Ce travail a été soutenu par NIH 1R01EY022901, le Prix de carrière développement de la recherche à prévenir la cécité (RPB), CMReeves &amp; MA Fondation Reeves, Fondation E. Matilda Ziegler pour les aveugles, la Fondation Templiers yeux, et une subvention sans restriction au ministère de la Ophtalmologie à l&#39;Université de l&#39;Utah de RPB. Nous remercions Balamurali Ambati d&#39;assistance technique sur la multi-modalité système d&#39;imagerie Spectralis et Tao Zhang pour des discussions et des commentaires sur le manuscrit.

Une. Préparation des instruments <ol><li> La procédure est réalisée dans un bain de procédure dans une animalerie. </li><li> Porter des masques, bonnets, blouses chirurgicales, pied-housses stériles et des gants avant de commencer l&#39;expérience. </li><li> Chauffer l&#39;eau dans un gobelet pour ~ 40 ° C sur une plaque chauffante. </li><li> Placez un coussin bleu stérile sur un coussin chauffant qui sera utilisé plus tard pour maintenir la température du corps de la souris lors de l&#39;imagerie. Mettez le coussin chauffant. </li><li> Préparer le système d&#39;imagerie: <ol><li> Retirez le couvercle de la poussière et allumer le laser. </li><li> Sortez la lentille de 55 ° et le monter sur la machine. </li><li> Ouvrez le logiciel d&#39;imagerie de l&#39;ordinateur et entrer les informations de la souris pour l&#39;imagerie sous la feuille d&#39;un nouveau patient (par exemple, génotype, âge, etc.). Sous la rubrique «type de dispositif», choisissez mode infrarouge (IR). </li></ol></li></ol> Remarque: Il a été rapporté que l&#39;utilisation of un à double lentille asphérique externe peut améliorer la qualité d&#39;image 17-20, même si nous n&#39;avons pas de problème à obtenir des images de haute qualité en IV-ICGA sans l&#39;aide de lentilles externes (voir résultats représentatifs, les figures 1-4). 2. L&#39;injection de la veine caudale ICG <ol><li> Dilater les yeux de souris avec 1% Tropicamide solution ophtalmique et attendre 5 min. </li><li> Peser la souris pour déterminer le montant de l&#39;anesthésie (kétamine / xylazine / acépromazine 65-100/10-20/1-3 mg / kg) nécessaire. </li><li> Récupérer un 1 ml seringue stérile avec une aiguille 32 G. Injecter la souris par voie intrapéritonéale avec les anesthésiques (13 mg / ml de kétamine, 2,6 mg / ml de xylazine 0,3 mg / ml dans du PBS stérile acépromazine). Attendez jusqu&#39;à ce que la souris est totalement anesthésié (~ 5 min). </li><li> Placez la queue de la souris dans 40 ° C l&#39;eau chaude pour provoquer une vasodilatation de la veine. </li><li> Récupérer une seringue de 1 ml avec une aiguille 32 G. Prélever la quantité voulue de l&#39;ICG, typiquement 50 ul de 1 mg / ml ICG, qui est stérilisée par filtration avec un filtre à seringue de 0,2 uM dans un tube stérile, pour une souris de 25 g (2 mg / kg). Veillez à ne pas introduire de l&#39;air dans la seringue. </li><li> Essuyez la queue avec un tampon d&#39;alcool pour stériliser la zone à injecter. </li><li> Tenez la queue d&#39;une main de sorte que la veine caudale latérale est à la hausse. Avec le biseau de l&#39;aiguille vers le haut, injecter l&#39;aiguille ~ 2 mm dans la veine à un angle minimal. Veillez à ne pas perforer la veine. Dessiner sur la seringue légèrement et retrouver des traces de la circulation sanguine dans le moyeu de l&#39;aiguille, ce qui indique que l&#39;aiguille a été insérée avec succès dans la veine. </li><li> Injecter lentement ICG dans la veine. Il devrait y avoir une résistance minimale lors de l&#39;injection. Retirer l&#39;aiguille et appliquer un tampon imbibé d&#39;alcool directement sur le site d&#39;injection pendant environ 5-10 secondes pour arrêter le saignement. La souris est ensuite prêt pour l&#39;imagerie. Afin de rattraper la première phase (0-4 min après l&#39;injection), il est essential à l&#39;image de la souris rapidement. </li></ol> Remarque: les yeux de souris peuvent facilement se sécher et peuvent développer une cataracte sous anesthésie. Il est important de garder l&#39;oeil humide en appliquant PBS stérile pendant la procédure. Essuyez l&#39;excès de PBS avec un coton-tige stérile avant l&#39;enregistrement ICG. D&#39;autres laboratoires ont utilisé une lentille de contact pour éviter la déshydratation de la cornée de 17 à 20. 3. Angiographie ICG <ol><li> Commencer à prendre des images 30-40 secondes après injection ICG, qui permet la capture de la première phase de remplissage de la choroïde jusqu&#39;à circulations rétiniennes et choroïdiennes sont à la luminosité maximale (0-4 min). La vascularisation rétinienne est mieux visualisé à discussion ~ 35-45 dioptries et vasculaire de la choroïde est visualisé à 10-15 dioptries. Remarque: Lors du premier examen d&#39;un modèle animal, il est recommandé de capturer des images sous tous les angles (nasales, temporelle, dorsale et ventrale) pour identifier tous abno possiblermalities dans le système vasculaire. Au cours de la première phase, les deux artères et des veines choroïdiennes moyennes et grandes sont bien visualisées. Dans le modèle animal utilisé dans ce protocole, les lésions de la choroïde (par exemple polypoïdale de dilatations) peuvent commencer à apparaître 1 min dans la première phase. </li><li> Régler la mise au point de l&#39;image sur le système vasculaire. Contrôle de la luminosité et de se concentrer à l&#39;aide du module de commande et le bouton de mise au point, respectivement. Ces valeurs sont réglables numériquement et sont facilement maintenus constants. Gardez la distance de l&#39;œil de la souris à la constante de lentille de la caméra pour assurer la qualité d&#39;image est reproductible en utilisant la technique qui suit. Remarque: Comme l&#39;appareil ne peut l&#39;image d&#39;une partie de l&#39;œil postérieur, nous essayons de garder le focus, la luminosité et la distance entre la lentille de la caméra et l&#39;oeil de la souris constante comme nous l&#39;image de la totalité de l&#39;œil postérieur sous des angles différents. La clé pour cela est d&#39;aligner la luminescence en forme de cercle émis par le GIC à travers l&#39;œil avec le domaine de la View de la caméra. Ceci est accompli en faisant de gauche à droite, des ajustements vers le haut et vers le bas, et en-and-out de la position de la caméra jusqu&#39;à ce que la totalité de l&#39;image n&#39;a pas de zones sombres. Lorsque la luminescence et le champ de vision de la caméra sont alignées, la distance de l&#39;œil à la lentille sera reproductible pour le prochain ensemble d&#39;images, ainsi que à une distance optimale pour une qualité d&#39;image. </li><li> Une fois le système vasculaire est mise au point, de capturer les trames d&#39;image en appuyant sur le bouton rond noir sur le module d&#39;acquisition. Le bouton rond noir peut également être utilisé pour réduire ou augmenter le signal de l&#39;ICG pour une meilleure qualité d&#39;image. </li><li> Déterminer l&#39;angle de vue optimal et profondeur de focalisation à des lésions de la choroïde de l&#39;image. Il est important de garder la position de l&#39;œil, la profondeur de focalisation, et d&#39;autres paramètres de l&#39;appareil fixe pour l&#39;ensemble de l&#39;ICGA temps bien sûr. Les images sont enregistrées en appuyant sur le bouton d&#39;acquérir sur le panneau de l&#39;écran tactile du module d&#39;acquisition. </li><li> Acquérir des images dans la phase intermédiaire à 6-15 min à l&#39;arrièreinjection er. Remarque: Les deux de la choroïde et vaisseaux rétiniens deviennent moins distinctes. Vascularisation de la choroïde apparaît fluorescence diffuse. Lésions choroïdiennes présentant hyperfluorescence émergent contrairement à la décoloration entourant la fluorescence de fond normal. </li><li> Acquérir des images dans la phase tardive à 17-25 minutes après l&#39;injection. Remarque: hyperfluorescents s&#39;estompe. Les deux navires de la choroïde et de la rétine ne sont plus visibles. La tête du nerf optique devient noir. Lésions de la choroïde hyperfluorescentes ont contraste maximal avec le fond à la décoloration. </li><li> Après avoir terminé l&#39;acquisition d&#39;images, appliquer un gel lubrifiant œil clair pour les yeux de souris et de laisser la souris sur un coussin chauffant pour la récupération. </li><li> Souris retourner dans leurs cages et zone d&#39;attente. Exporter des images sous forme de fichiers TIFF ou JPEG pour une analyse plus approfondie. </li></ol> Remarque: Le calendrier de chaque phase n&#39;est pas absolue. Nous avons constaté que le calendrier de chphase de ch pourrait changer en fonction de la quantité de ICG injectée. Plus ICG tend à prolonger chaque phase. La meilleure façon de définir une phase est fonction des caractéristiques principales de chacune des phases énumérées ci-dessus.

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YF est un inventeur de deux brevets en instance qui sont pertinents pour le modèle de la souris AMD utilisée dans ce travail. SK, ZB, et ADJ n&#39;ont rien à divulguer.

Dans cette étude, nous avons démontré l&#39;utilisation de ICG à l&#39;image des lésions choroïdiennes chez des souris transgéniques HtrA1. Les caractéristiques du début, au milieu, et les phases tardives de ICG dans notre modèle de souris correspondent le cours du temps et dans les études humaines 1. Il est important de prendre de meilleures comparaisons entre pathologie et animales phénotypes humains, qui ont une valeur inestimable pour la recherche sur les mécanismes physiopathologiques et des...

Indocyanine angiographie au vert (ICG) est un test de diagnostic de problèmes d&#39;image liés aux vaisseaux sanguins dans l&#39;oeil. Le spectre d&#39;absorption de l&#39;ICG varie de 790 à 805 nm alors que le spectre d&#39;émission s&#39;étend de 770 à 880 nm avec le pic d&#39;émission à 835 nm 1. Ceci est différent de l&#39;autre colorant populaire, la fluorescéine de sodium, dont le spectre se situe dans la gamme visible. Le spectre infrarouge permet ICG à pénétrer à travers l&#39;épithélium rétinien pigmentaire (RPE), fluide sérosanguineux, et les exsudats lipidiques, qui peut facilement bloquer la visualisation par base de sodium de fluorescéine angiographie à la fluorescéine (FA). ICG est de 98% liée aux protéines dans le système vasculaire résultant en moins d&#39;extravasation, permettant une meilleure imagerie des vaisseaux de la choroïde et de lésions de la choroïde 1,2. ICG est presque le seul choix pour visualiser la vascularisation de la choroïde, qui est postérieure à l&#39;EPR. Figure 1 montre la comparaison des ICG et FA en imagerie vasculaire dans les yeux de souris. FA peut be utilisé pour l&#39;image de la vascularisation de la rétine bien mais pas la vascularisation de la choroïde. En revanche, ICG peut être utilisé à la fois l&#39;image de la rétine et de la vascularisation de la choroïde. ICG est réalisée avec haute résolution de systèmes d&#39;imagerie numérique ou ophtalmoscope laser à balayage (SLO) ainsi que dans l&#39;infrarouge des caméras vidéo, que nous utiliserons dans cette étude. Dans la clinique, ICG a été recommandée dans le diagnostic d&#39;un certain nombre de troubles choriorétiniennes impliquant le système vasculaire de la choroïde, y compris polypoïdale choroïde vasculopathie (PCV), de la rétine angiomateux prolifération (RAP), stries angioïdes, vitelliforme dystrophie maculaire, CRSC, hémangiome choroïdien, la rétine hémorragie macroaneurysms artériolaires, les tumeurs de la choroïde, et certaines formes de uvéite postérieure 1,3. La combinaison de ICG avec FA et la tomographie par cohérence optique (OCT) fournissent des outils puissants pour les cliniciens dans le diagnostic et la gestion des exsudative maculaire liée à l&#39;âgedégénérescence (AMD) 4-10. ICG est particulièrement utile pour des conditions impliquant la choroïde diagnostic. En fait, ICG est considéré comme l&#39;étalon-or pour le diagnostic PCV, une variante de la DMLA exsudative 11-13. PCV est caractérisé par un réseau de vaisseaux de ramification avec des dilatations polypoïdale terminaux dans le système vasculaire de la choroïde 11-13. PCV est fréquemment associée à des détachements sérosanguineux récurrents de l&#39;EPR et de la rétine avec des fuites et des saignements des composants polypoïdale 11,14,15. Nous avons récemment rapporté la génération du premier modèle animal de PCV par voie transgénique exprimant HTRA1 humain, une serine protease multi-fonctionnel, chez la souris l&#39;épithélium pigmentaire rétinien (EPR) 16. Nous avons montré que l&#39;augmentation induite HTRA1 caractéristiques de PCV, par exemple lésions polypoïdale. Ici, nous avons démontré l&#39;utilisation du temps-cours ICGA par injection dans la veine de la queue à la recherche d&#39;AMD en utilisant notre modèle de souris de HTRA1. Nos données suggèrent queIV-ICG est supérieure à l&#39;IP (ou sous-cutanée (SC))-ICG qui sont actuellement utilisés dans le domaine de 17,18 pour la caractérisation des lésions dans la choroïde. Déclaration sur la recherche sur les animaux Les expérimentations animales ont été menées selon des protocoles approuvés par des institutionnels de protection des animaux et l&#39;utilisation Comité (IACUC), et ont été réalisées conformément à la Déclaration ARVO pour l&#39;utilisation d&#39;animaux dans ophtalmique et Vision Research.

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	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

Vert d&#39;indocyanine angiographie (ou ICG) est une technique réalisée par les ophtalmologistes pour diagnostiquer des anomalies de la choroïde et le système vasculaire rétinien de diverses maladies oculaires telles que la dégénérescence maculaire liée à l&#39;âge (DMLA). ICG est particulièrement utile pour l&#39;imagerie du système vasculaire choroïdienne postérieure de l&#39;oeil dû à sa capacité de pénétrer à travers la couche pigmentée avec son spectre infrarouge. ICG cours du temps peut être divisé en début, milieu et fin phases. Les trois phases fournissent de précieuses informations sur la pathologie de problèmes oculaires. Bien que le temps bien sûr ICGA par voie intraveineuse (IV) l&#39;injection est largement utilisé dans la clinique pour le diagnostic et la gestion des problèmes choroïde, ICG par injection intrapéritonéale (IP) est couramment utilisé dans la recherche animale. Ici, nous avons démontré la technique pour obtenir des images haute résolution ICGA temps-cours chez la souris par injection de queue veineuse et ophtalmoscopie confocale à balayage laser. Nous avons utilisé cette technique à l&#39;image de la choroïde lesions dans un modèle de souris de la dégénérescence maculaire liée à l&#39;âge. Bien qu&#39;il soit beaucoup plus facile d&#39;introduire ICG à la vascularisation de la souris par IP, nos données indiquent qu&#39;il est difficile d&#39;obtenir des images reproductibles ICGA cours du temps par IP-ICG. En revanche, ICG par injection dans la veine caudale fournit ICGA images temps de cours de qualité comparables à des études humaines. En outre, nous avons montré que ICGA effectuée sur des souris albinos donne des images plus claires de vaisseaux choroïdiens que celle réalisée sur des souris pigmentées. Nous suggérons que le temps bien sûr IV-ICG devrait devenir une pratique courante dans la recherche AMD basée sur des modèles animaux.

Nous avons effectué ICG cours du temps dans HtrA1 des souris transgéniques de la même portée et de WT de commande, qui sont tous deux sur le fond CD1. Le CD1 fond albinos a été choisi pour faciliter l&#39;angiographie au vert d&#39;indocyanine (ICG) d&#39;imagerie (voir la discussion). Certains anévrisme comme dilatations ont commencé à apparaître dans la phase précoce chez la souris HTRA1 (Figure 2, une flèche rouge indique la dilatation à l&#39;extrémité d&#39;un navire et un cercle rou...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Vert d&#39;indocyanine (angiographie ou ICG) réalisée par injection dans la veine caudale fournit des images ICGA de cours de temps de qualité pour caractériser des anomalies dans la choroïde de la souris.

Détecter des anomalies dans les vaisseaux sanguins de la choroïde dans un modèle de souris de la dégénérescence maculaire liée à l&#39;âge par Time-sûr indocyanine vert angiographie

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Research

JoVE Journal

Medicine

13.5K vues.  University of Utah Health Sciences Center.  Vert d'indocyanine (angiographie ou ICG) réalisée par injection dans la veine caudale fournit des images ICGA de cours de temps de qualité pour caractériser des anomalies dans la choroïde de la souris. 

Vidéo: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Recherche

Médecine

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Évaluation non invasive de la Cardiac Malformations en auto-immune expérimentale myocardite par résonance magnétique microscopie imagerie chez la souris

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Un modèle de souris pour induite par laser néovascularisation choroïdienne

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

Le modèle monoïodacétate de douleur arthrosique chez la souris

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Détection de Anastasis In Vivo par CaspaseTracker biocapteur

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Une thérapie régénératrice par autogreffe de cellules Suprachoroidal à sec Age-related Macular Degeneration : préliminaire In Vivo rapport

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Un modèle murin BALB/c néonatal d’entérocolite nécrosante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

Hépatectomie centrale laparoscopique fluorescente pour le cancer du foie à l’aide d’une coloration négative au vert d’indocyanine

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

Exploration de l’autofluorescence quantitative dans la dégénérescence maculaire liée à l’âge

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Pleins feux sur l’auteur : Comprendre la physiopathologie de la dégénérescence maculaire liée à l’âge avec le flux de travail QAF 

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Un flux de travail pour déterminer quantitativement les variations spécifiques de la lésion de la dégénérescence maculaire liée à l’âge dans l’autofluorescence du fond d’œil

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...