Ce travail a été partiellement financé par le NIH RO1-EY019951 (AAF), Illinois Société pour la Prévention de la Cécité (JCC, AAF), les fonds non affectés au Département d&#39;ophtalmologie de la recherche pour prévenir la cécité (RPB), New York et RPB étudiant en médecine Fellowship Grant (JCC).

1. Préparation rétinienne <ol><li> Énucléer l&#39;oeil de la souris. En utilisant une main, ouvrir les paupières de sorte que l&#39;œil est visible. Avec l&#39;autre main, à l&#39;aide d&#39;une pince courbes (courbes vers le haut), appliquer une pression sur les aspects supérieurs et inférieurs de l&#39;orbite jusqu&#39;à ce que les dépasse globe. Fermez doucement forceps à la partie postérieure de l&#39;œil et de soulever dans un mouvement continu pour énucléer l&#39;œil. </li><li> Corrigez les yeux avec 10% de formol tamponné neutre pendant au moins 24 heures. </li><li> Lieu oeil dans une solution de PBS dans une petite boîte de Pétri. </li><li> Disséquer rétine soigneusement sous un microscope, en prenant soin d&#39;éviter de provoquer de grosses larmes. Faire une coupe initiale dans la cornée avec des ciseaux de dissection. Puis, tout en maintenant la zone de coupe avec des pinces droites, utilisez des ciseaux pour couper le long de la frontière de la cornée et la sclérotique de retirer la cornée. En utilisant des pinces courbes et droites fines, détachez soigneusement la sclérotique et la choroïde loin de la rétine vers le nerf optique. Cette étape exige de la patience commelibérant rapidement la rétine peut entraîner des larmes. Retirez l&#39;objectif et tout excédent iris et les débris de la rétine 16. </li></ol> 2. Eau lave <ol><li> Lieu rétine dans un plat de 24 puits et couvrir d&#39;eau (environ 500 pi, filtrée avec au moins un filtre de 45 um). </li><li> Changez l&#39;eau toutes les 30 min-1 heure, au moins 4-5 fois. </li><li> Laissez sécher une nuit dans de l&#39;eau avec agitation douce à température ambiante. </li></ol> Remarque: Les étapes de lavage sont très importants car ils permettent de séparer les couches de la rétine de neurones à partir des vaisseaux sanguins. 3. Trypsine Digest <ol><li> Enlevez l&#39;eau et de la rétine incuber dans une solution de 3% de trypsine (Difco 1:250) dans 0,1 M de tampon Tris (pH 7,8) à 37 ° C avec douceur à aucune secousse. Trypsine digestion est terminée lorsque le tissu commence à montrer des signes de désintégration (environ 1,5 heures). Note: Eviter d&#39;agiter rapidecar cela peut endommager le système vasculaire. </li><li> Soigneusement, une pipette à la trypsine dans un puits séparé en utilisant un microscope pour éviter de toucher la rétine. Cet excès de trypsine peut être utilisé plus tard, lors de la manipulation des vaisseaux sous le microscope, à l&#39;étape 4.3. </li></ol> 4. Séparer le système vasculaire <ol><li> Ajouter de l&#39;eau filtrée à la rétine. Ensuite, tenter de décoller la membrane limitante interne avec une pince, en utilisant des ciseaux si nécessaire. Agiter avec une légère agitation pendant 5 min. Soigneusement, une pipette à l&#39;eau sous le microscope pour éviter d&#39;endommager la rétine. </li><li> Répétez lavages à l&#39;eau (pendant 5 minutes chacun) pour aider à libérer le réseau de vaisseaux du tissu rétinien adhérente. lavages à l&#39;eau sont terminés quand il ya peu ou pas de débris restant dans l&#39;eau après le lavage. </li><li> Manipulations soigneuses sous un microscope de dissection sont maintenant nécessaires pour libérer davantage le réseau de vaisseaux. Voici quelques techniques utiles, qui peuvent être utilisés dans l&#39;ordre ou individually pour atteindre le résultat souhaité en fonction des préférences technique: <ol class="lalpha"><li> Avec une pipette 200 pi, une pipette soigneusement l&#39;eau de haut en bas à côté de bateaux, l&#39;eau soufflant à bateaux, provoquant une agitation douce. </li><li> Utilisation de la pipette 200 ul, Pipette la trypsine de haut en bas pour aider les parois de manteau de la pipette. Ensuite, ATTENTIVEMENT pipette le réseau de vaisseaux tout haut et en bas une fois pour aider à briser le tissu non vasculaire. Note: Le centrage de la pointe de la pipette au niveau du nerf optique plutôt que sur les bords du réseau vasculaire peut également empêcher la vascularisation de coller à la pointe. </li><li> Soulevez doucement vasculaire avec une pince fine hors de l&#39;eau pour déloger les composants non-vasculaires. </li><li> Si les étapes précédentes ne fonctionnent pas, ajoutez la trypsine de la rétine pour aider à briser le tissu et incuber à 37 ° C pendant plusieurs minutes. Ensuite, retirez la trypsine et répétez les étapes 4.1-4.3. </li></ol></li></ol> &lt;strong&gt; Note: Assurez-vous de bien enrober tous les instruments qui entrent en contact avec les navires de la trypsine (par exemple la pointe de pipette, forceps). Cela permettra d&#39;éviter l&#39;adhérence de la vascularisation de l&#39;équipement. 5. Visualiser la vascularisation <ol><li> Déposer une goutte d&#39;eau sur une lame propre. Avec une pipette ou une pince 200 pi, transférer les vaisseaux digérés dans l&#39;eau. Assurez-vous que la plate-mount vasculaire est déplié. La tension superficielle de l&#39;eau permet de déplier le réseau vasculaire maintenant diaphane. Si le tissu ne se déroule pas, ajouter de l&#39;eau et secouez doucement la lame pour faciliter le déroulement. </li><li> Retirer l&#39;excès d&#39;eau très lentement par la pipette ou en utilisant une serviette en papier. </li><li> Laisser les lames sécher complètement. </li><li> Colorer diapositives via le protocole approprié avec soit PAS / hématoxyline ou H &amp; E tache. Note: PAS / hématoxyline est utile pour identifier paroi de la cuve et des noyaux cellulaires, H &amp; E est utile pour da démonstration de RBC ainsi. </li><li> Déshydrater et monter (Moyen Permount montage). </li></ol>

<ol>
	<li>Kuwabara, T., Cogan, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+retinal+vascular+patterns.+I.+Normal+architecture.">Studies of retinal vascular patterns. I. Normal architecture.</a> Arch. Ophthalmol. 64, 904-911 (1960).</li><li>Kuwabara, T., Cogan, D. G. <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+patterns.+VII.+Acellular+change.">Retinal vascular patterns. VII. Acellular change.</a> Invest. Ophthalmol. 4, 1049-1064 (1965).</li><li>Bell, W. R., Green, W. R., Goldberg, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathologic+and+trypsin+digestion+studies+of+the+retina+in+incontinentia+pigmenti.">Histopathologic and trypsin digestion studies of the retina in incontinentia pigmenti.</a> Ophthalmology. 115, 893-897 (2008).</li><li>Danis, R. P., Wallow, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HRP/trypsin+technique+for+studies+of+the+retinal+vasculature.">HRP/trypsin technique for studies of the retinal vasculature.</a> Invest. Ophthalmol. Vis. Sci. 27, 434-437 (1986).</li><li>Kim, S. H., Chu, Y. K., Kwon, O. W., McCune, S. A., Davidorf, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+studies+of+the+retina+in+a+new+diabetic+model;+SHR/N:Mcc-cp+rat.">Morphologic studies of the retina in a new diabetic model; SHR/N:Mcc-cp rat.</a> Yonsei Med. J. 39, 453-462 (1998).</li><li>Cuthbertson, R. A., Mandel, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+the+mouse+retina.+Endothelial+cell-pericyte+ratio+and+capillary+distribution.">Anatomy of the mouse retina. Endothelial cell-pericyte ratio and capillary distribution.</a> Invest. Ophthalmol. Vis. Sci. 27, 1659-1664 (1986).</li><li>Laver, N. M., Robison, W. G., Pfeffer, B. 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Endothelial cell-pericyte ratio.</a> Ophthalmic Res. 21, 106-111 (1989).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+30.">Chapter 30.</a> Diabetic Retinopathy. , (2005).</li><li>Rosenblatt, B., Benson, W. E., Yannoff, M., Duker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.19+Diabetic+Retinopathy.">Chapter 6.19 Diabetic Retinopathy.</a> Ophthalmology. , (2008).</li><li>Friedenwald, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+some+problems+of+retinal+vascular+disease.">A new approach to some problems of retinal vascular disease.</a> Am. J. Ophthalmol. 32, 487-498 (1949).</li><li>Podesta, 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=Bax+is+increased+in+the+retina+of+diabetic+subjects+and+is+associated+with+pericyte+apoptosis+in+vivo+and+in+vitro.">Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.</a> Am. J. Pathol. 156 (10), 1025-1032 (2000).</li><li>Dagher, Z., 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=Studies+of+rat+and+human+retinas+predict+a+role+for+the+polyol+pathway+in+human+diabetic+retinopathy.">Studies of rat and human retinas predict a role for the polyol pathway in human diabetic retinopathy.</a> Diabetes. 53, 2404-2411 (2004).</li><li>Puro, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinovascular+physiology+and+pathophysiology:+new+experimental+approach/new+insights.">Retinovascular physiology and pathophysiology: new experimental approach/new insights.</a> Prog. Retin. Eye Res. 31, 258-270 (2012).</li><li>Roy, S., Nasser, S., Yee, M., Graves, D. T., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long-term+siRNA+strategy+regulates+fibronectin+overexpression+and+improves+vascular+lesions+in+retinas+of+diabetic+rats.">A long-term siRNA strategy regulates fibronectin overexpression and improves vascular lesions in retinas of diabetic rats.</a> Mol. 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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Trypsine digest est une méthode standard pour évaluer la vascularisation de la rétine. Malheureusement, il est techniquement difficile et peut entraîner des taux de perte de spécimen s&#39;il n&#39;est pas effectué correctement. En outre, la procédure est particulièrement difficile chez la souris, ce qui peut limiter l&#39;application de cette technique dans les modèles animaux génétiques couramment utilisés de maladies oculaires. Ce document fournit des conseils sur la façon d&#39;exécuter de manière eff...

Visualiser la vascularisation de la rétine est une approche extrêmement important de disséquer les mécanismes de diverses maladies oculaires comme la rétinopathie diabétique. Il permet d&#39;évaluer les anomalies vasculaires premières, y compris les micro-anévrismes, la dégénérescence et la perte capillaire pericyte 8,9. À ce jour, il ya eu plusieurs techniques mises au point pour analyser la vascularisation rétinienne. Perfusion de divers colorants a été utilisé pour mettre en évidence les vaisseaux, mais tous ont partagé les mêmes limites. Injection met rarement toute la vascularisation rétinienne moins donné à une haute pression, ce qui risque de rupture et d&#39;endommager les navires 1. Immunocoloration de l&#39;endothélium vasculaire avec fluorophore marqué G isolectine B4 (Alexa Fluor 594 conjugué; I21413; Invitrogen; dilution 1:100) et les supports plats rétine peut mettre en évidence l&#39;architecture globale des vaisseaux, mais sans visualisation détaillée des capillaires, les membranes basales et pericytes . Il a été noté parFriedenwald que les navires peuvent être mis en évidence par coloration plate-monts de rétine avec de l&#39;acide périodique Schiff (PAS) ou hématoxyline et l&#39;éosine (H &amp; E) coloration 10. Cependant, la coloration était non spécifique aux vaisseaux et a souligné le tissu non vasculaire ainsi, il est difficile de différencier les vaisseaux. Dans les années 1960, Cogan et Kuwabara développé la technique la trypsine digest qui a rendu plus facile de visualiser la vascularisation rétinienne en digérant les composantes non vasculaires de la rétine 1. Depuis ce temps, la trypsine digest est devenue la méthode de référence dans l&#39;analyse de la vascularisation de la rétine 2-5. Cependant, il est important de noter que d&#39;autres alternatives techniques pour isoler le système vasculaire ont été décrits. L&#39;utilisation de lyse osmotique a été utilisée pour isoler le système vasculaire et permettre des études biochimiques du tissu 11,12, mais la procédure n&#39;a pas été utilisé comme méthode principale pour l&#39;étude anatomique. Procédé d&#39;impression de tissu estutilisée pour isoler de larges segments de microvascularisation et permet la possibilité d&#39;étudier l&#39;architecture électrotonique de la vascularisation 13. En théorie, cette technique pourrait également être utilisée pour étudier les changements anatomiques, comme la qualité des vaisseaux est élevé. Cependant, il est seulement capable d&#39;isoler les segments de l&#39;ensemble du réseau vasculaire. Bien que ces méthodes ne peuvent pas remplacer la trypsine digérer, il est important de noter qu&#39;ils ont des avantages et des inconvénients et sont complémentaires à cet égard. La méthode digest trypsine est techniquement difficile et il est difficile de performer de façon constante 6,7. En outre, il a été noté que la trypsine digest est particulièrement difficile sur un modèle de souris, en particulier si l&#39;on veut préserver l&#39;architecture vasculaire global de la rétine 6,7. Les défis incluent (1) sur-digestion de la rétine qui entraîne une perte à la fois du système vasculaire ainsi que le tissu non vasculaire, (2) sous-digestion, exigeantdissection étendue mécanique qui pourrait à son tour conduire à des lésions de la chambre de cuve (3), une mauvaise séparation des tissus non vasculaires du système vasculaire qui conduit à une coloration non spécifique. Plusieurs manuscrits ont mis en évidence ces défis, mais aucune n&#39;a fourni un protocole détaillé et cohérent pour les surmonter 14,15. Ce manuscrit va introduire une méthodologie étape par étape décrivant la technique pour effectuer la trypsine digest sur la souris et le rat rétines avec des conseils spécifiques sur la manipulation des étapes particulièrement difficiles. Un aperçu schématique est illustré à la figure 1.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fischer Scientific</td>
			<td>SF100-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin (1:250)</td>
			<td>Amresco</td>
			<td>0458-50G</td>
			<td>Make 3% trypsin in 0.1 M Tris</td>
		</tr>
		<tr>
			<td>TRIZMA base</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Create 0.1 M Tris Buffer (pH 7.8 @ 37 &deg;C)</td>
		</tr>
		<tr>
			<td>TRIZMA</td>
			<td>Sigma</td>
			<td>T3253-500G</td>
		</tr>
		<tr>
			<td>Steritop sterile vacuum bottle</td>
			<td>Millipore</td>
			<td>SCGPS05RE</td>
			<td>Create filtered water</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Any microscope that allows good visualization of the retina is adequate. (10x is sufficient with working distance of 24 mm)</td>
		</tr>
		<tr>
			<td>Straight scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td>Cutting edge: 8 mm; Tip diameter: 0.2 mm; 10 cm</td>
		</tr>
		<tr>
			<td>Straight Forceps (Inox)</td>
			<td>Dumont #5</td>
			<td>11252-20</td>
			<td>Biologie tip; 0.05 x 0.02 mm; 11 cm</td>
		</tr>
		<tr>
			<td>Curved Forcepts (Dumostar)</td>
			<td>Dumont #7</td>
			<td>11297-10</td>
			<td>Biologie tip; 0.07 x 0.04 mm; 11.5 cm</td>
		</tr>
		<tr>
			<td>Dubnoff Metabolic Shaker Incubator</td>
			<td>Precision Scientific</td>
			<td>BDG59442</td>
			<td>Shaker optional</td>
		</tr>
		<tr>
			<td>24-well dish</td>
			<td>Falcon</td>
			<td>353047</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>VWR</td>
			<td>82027-132</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

trypsin digest protocol to analyze the retinal vasculature of a mouse model

Trypsine digest est la méthode de référence pour analyser la vascularisation rétinienne 1-5. Il permet de visualiser l&#39;ensemble du réseau de vaisseaux complexes tridimensionnels sanguins et des capillaires rétiniens en créant une plate-montage en deux dimensions des canaux vasculaires interconnectés après digestion des composants non-vasculaires de la rétine. Cela permet d&#39;étudier diverses modifications pathologiques vasculaires, tels que les micro-anévrismes, la dégénérescence capillaire et anormal endothéliales des ratios pericyte. Cependant, la méthode est techniquement difficile, surtout chez la souris, qui sont devenus le modèle animal le plus largement disponible pour étudier la rétine en raison de la facilité de manipulations génétiques 6,7. Dans l&#39;oeil de la souris, il est particulièrement difficile de supprimer complètement les composants non-vasculaires tout en conservant l&#39;architecture globale des vaisseaux sanguins de la rétine. À ce jour, il ya une pénurie de littérature qui décrit la technique de digérer la trypsine en détail in la souris. Ce manuscrit fournit une méthodologie détaillée de la trypsine digest dans la rétine de souris, étape par étape, tout en fournissant des conseils sur les étapes de dépannage difficiles.

Le produit final d&#39;une procédure réussie est une plate-montage de l&#39;ensemble du réseau de la vascularisation rétinienne de la souris, avec l&#39;architecture maintenu, colorées avec soit PAS / hématoxyline ou H &amp; E, comme indiqué dans les figures 2-4. Effacer la différenciation des cellules endothéliales et des péricytes peut être considéré comme le montre la figure 3. Dans la rétine, les noyaux des cellules endothéliales sont de forme ovale ou allongée et se...

Watch this Scientific Journal Video about Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model at JoVE.com

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsine digest est l&#39;une des méthodes les plus couramment utilisées pour analyser la vascularisation rétinienne. Ce manuscrit décrit la méthode en détail, y compris les modifications clés pour optimiser la technique et retirer le tissu non vasculaire tout en préservant l&#39;architecture d&#39;ensemble des navires.

Trypsine protocole Digest pour analyser la vascularisation rétinienne d&#39;un modèle de souris

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

trypsin-digest-protocol-to-analyze-retinal-vasculature-mouse

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Neuroscience

18.2K vues.  Northwestern University Feinberg School of Medicine. Trypsine digest est l'une des méthodes les plus couramment utilisées pour analyser la vascularisation rétinienne. Ce manuscrit décrit la méthode en détail, y compris les modifications clés pour optimiser la technique et retirer le tissu non vasculaire tout en préservant l'architecture d'ensemble des navires.

Vidéo: Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Enregistrements Patch Clamp à partir de neurones rétiniens souris dans une préparation adaptés à l&#39;obscurité Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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Neurosciences

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissection d&#39;un oeil de la souris pour un mont entiers de l&#39;épithélium pigmentaire rétinien

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Un modèle Optic Nerve Crush blessures murins pour étudier la rétine de survie des cellules ganglionnaires

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Morphometric Analyses of Retinal Sections

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the retinal ganglion cell layer (RGCL) in rat models with N-methyl-D-aspartate (NMDA)&#x2013;induced excitotoxicity1, retinal ischemia-reperfusion injury2 and glaucoma3. Reduction of INL and inner plexiform layer (IPL) thicknesses were reversed with citicoline treatment in rats' eyes subjected to kainic acid-mediated glutamate excitotoxicity4. Alteration of RGC density and soma sizes were observed with different drug treatments in eyes with elevated intraocular pressure3,5,6. Therefore, having objective methods of analyzing the retinal morphometries may be of great significance in evaluating retinal pathologies and the effectiveness of therapeutic strategies.
The retinal structure is multi-layers and several different kinds of neurons exist in the retina. The morphometric parameters of retina such as cell number, cell size and thickness of different layers are more complex than the cell culture system. Early on, these parameters can be detected using other commercial imaging software. The values are normally of relative value, and changing to the precise value may need further accurate calculation. Also, the tracing of the cell size and morphology may not be accurate and sensitive enough for statistic analysis, especially in the chronic glaucoma model. The measurements used in this protocol provided a more precise and easy way. And the absolute length of the line and size of the cell can be reported directly and easy to be copied to other files. For example, we traced the margin of the inner and outer most nuclei in the INL and formed a line then using the software to draw a 90 degree angle to measure the thickness. While without the help of the software, the line maybe oblique and the changing of retinal thickness may not be repeatable among individual observers. In addition, the number and density of RGCs can also be quantified. This protocol successfully decreases the variability in quantitating features of the retina, increases the sensitivity in detecting minimal changes.
 	This video will demonstrate three types of morphometric analyses of the retinal sections. They include measuring the INL thickness, quantifying the number of RGCs and measuring the sizes of RGCs in absolute value. These three analyses are carried out with Stereo Investigator (MBF Bioscience &mdash; MicroBrightField, Inc.). The technique can offer a simple but scientific platform for morphometric analyses.

This video demonstrates three types of morphometric analyses of the retina, which include measuring the inner nuclear layer thickness, quantifying the number of retinal ganglion cells (RGCs) and measuring the sizes of RGCs. The technique can offer a simple but scientific platform for morphometric analyses.

Analyses morphométriques des sections rétiniennes

Morphometric analyses of retinal sections have been used in examining retinal diseases. For examples, neuronal cells were significantly lost in the ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Une description visuelle de la dissection de la vascularisation cérébrale et de surface associées Méninges et le plexus choroïde du cerveau de rat

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

La mise en œuvre Clamp dynamique avec conductances synaptiques et artificielle chez la souris cellules ganglionnaires rétiniennes

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Imagerie de la vidéo et cartes spatiotemporelles d&#39;analyser motilité gastro-intestinale chez la souris

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Protocol for Isolating the Mouse Circle of Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.

We describe here a reproducible protocol for isolating the mouse circle of Willis.

Protocole pour Isoler le Cercle de souris de Willis

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Préparation de la souris Retinal Cryo-sections pour l'immunohistochimie

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Optimization of the Retinal Vein Occlusion Mouse Model to Limit Variability

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a detailed method pointing out critical steps is provided with recommendations for optimization to achieve consistently successful occlusion rates across different genetically modified mouse strains. The RVO mouse model consists primarily of the intravenous administration of a photosensitizer dye followed by laser photocoagulation using a retinal imaging microscope attached to an ophthalmic guided laser. Three variables were identified as determinants of occlusion consistency. By adjusting the wait time after rose bengal administration and balancing the baseline and experimental laser output, the variability across experiments can be limited and a higher success rate of occlusions achieved. This method can be used to study retinal diseases that are characterized by retinal edema and hypoxic-ischemic injury. Additionally, as this model induces vascular injury, it can also be applied to study the neurovasculature, neuronal death, and inflammation.

Here, we describe an optimized protocol for retinal vein occlusion using rose bengal and a laser-guided retinal imaging microscope system with recommendations to maximize its reproducibility in genetically modified strains.

Optimisation du modèle murin d’occlusion veineuse rétinienne pour limiter la variabilité

Mouse models of retinal vein occlusion (RVO) are often used in ophthalmology to study hypoxic-ischemic injury in the neural retina. In this report, a ...