The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

Tutti gli animali sono trattati in conformità con la guida della cura e l&#39;uso di animali da laboratorio 2013 Edition, l&#39;Associazione per la Ricerca e la Visione e Ophthalmology (ARVO) Dichiarazione per l&#39;uso di animali in Oftalmica e Vision Research, e approvato dal Animal Istituzionale Cura e Comitato Usa per la Northwestern University. Nota: La seguente procedura può essere eseguita interamente con un operatore; tuttavia, è molto più efficiente condotto con due operatori ai compiti divisi conseguenza. 1. Preparare stazione laser e pre-laser <ol><li> Posizionare il laser e lampada a fessura dove può essere facilmente accessibile. Accendere il laser e impostato su parametri pre-determinato (ad esempio, 75 dimensioni micron posto, 100 mW di potenza, 100 durata msec). ATTENZIONE: Assicurarsi operatore porta tutti i segni di sicurezza laser animale e dispositivi di protezione individuale e pertinente sicurezza laser vengono visualizzati al di fuori della stanza procedimento. <ol><li> Prima di utilizzare eventuali animali da esperimento, trovare parametri ideali utilizzando una calibrazione, mouse non sperimentale. Parametri laser dipenderanno dal laser utilizzato. Parametri ideali sono definiti dall&#39;impostazione potenza del laser più basso che provoca costantemente la formazione di &quot;bolla&quot; se correttamente a fuoco. Guarda il video per esempio di lesione con una corretta &quot;bolla&quot; di formazione. </li></ol></li><li> Preparare la stazione di pre-laser in modo che l&#39;anestesia, animale più caldo, salviette di tessuto, e tutte le gocce per gli occhi (tropicamide, tetracaina, e lacrime artificiali) sono facilmente a portata di mano all&#39;operatore. <ol><li> Assicurarsi che animale caldo viene preriscaldata per correggere la temperatura (37 ° C) prima dell&#39;iniezione di anestetico in primo mouse per evitare l&#39;ipotermia indotta anestesia. </li></ol></li><li> Posizionare fase mouse su più caldo in modo che possa trattenere il calore e rimanere al caldo una volta procedura laser inizia. </li></ol> 2. Topo Anestesia e preparazione pre-laser <ol><li> Prima di iniettareing anestesia, ispezionare l&#39;occhio macroscopicamente per assicurarsi che non ci sono malformazioni o anomalie che diminuiscono la chiarezza della cornea (es cataratta). </li><li> Pesare mouse. </li><li> Record peso, il sesso, e il numero ID animale. </li><li> Utilizzando peso, calcolare giusta quantità di anestetico da utilizzare sulla base di indicazioni fornite da istituto (ad esempio 100 mg / kg di ketamina cloridrato, 10 mg / xylazina kg O tribromoethanol 250 mg / kg; per un 20 g di mouse iniettare 0,20 ml xilazina / ketamina cocktail O 0,25 ml di tribromoethanol). Avere una tabella di dosaggi precalcolati per peso in incrementi di 1 g, al fine di ridurre gli errori matematici. </li><li> Scruff mouse e iniettare anestetico per via intraperitoneale basano su calcoli al punto 2.4. </li><li> Posizionare il mouse su animali più caldo e aspettare fino a quando il mouse è completamente anestetizzato controllando il riflesso pedale via pizzico piedi (circa 3-5 minuti). </li><li> Arrotolate un tessuto pulire e proteggere zona nasale del mouse per evitare che aspirationi di liquido di roll-off. Mouse rotolo su un lato e una goccia (circa 30 ml) di tetracaina cloridrato in ciascun occhio per l&#39;anestesia topica. Attendere 2 minuti per soluzione abbia effetto. </li><li> Ripetere il passaggio 2.7 con una goccia di Tropicamide d&#39;attualità per dilatazione pupillare. In alternativa, utilizzare fenilefrina cloridrato (2,5%) per la dilatazione. </li><li> Attendere 2 minuti per le soluzioni per rendere effettive; tenere animali su più caldo durante questo periodo. </li><li> Trascorso il tempo necessario, posizionare rapidamente il mouse sul palco del mouse e posizionare il palcoscenico sul mento resto della lampada a fessura. </li><li> Accendere lampada a fessura per la luminosità della luce più bassa e verificare il grado di dilatazione pupillare. Se alunno non è adeguatamente dilatato (circa 2,5-3 mm), tornare mouse per animali più caldo e aspettare. In alternativa, l&#39;amministrazione un&#39;altra goccia di Tropicamide. Una volta che l&#39;occhio è sufficientemente dilatato, passare alla procedura laser. </li></ol> 3. Procedura Laser Nota: assicurarsi altri persone nella stanza indossare occhiali protettivi quando si è lontani da protetto laser lampada a fessura oculare <ol><li> Regolare la posizione del mouse sul mouse fase, in modo che sia in posizione ideale per la visualizzazione del nervo ottico (vedi 3.1.2). <ol><li> Orientare il mouse sul supporto in modo che è orizzontale, perpendicolare al fascio lampada a fessura, con la testa da un lato e la coda all&#39;altra. Posizionamento ideale del mouse farà la visualizzazione del nervo ottico molto più facile una volta che viene applicato vetrino. </li><li> Ruotare leggermente il mouse quindi è ad un angolo di 170 ° ~ con la testa più vicino all&#39;operatore laser. </li><li> Assicurarsi che il mouse sia il più vicino possibile lampada a fessura, ma ancora in una posizione in cui è stabile e dove la mano dell&#39;operatore avrà spazio sufficiente per la manipolazione fine. </li></ol></li><li> Dopo che il mouse è in posizione ideale, mettete una goccia di soluzione di lacrima artificiale su un x 25 mm coprioggetto vetro da 25 mm. <ol><li> Mettere una goccia di soluzione di lacrime artificiali sul opp del topoocchio osite - questo occhio assicurerà è idratata e la formazione di aiuto ritardo di cataratta. </li></ol></li><li> Tenere angolo del vetrino tra il pollice e le dita puntatore; posizione in modo che il vetro viene schiacciato tra punte di entrambe le dita. </li><li> Avvolgere delicatamente le altre tre dita intorno corpo dell&#39;animale per il sostegno e la stabilizzazione mano. Posizione della mano in modo che il vetrino di vetro può essere facilmente posizionato sul occhio del mouse. <ol><li> Assicurarsi che il polso è stabilizzato su una superficie stabile in modo da ridurre il tremore della mano. </li></ol></li><li> Una volta ottenuta la posizione stabile, premere delicatamente coprioggetto in vetro (con goccia di lacrima artificiale ancora aderito) sul occhio del mouse. <ol><li> Assicurarsi che il vetrino sia posizionato il più possibile perpendicolare al fascio laser al fine di evitare fascio laser dispersione o riflessione. Il vetrino agisce come una lente a contatto per appiattire la cornea. </li></ol></li><li> Guardare attraverso lampada a fessura e con la mano libera ginocchiera concentrarsi untIL retina possono essere visualizzati. La retina avrà un / colore rosso giallo chiaro a seconda della posizione visualizzato, distinti, vasi rossi saranno visibili. </li><li> Lentamente e con attenzione manipolare testa mouse e / o coprioggetto fino a visualizzare il nervo ottico. Il nervo ottico sarà di colore giallo con più navi che si irradiano da esso. </li><li> Una volta operatore ha confermato la visualizzazione di nervo ottico, accendere il laser focalizzazione del fascio. </li><li> Una volta fascio laser è stata accesa, manovrare laser focalizzazione del fascio nella posizione desiderata (circa 1 diametro del disco del nervo ottico). </li><li> Fuoco del raggio laser sul RPE del fondo dell&#39;occhio. Messa a fuoco appropriata si ottiene con il raggio laser più nitida e chiara. Se l&#39;obiettivo fascio appare ovale o fuori fuoco, ginocchiera fuoco lampada a fessura o riposizionare vetro coprioggetto. </li><li> Una volta che il fascio di puntamento è focalizzata sulla RPE, avviare somministrazione laser utilizzando grilletto piede del laser. <ol><li> Essere sicuri di evitare vasi retinici per prevenire intraoculare luimorrhage. </li></ol></li><li> Osservare per la comparsa di una bolla subito dopo la somministrazione laser. Il contorno del tiro laser dovrebbe essere chiara e non confuso in alcun modo. <ol><li> Se il colpo del laser non provoca la formazione di bolle, o area di impatto sembra confuso (aspetto torbido con mal definito confine circolare contro chiaro, confine ben definito di un impatto di successo), o se l&#39;emorragia è visto dopo la somministrazione del laser, non lo fanno includere queste lesioni per le analisi future. </li></ol></li><li> Ripetere i passaggi 3,10-3,12 per tutte le posizioni volute CNV (di solito a 3, 6, 9, e 12 posizioni intorno nervo ottico). Se induzioni laser sono applicati a circa stessa distanza dal nervo ottico, rifocalizzazione non dovrebbe essere necessario. Tuttavia, a causa del forte curvatura dell&#39;occhio mouse e piccole variazioni che possono esistere nella retina, ricentrare la trave può essere necessario tra le amministrazioni laser consecutivi. </li><li> Record in un notebook della posizione e risultato di postaamministrazione tiro laser ach e risultato (buon fine, nebbioso, emorragia, ecc.) di ogni colpo somministrato per l&#39;occhio. Assicurarsi di posizionare il laser in modalità stand-by quando non è in uso. </li><li> Ripetere 3,1-3,14 per altro occhio del mouse, se necessario, utilizzando la mano opposta per la stabilizzazione e un nuovo vetrino. </li><li> Dopo che tutti i colpi di laser desiderati vengono somministrati, spegnere laser e lampada a fessura. </li><li> Eliminare vetrino e posto del mouse su più caldo per il recupero dall&#39;anestesia. Macroscopicamente ispezionare occhio per eventuali infortuni e una goccia di soluzione di lacrime artificiali per mantenere l&#39;occhio idratato e potenzialmente impedire il futuro sviluppo della cataratta. Una volta che il mouse si riprende dall&#39;anestesia, tornare alla gabbia. </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td></td><td> AVERTIN </td><td> AVERTIN </td><td> AVERTIN </td><td> XYL / KET </td><td> XYL / KET </td></tr><tr><td> Mouse Peso (g) </td><td> Dose (mg / kg) </td><td&gt; Soluzione Concentrazione (mg / ml) </td><td> Anestetico Dose (ml) </td><td> Dose (mg / kg) </td><td> Anestetico Dose (ml) </td></tr><tr><td> 15 </td><td> 250 </td><td> 20 </td><td> 0.1875 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.15 </td></tr><tr><td> 16 </td><td> 250 </td><td> 20 </td><td> 0.2 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.16 </td></tr><tr><td> 17 </td><td> 250 </td><td> 20 </td><td> 0,2125 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.17 </td></tr><tr><td> 18 </td><td> 250 </td><td> 20 </td><td> 0.225 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.18 </td></tr><tr><td> 19 </td><td> 250 </td><td> 20 </td><td> 0,2375 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.19 </td></tr><tr><td> 20 </td><td> 250 </td><td> 20 </td><td> 0.25</td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.2 </td></tr><tr><td> 21 </td><td> 250 </td><td> 20 </td><td> 0,2625 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.21 </td></tr><tr><td> 22 </td><td> 250 </td><td> 20 </td><td> 0,275 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.22 </td></tr><tr><td> 23 </td><td> 250 </td><td> 20 </td><td> 0,2875 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.23 </td></tr><tr><td> 24 </td><td> 250 </td><td> 20 </td><td> 0.3 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.24 </td></tr><tr><td> 25 </td><td> 250 </td><td> 20 </td><td> 0,3125 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.25 </td></tr><tr><td> 26 </td><td> 250 </td><td> 20 </td><td> 0,325 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.26 </td></tr><tr><td> 27 </td><td> 250 </td><td&gt; 20 </td><td> 0,3375 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.27 </td></tr><tr><td> 28 </td><td> 250 </td><td> 20 </td><td> 0.35 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.28 </td></tr><tr><td> 29 </td><td> 250 </td><td> 20 </td><td> 0,3625 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.29 </td></tr><tr><td> 30 </td><td> 250 </td><td> 20 </td><td> 0.375 </td><td> 100 mg / kg di ketamina; 10 mg / kg xilazina </td><td> 0.3 </td></tr></tbody></table> Tabella 1: XyIKet Dosaggio Chart.

<ol>
	<li>Bressler, N. M., Bressler, S. B., Fine, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+related+macular+degeneration.">Age related macular degeneration.</a> Surv Ophthalmol. 32, 375-413 (1988).</li><li>Congdon, N., 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=Causes+and+Prevalence+of+Visual+Impairment+Among+Adults+in+the+United+States.">Causes and Prevalence of Visual Impairment Among Adults in the United States.</a> Arch Ophthalmol. 122, 477-485 (2004).</li><li>Jager, R. D., Mieler, W. F., Miller, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Macular+Degeneration.">Age-Related Macular Degeneration.</a> NEJM. 358, 2606-2617 (2008).</li><li>Poor, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+the+Mouse+Model+of+Choroidal+Neovascularization+Induced+by+Laser+Photocoagulation.">Reliability of the Mouse Model of Choroidal Neovascularization Induced by Laser Photocoagulation.</a> IOVS. 55, 6525-6534 (2014).</li><li>Ryan, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+an+experimental+model+of+subretinal+neovascularization+in+disciform+macular+degeneration.">The development of an experimental model of subretinal neovascularization in disciform macular degeneration.</a> Trans Am Ophthalmol Soc. 77, 707-745 (1979).</li><li>Miller, H., Miller, B., Ishibashi, T., Ryan, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+laser+induced+choroidal+subretinal+neovascularization.">Pathogenesis of laser induced choroidal subretinal neovascularization.</a> IOVS. 31, 899-908 (1990).</li><li>Pennesi, M. E., Neuringer, M., Courtney, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+age+related+macular+degeneration.">Animal models of age related macular degeneration.</a> Molr Aspects Mede. 33, 487-509 (2012).</li><li>Grossniklaus, H. E., Kang, S. J., Berglin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Choroidal+and+Retinal+Neovascularization.">Animal Models of Choroidal and Retinal Neovascularization.</a> Prog Retin Eye Res. 29, 500-519 (2010).</li><li>Zeiss, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REVIEW+PAPER:+Animals+as+Models+of+Age+Related+Macular+Degeneration+An+Imperfect+Measure+of+the+Truth.">REVIEW PAPER: Animals as Models of Age Related Macular Degeneration An Imperfect Measure of the Truth.</a> Vet Pathol Online. 47, 396-413 (2010).</li><li>Tobe, T., 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=Targeted+Disruption+of+the+FGF2+Gene+Does+Not+Prevent+Choroidal+Neovascularization+in+a+Murine+Model.">Targeted Disruption of the FGF2 Gene Does Not Prevent Choroidal Neovascularization in a Murine Model.</a> Am J Pathol. 153, 1641-1646 (1998).</li><li>He, L., Marneros, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+Are+Essential+for+the+Early+Wound+Healing+Response+and+the+Formation+of+a+Fibrovascular+Scar.">Macrophages Are Essential for the Early Wound Healing Response and the Formation of a Fibrovascular Scar.</a> Am J Pathol. 182, 2407-2417 (2013).</li><li>Hoerster, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+ex+vivo+characterization+of+laser+induced+choroidal+neovascularization+variability+in+mice.">In vivo and ex vivo characterization of laser induced choroidal neovascularization variability in mice.</a> Graefes Arch Clin Exp Ophthalmol. 250, 1579-1586 (2012).</li><li>Jawad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Macrophage+Class+A+Scavenger+Receptors+in+a+Laser+Induced+Murine+Choroidal+Neovascularization+Model.">The Role of Macrophage Class A Scavenger Receptors in a Laser Induced Murine Choroidal Neovascularization Model.</a> IOVS. 54, 5959-5970 (2013).</li><li>Lambert, V., 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=Laser+induced+choroidal+neovascularization+model+to+study+age+related+macular+degeneration+in+mice.">Laser induced choroidal neovascularization model to study age related macular degeneration in mice.</a> Nat Protocols. 8, 2197-2211 (2013).</li><li>Sakurai, E., Anand, A., Ambati, B. K., van Rooijen, N., Ambati, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+Depletion+Inhibits+Experimental+Choroidal+Neovascularization.">Macrophage Depletion Inhibits Experimental Choroidal Neovascularization.</a> IOVS. 44, 3578-3585 (2003).</li><li>Espinosa Heidmann, D. G., 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=Macrophage+Depletion+Diminishes+Lesion+Size+and+Severity+in+Experimental+Choroidal+Neovascularization.">Macrophage Depletion Diminishes Lesion Size and Severity in Experimental Choroidal Neovascularization.</a> IOVS. 44, 3586-3592 (2003).</li><li>Giani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Evaluation+of+Laser+Induced+Choroidal+Neovascularization+Using+Spectral+Domain+Optical+Coherence+Tomography.">In Vivo Evaluation of Laser Induced Choroidal Neovascularization Using Spectral Domain Optical Coherence Tomography.</a> IOVS. 52, 3880-3887 (2011).</li><li>Kwak, N., Okamoto, N., Wood, J. M., Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+Is+Major+Stimulator+in+Model+of+Choroidal+Neovascularization.">VEGF Is Major Stimulator in Model of Choroidal Neovascularization.</a> IOVS. 41, 3158-3164 (2000).</li><li>Reich, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+interfering+RNA+(siRNA)+targeting+VEGF+effectively+inhibits+ocular+neovascularization+in+a+mouse+model.">Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.</a> Molr Vis. 9, 210-216 (2003).</li><li>Baffi, J., Byrnes, G., Chan, C. C., Csaky, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroidal+Neovascularization+in+the+Rat+Induced+by+Adenovirus+Mediated+Expression+of+Vascular+Endothelial+Growth+Factor.">Choroidal Neovascularization in the Rat Induced by Adenovirus Mediated Expression of Vascular Endothelial Growth Factor.</a> IOVS. 41, 3582-3589 (2000).</li><li>Claybon, A., Bishop, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Mouse+Eye+for+a+Whole+Mount+of+the+Retinal+Pigment+Epithelium.">Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium.</a> JoVE. , 2563 (2011).</li><li>Espinosa-Heidmann, D. G., 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=Age+as+an+Independent+Risk+Factor+for+Severity+of+Experimental+Choroidal+Neovascularization.">Age as an Independent Risk Factor for Severity of Experimental Choroidal Neovascularization.</a> IOVS. 43, 1567-1573 (2002).</li><li>Zhu, Y., 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=Improvement+and+Optimization+of+Standards+for+a+Preclinical+Animal+Test+Model+of+Laser+Induced+Choroidal+Neovascularization.">Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization.</a> PLoS ONE. 9, e94743 (2014).</li><li>Weinstock, M., Stewart, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+in+rodents+of+reversible+drug+induced+opacities+of+the+lens.">Occurrence in rodents of reversible drug induced opacities of the lens.</a> British J Ophthalmol. 45, 408-414 (1961).</li><li>Ridder III, W. H., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+Cataract+Development+in+Anesthetized+Mice.">Causes of Cataract Development in Anesthetized Mice.</a> Exp Eye Res. 75, 365-370 (2002).</li></ol>

The authors declare they have no competing financial interests.

Ci sono diversi fattori che possono influenzare la consegna del laser e conseguente sviluppo CNV dopo il successo del laser-induzione. Questi fattori devono essere controllati e standardizzati per avere i risultati più affidabili. Il più pertinente di questi fattori sono la selezione del mouse (genotipo, età e sesso), la selezione anestetico, e le impostazioni di laser. Il modello di topo specifico utilizzato può avere un effetto significativo sul corso dello sviluppo CNV. Il genotipo pi...

Età degenerazione maculare (AMD) è una delle principali cause di cecità negli individui di età superiore ai 50 1-3. AMD possono essere classificati in due forme: atrofica (&quot;a secco&quot;) e AMD neovascolare (&quot;wet&quot;) di AMD. La prima è caratterizzata da atrofia geografica dell&#39;epitelio pigmentato retinico (RPE), coriocapillare e fotorecettori, mentre il secondo è caratterizzato dalla invasione dei vasi anomali della coroide negli strati retinici esterni causando perdite, emorragia, e la fibrosi, e in ultima analisi, porta alla cecità 1,2. Delle due forme, AMD neovascolare rappresenta la maggior parte della perdita della vista 1. Fortunatamente, questa forma ha numerose opzioni efficaci per la gestione farmacologica, mentre la sua controparte atrofica attualmente con dimostrato trattamenti medici 3. Inoltre, perché la forma neovascolare è stato facilmente ri-capitolò in un modello animale, è stato più ampiamente accessibili a un baseRicerca MD esplorare i meccanismi patologici alla base per lo sviluppo di nuove terapie 4. Il primo modello animale di neovascolarizzazione coroidale sperimentale (CNV) è stato sviluppato da Ryan et al. nei primati non umani 5. Questo modello di rottura indotta della membrana di Bruch con la fotocoagulazione laser, che ha causato una risposta infiammatoria locale con conseguente angiogenesi simile a quello visto in AMD neovascolare. La progressione istopatologica dell&#39;angiogenesi induzione post-laser è stato trovato per imitare AMD neovascolare, che ha confermato la validità del modello 6. Primati non umani offrire l&#39;anatomia più simili agli esseri umani, ma purtroppo, sono costosi da mantenere, non può essere facilmente manipolati geneticamente, e hanno un decorso lento della progressione della malattia 7. Contrastingly, modelli di roditori sono molto più costo-efficace per mantenere, può essere geneticamente manipolati con relativa facilità, e hanno un molto più veloce course di progressione della malattia (esperimenti possono essere condotti su una scala temporale di settimane rispetto a mesi). Questi esperimenti dovrebbero essere condotti solo in roditori pigmentati in quanto è molto difficile da visualizzare in animali albini. Il modello di CNV mouse laser-indotta, sviluppato dal gruppo di Campochiaro alla fine degli anni 90 del 10, è cresciuto fino a essere il modello animale dominante nella maggior parte dei recenti studi 11-16. A causa della patogenesi complessa e ancora chiaro di CNV, il modello laser è stato applicato in tutti gli aspetti della ricerca AMD umida che vanno dallo studio dei meccanismi molecolari dell&#39;angiogenesi guida per valutare nuove modalità di trattamento per il futuro uso umano. Ad esempio, Sakurai et al. e-Espinosa Heidmann et al. utilizzato il modello laser per studiare l&#39;effetto di macrofagi sullo sviluppo di CNV utilizzando topi transgenici e trattamenti farmacologici deplezione 15, 16. Giani et al. e Hoerster et al. usato ottica tomografia a coerenza (OCT) per l&#39;immagine del CNV, nel tentativo di caratterizzare la progressione della CNV e confrontare i risultati istopatologici i risultati visti in ottobre di imaging 12,17 indotta da laser. Infine, gli studi che coinvolgono iniezione intravitreale di agenti anti-angiogenici sono stati usati come prerequisiti per la sperimentazione umana e sono stati di vitale importanza nello sviluppo della prima generazione di agenti anti-VEGF utilizzate nella gestione di DMLE neovascolare oggi 10,18,19. Modelli per CNV sperimentale alternativi utilizzano metodi chirurgici per indurre CNV. Questa procedura comporta l&#39;iniezione di sostanze pro-angiogenici (ad esempio vettori virali ricombinanti overexpressing VEGF, iniezione sottoretinica di cellule RPE e / o perline di polistirene) per imitare l&#39;aumentata espressione di VEGF visto in DMLE neovascolare, con l&#39;obiettivo di causare angiogenesi 8,20. Tuttavia, questo metodo si ottiene un drasticamente minore incidenza di neovascolarizzazione; questi studi hanno dimostrato che in CNVC57 / BL6 topi si verifica nel 31% delle iniezioni rispetto al tasso di successo ~ 70% visto nel metodo fotocoagulazione laser nello stesso ceppo di topi 8,14. Per queste ragioni, e dati i vantaggi dell&#39;utilizzo di roditori rispetto a primati non umani, il modello murino di indotta da laser CNV è diventato il modello animale standard CNV per la maggior parte neovascolare esperimenti di studio AMD 8. L&#39;occhio mouse è un minuscolo, delicato dei tessuti con cui lavorare. Manovre dell&#39;occhio per visualizzare la retina è difficile e richiede molta pratica fino a raggiungere la padronanza. Questo compito è complicato dal fatto che deve essere appreso con la mano dominante e non dominante. Inoltre, dopo che i movimenti fini richieste per visualizzare la retina sono stati tratti, il coordinamento tra entrambe le mani e il pedale funzionamento del laser sono importanti. In questo lavoro, abbiamo cercato di distillare le sfide di apprendimento di tutte le manipolazioni fisiche coinvolte nel indotta da laser CNV procedure in una guida che avrebbe aiutato gli operatori a raggiungere un rapido successo con questo modello.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

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.

Quantificazione del CNV può essere effettuata attraverso l&#39;analisi dei coroide piatto montato con immunofluorescenza per etichettare i vasi CNV. I due metodi più frequentemente utilizzati di preparazione dei tessuti sono etichettatura FITC-destrano, fatto tramite perfusione immediatamente prima del sacrificio degli animali, o post mortem immuno-colorazione con un marker delle cellule endoteliali. Entrambi questi metodi sono stati descritti in precedenza in dettaglio 13,14,21; figure</str...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

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 modello di topo per Laser-indotta della coroide neovascolarizzazione

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

a-mouse-model-for-laser-induced-choroidal-neovascularization

Research

JoVE Journal

Medicine

17.4K Views.  Northwestern University Feinberg School of Medicine. 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.

Video: A Mouse Model for Laser-induced Choroidal Neovascularization

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

Un modello di topo indotta da laser di cronica ipertensione oculare per caratterizzare difetti visivi

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

Ricerca

Medicina

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

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

Rilevamento Anomalie nella coroide Vasculature in un modello murino di età degenerazione maculare da Time-course verde indocianina angiografia

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

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Un Injury Modello alcali-burn di neovascolarizzazione corneale nel mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un Mouse fetale Modello Skin of Wound Repair scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Un neonatale mouse Spinal Cord Injury compressione Modello

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Puntura-indotta di Neovascularization dell'iride come un modello murino di Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

Un nuovo approccio al monitoraggio dell'innesto neovascolarizzazione nella gengiva umana

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Un modello murino per la pancreatite cronica tramite infusione di TNBS del dotto biliare

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Lesioni indotte da laser semi-automatizzate controllate per lo studio della rigenerazione del midollo spinale nelle larve di zebrafish

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...

Methods Article

Un modello di topo per Laser-indotta della coroide neovascolarizzazione