Design Limited</td>
			<td style="width:137px;"></td>
			<td>Software package that works with the Power1401</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Audio monitor</td>
			<td>Natus</td>
			<td style="width:137px;">Am 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Square pulse stimulator</td>
			<td>Natus</td>
			<td style="width:137px;">S48</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Photoelectric isolation unit</td>
			<td>Natus</td>
			<td style="width:137px;">PSIU6</td>
			<td>Stimulus isolation to reduce noise</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">Concentric bipolar microelectrode</td>
			<td style="width:291px;">FHC Inc.</td>
			<td style="width:137px;">CBFFG75</td>
			<td>To deliver electrical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dual-mode lever system</td>
			<td>Aurora Scientific Inc.</td>
			<td style="width:137px;">Series 300C&#160;</td>
			<td>To deliver mechanical stimuli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td>forceps with fine tips</td>
		</tr>
	</tbody>
</table>

in vitro functional characterization of mouse colorectal afferent endings

Cette vidéo montre en détail une seule fibre protocole d&#39;enregistrement électrophysiologique in vitro en utilisant une préparation souris rectum nerf. L&#39;approche permet l&#39;identification et la caractérisation fonctionnelle impartiale des afférences colorectal individuels. Enregistrements extracellulaires des potentiels d&#39;action (PA) à multiplication qui proviennent d&#39;une ou de quelques afférents (par exemple, une seule fibre) champs récepteurs (RFS) dans le rectum sont fabriqués à partir taquiné faisceaux de fibres nerveuses. Le cancer colorectal est retiré avec soit le pelvienne (PN) ou lombaire splanchnique (LSN) nerf attachée et ouvert longitudinalement. Le tissu est placé dans une chambre d&#39;enregistrement, goupillé plat et perfusé avec une solution oxygénée de Krebs. Stimulation électrique focale est utilisé pour localiser les terminaisons afférentes colorectaux, qui sont ensuite testés par trois stimuli mécaniques distinctes (émousser sondage, muqueuse caresser et de se étirer circonférentielle) de classer fonctionnellement les afférences en cinq mechanosensiles classes tifs. Fins répondu à aucune de ces stimuli mécaniques sont classées comme afférences mécaniquement insensibles (SIAM). Les deux mécanosensible et MIAs peuvent être évalués pour la sensibilisation (par exemple en réponse améliorée, seuil réduit, et / ou l&#39;acquisition d&#39;mécanosensibilité) par l&#39;exposition localisée de la RFS aux produits chimiques (par exemple, la soupe inflammatoire (IS), la capsaïcine, l&#39;adénosine triphosphate (ATP)). Nous décrivons l&#39;équipement et la préparation d&#39;enregistrement rectum nerf, la récolte du côlon et du rectum avec PN ci-joint ou LSN, l&#39;identification de la RFS dans le rectum, l&#39;enregistrement mono-fibre à partir de faisceaux nerveux, et l&#39;application localisée de produits chimiques à la RF. En outre, les défis de la préparation et l&#39;application de la stimulation mécanique normalisée sont également discutés.

La configuration est illustrée sur la figure 1. Il comprend une chambre de tissu sur mesure qui abrite le rectum dans un compartiment de bain silicone doublée et le nerf fixé dans un compartiment rempli d&#39;huile minérale contiguë. La chambre à deux compartiments a été usiné à partir d&#39;un bloc de plastique acrylique par une machine CNC; le fond des deux compartiments a ensuite été doublé avec de la silicone solide pour permettre à la broche facile bas du tissu colorectal. AP extracel...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro caractérisation fonctionnelle de la souris colorectal afférente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K vues.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

Vidéo: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

L&#39;utilisation de fibroblastes humains primaires pour la surveillance des phénotypes mitochondriales dans le domaine de la maladie de Parkinson

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Recherche

Médecine

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro Organoid Culture des primaires tumeurs du côlon de souris

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

L&#39;isolement et la caractérisation fonctionnelle de Human ventriculaire cardiomyocytes à partir des échantillons chirurgicaux frais

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Création et caractérisation de l&#39;UTI et Cauti dans un modèle de souris

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Évaluation des cardiaques morphologiques et fonctionnelles changements dans le modèle de souris de Transverse Aortic Constriction par échocardiographie Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Longitudinal<em&gt; In Vivo</em&gt; Imagerie du Cerebrovasculature: Pertinence pour les maladies du système nerveux central

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

préparation et<em&gt; In vitro</em&gt; Caractérisation des aimanté miR-modifié cellules endothéliales

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Développement et caractérisation fonctionnelle des cellules dendritiques tolérogène murin

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Caractérisation fonctionnelle des variantes de RYR1 humaines exprimées de manière endogène

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).