Gli autori vorrei ringraziare Yuliya Naumchuk (Loyola University Chicago) e Agne Žiniauskaitė (Experimentica Ltd.) per un eccellente supporto tecnico e videografiche. Programma di ricerca del Dr. Kaja è supportato dal Dr. John P. e Therese E. Mulcahy dotato Professorship in Oftalmologia presso la Loyola University di Chicago.

Tutti gli animali sono stati trattati in conformità con la dichiarazione di ARVO per l'uso degli animali in oftalmica e Vision Research e CE direttiva 86/609/CEE per gli esperimenti sugli animali, utilizzando protocolli approvati e monitorati dal Consiglio esperimento animale della Finlandia.
1. indotto da laser mouse CNV modello 5
<ol><li>Ispezionare gli occhi dell'animale in modo macroscopico per eventuali anomalie.</li>
	<li>Pesare il mouse.</li>
	<li>Calcolare e preparare un'appropriata quantità di anestetici da utilizzare, in base al peso dell'animale, ad esempio una miscela di medetomidina (1 mg/kg), ketamina (75 mg/kg) e acqua distillata (soluzione di NaCl 0,9%) a un rapporto di 1:1.5:2.5, o ketamina (40-75 mg/kg), xilazina (5 mg/kg) e acqua distillata (soluzione di NaCl 0,9%) a un rapporto di 1:2. 5:1; per un mouse 20g, iniettare 0,1 mL della miscela.</li>
	<li>Iniettare anestetico per via intraperitoneale.</li>
	<li>Posizionare il mouse nuovamente dentro la gabbia e aspettare fino a quando l'animale è anestetizzato. Confermare che il mouse è adeguatamente anestetizzato dalla mancanza di una reflex pedale.</li>
	<li>Garantire l'uso di dispositivi di protezione individuale sicurezza laser.</li>
	<li>Accendere una lampada a fessura e un laser a diodo 532 nm.</li>
	<li>Rimuovere il mouse dalla gabbia e posto il termoforo.</li>
	<li>Applicare una goccia di Tropicamide per dilatazione pupillare. Attendere 3-5 min per la dilatazione pupillary pieno (3 mm).</li>
	<li>Posizionare il mouse sul palco della lampada a fessura.</li>
	<li>Depositare una goccia di gel liquido opthalmic su un vetrino coprioggetto per applanate la cornea.</li>
	<li>Orientare l'occhio del mouse con la testa del nervo ottico nel centro.</li>
	<li>Impostare la potenza del laser a 100 mW, la durata di 100 ms e la dimensione dello spot a 50 µm.</li>
	<li>Focalizzare il fascio laser sull'epitelio pigmentato retinico (RPE).</li>
	<li>Fare tre colpi di laser in un occhio evitando vasi sanguigni retinici idealmente a 4, 8 e 12 posizioni intorno al nervo ottico, rispettivamente. Ispezionare il fondo dell'occhio dopo tutti i laser scatti per assenza di emorragia retinica. L'occhio controlaterale serve come controllo non laserato.</li>
	<li>Scartare il mouse vetrino coprioggetti e posto indietro il termoforo.</li>
	<li>Applicare una goccia di PEG gel gocce su entrambi gli occhi.</li>
</ol>2. SD-OCT 6,7
<ol><li>Posizionare il mouse in fase di allineamento del roditore e immobilizzare la testa.</li>
	<li>Allineare la lente del sistema SD-OCT (ad es., Bioptigen/Leica Envisu R2200) ad affrontare l'occhio per l'imaging in vivo utilizzando controller X - e Y-stage.</li>
	<li>Eseguire scansioni di SD-OCT per verificare rotture della membrana di Bruch: una volta che il SD-OCT scansiona l'intero occhio, spostare manualmente la linea di riferimento sui siti laserati. Rotture della membrana di Bruch devono essere chiaramente visibile nelle aree laserate (Vedi Figura 1).</li>
</ol>3. fluoresceina angiografia 7,8,9
<ol><li>Rimuovere il mouse con il titolare e posizionarlo sul sistema FA (ad es., Heidelberg Spectralis HRA2).</li>
	<li>Messa a fuoco laser masterizzare aree del fondo dell'occhio utilizzando la modalità a raggi infrarossi riflettanza con la testa del nervo ottico al centro della finestra di visualizzazione.</li>
	<li>Iniettare 0,1 mL di sale di sodio di 5% della fluorescina per un mouse di 20 g per via sottocutanea o per via intraperitoneale.</li>
	<li>Concentrarsi sul livello della coroide.</li>
	<li>Prendere un'immagine dal livello di messa a fuoco della coroide.</li>
	<li>Rimettere a fuoco a livello della retina e prendere un'immagine.</li>
	<li>Attendere per 30 s e ripetere passaggi 3.4-3.6.</li>
	<li>Rimuovere il mouse dal supporto e posizionarlo sul rilievo di riscaldamento.</li>
	<li>Invertire l'anestesia α2-antagonista per medetomidina, atipamezolo (0,5 mg/kg, i.p.) o attesa per animale recupero dall'anestesia.</li>
	<li>Ripetere in vivo SD-OCT e FA imaging in animali anestetizzati sui giorni di follow-up 5, 10 e 14.</li>
</ol>4. CNV classificazione
<ol><li>Grado il danno della membrana di Bruch da immagini di ottobre e coroidica FA immagini scattate subito dopo la lavorazione al laser il giorno 0 come segue: 0 - membrana di Bruch non è stata danneggiata 1 - successo danno della membrana di Bruch</li>
	<li>Grado la presenza di CNV da laserata macchie che aveva perdite come osservato confrontando la dinamica del segnale della fluorescina in una serie di immagini retiniche FA come segue: 0 - aspetto normale della retina 0,5 - macchiatura debole di perdita 1.0 - aree CNV che perde Nota: Utilizzare l'imaging OCT per ulteriore conferma di CNV o in discutibile FA dove la presenza di liquido intraretinico in immagini di ottobre vorrei suggerire CNV classificazione.</li>
</ol>5. misure di spessore retinico
<ol><li>Utilizzare un software di segmentazione automatizzata per le misurazioni dello spessore retinico. Assicurarsi che lo spessore totale della retina è considerato, come lo spessore degli strati tutti da strato delle fibre nervose di RPE (siti di misura sano) o di una linea immaginaria che collega RPE intorno al sito di danno (laserate siti) (Vedi anche Figura 7).</li>
</ol>

<ol>
	<li>Dobi, E. T., Puliafito, C. A., Destro, M. <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+model+of+experimental+choroidal+neovascularization+in+the+rat.">A new model of experimental choroidal neovascularization in the rat.</a> Arch. Ophthalmol. Chic. Ill 1960. 107, 264-269 (1989).</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=Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors.">Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors.</a> Invest. Ophthalmol. Vis. Sci. 39, 180-188 (1998).</li><li>Seo, M. 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=Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor.">Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.</a> Am. J. Pathol. 154, 1743-1753 (1999).</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>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> J Vis Exp. (106), e53502 (2015).</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> Invest. Ophthalmol. Vis. Sci. 52, 3880-3887 (2011).</li><li>Gong, 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=Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice.">Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice.</a> PloS One. 10, e0132643 (2015).</li><li>Sheets, K. 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=Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse.">Neuroprotectin D1 attenuates laser-induced choroidal neovascularization in mouse.</a> Mol. Vis. 16, 320-329 (2010).</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. Albrecht Von Graefes Arch. Klin. Exp. Ophthalmol. 250, 1579-1586 (2012).</li><li>Sulaiman, R. 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=A+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization.">A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.</a> J. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. Ther. Off. J. Assoc. Ocul. Pharmacol. 31, 447-454 (2015).</li></ol>

L'autore Symantas Ragauskas, pH.d. è un dipendente (ricercatore) e azionista di Experimentica Ltd. che offre servizi di ricerca che impiegano il modello preclinico di CNV utilizzato in questo articolo del contratto.
L'autore Eva Kielczewski è un impiegato (Ricerca applicazioni engineer, OCT) di Leica Microsystems che produce sistemi di SD-OCT utilizzati in questo articolo.
L'autore Joseph Vance è un dipendente (NA OCT direttore vendite) di Leica Microsystems che produce sistemi di SD-OCT utilizzati in questo articolo. Joseph Vance è anche Presidente e amministratore delegato di spettiva, LLC.
L'autore Simon Kaja, pH.d. è consulente Chief Scientific Officer e azionista di Experimentica Ltd., un'organizzazione di ricerca preclinica contratto che offre servizi di ricerca, contraggono incl. il modello preclinico di CNV utilizzato in questo articolo. Simon Kaja, pH.d. è anche CEO di K & P scientifica, LLC, una consulenza, Scienze della vita e serve come il Dr. John P. e Therese E. Mulcahy dotato professore di Oftalmologia presso la Loyola University Chicago, Stritch School of Medicine. I termini di questo accordo sono stati esaminati e approvati dal Loyola University Chicago in conformità alla sua politica di conflitto di interessi.
L'autore Giedrius Kalesnykas, pH.d. è un dipendente (CEO) e azionista di Experimentica Ltd. che offre servizi di ricerca che impiegano il modello preclinico di CNV utilizzato in questo articolo del contratto.

Imaging multimodale offre preziosi strumenti per la valutazione di patologia CNV. Qui abbiamo presentato un protocollo imaging composto da FA, SD-OCT e segmentazione automatica per la valutazione rapida, riproducibile e affidabile di patologia CNV. Una rottura della membrana di Bruch dopo amministrazione del laser è stato confermato. Inoltre, l'uso di SD-OCT in questa fase anche ha permesso la visualizzazione immediata delle possibili emorragie intraretiniche e subretinal, che può confondere l'interpretazione dei risul...

Il primo tentativo riuscito di imitare la patologia di CNV umana nei roditori è stato dimostrato quasi tre decenni fa con un laser di krypton a lungo Evans ratti1. Da allora in poi, un laser di krypton è stato utilizzato per rompere la membrana di Bruch nel ceppo più popolare del mouse C57BL/6J2,3,4. Il tasso di successo dell'induzione di CNV è stato verificato con FA e macchie istologiche. Un rapido sviluppo di modalità di imaging non invasivo, ad esempio OCT, ha favorito la crescita del campo di modelli preclinici del roditore. La possibilità di monitorare i cambiamenti morfologici nella retina a più punti di tempo nello stesso occhio significativamente contribuisce alla riduzione dell'uso di animali e aumenta l'efficienza negli studi sperimentali. La valutazione istologica delle lesioni CNV è piuttosto semplice e richiede etichettatura di crescita vascolare anormale intorno al sito di amministrazione di laser, acquisizione di immagini e stima di superficie/volume utilizzando un software di analisi di immagine. Al contrario, la modalità di imaging in vivo introdurre più complesse analisi di CNV patologia e sua interpretazione.
Qui presentiamo un metodo semplice e relativamente veloce per induzione di grado, la progressione e regressione di CNV utilizzando FA, SD-OCT, e il metodo di segmentazione automatizzata nel topo indotta da laser CNV modello.

<table><tbody><tr><td>Medetomidine (commercial name Domitor)</td><td>Orion</td><td>Vnr 01 56 02</td><td>Anesthesia</td></tr><tr><td>Ketamine</td><td>Intervet</td><td>Vnr 51 14 85</td><td>Anesthesia</td></tr><tr><td>0,9% NaCl</td><td>B Braun</td><td>357 0340</td><td>Anesthesia</td></tr><tr><td>Xylazine (commercial name Rompun vet)</td><td>Bayer</td><td>vnr 14 89 99</td><td>Anesthesia</td></tr><tr><td>Tropicamide</td><td>Santen</td><td>Vnr 04 12 36</td><td>Mydriatic agent</td></tr><tr><td>Viscotears</td><td>Alcon</td><td>Vnr 44 54 81</td><td>Lubricant</td></tr><tr><td>Systane</td><td>Alcon</td><td>&amp;nbsp;-</td><td>Lubricant</td></tr><tr><td>5% Fluorescein sodium salt</td><td>Sigma Aldrich</td><td>F6377-100G</td><td>Fluoresent agent</td></tr><tr><td>Atipamezole (commercial name Antisedan)</td><td>Orion</td><td>Vnr 47 19 53</td><td>Anesthesia</td></tr></tbody></table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

Indotta da laser il neovascularization coroidico (CNV) è una modella ben affermata per imitare la forma umida di degenerazione maculare senile (AMD). In questo protocollo, ci proponiamo di guidare il lettore non semplicemente attraverso le considerazioni tecniche di generare lesioni indotte da laser per innescare processi neovascolare, ma piuttosto concentrarsi sulle informazioni potenti che possono essere ottenute da multimodal longitudinale in vivo imaging durante tutto il periodo di follow-up.
Il topo indotta da laser modello CNV è stato generato da un'amministrazione di laser del diodo. Tecniche di imaging multimodale in vivo sono stati utilizzati per monitorare la CNV induzione, progressione e regressione. Primo, tomografia a coerenza ottica dominio spettrale (SD-OCT) è stata effettuata immediatamente dopo la lavorazione laser per verificare una rottura della membrana di Bruch. Successivi in vivo imaging mediante angiografia della fluorescina (FA) ha confermato il successo danno della membrana di Bruch da seriale immagini acquisite a livello della coroide. Follow-up longitudinale di proliferazione di CNV e regressione nei giorni 5, 10 e 14 dopo la lavorazione laser è stato effettuato usando sia SD-OCT e FA. Semplice e affidabile di classificazione di leasions di CNV che perde da FA immagini è presentato. Segmentazione automatizzata per la misurazione dello spessore retinico totale, combinato con applicazione manuale calibro per la misurazione dello spessore retinico a siti CNV, consentire la valutazione imparziale della presenza dell'edema. Infine, verifica istologica di CNV viene eseguita utilizzando isolectin GS-IB4 colorazione sul flatmounts coroidico. La colorazione è con soglia, e viene calcolata l'area isolectin-positivi con ImageJ.
Questo protocollo è particolarmente utile negli studi di terapeutica che richiedono alta-velocità di trasmissione-come proiezione di patologia CNV quanto consente veloce, multimodale e affidabile classificazione dell'edema di patologia e retinico CNV. Inoltre, ad alta risoluzione SD-OCT consente la registrazione di altre caratteristiche patologiche, come l'accumulazione di liquido subretinal o intraretinico. Tuttavia, questo metodo non fornisce la possibilità di automatizzare l'analisi di volume CNV da immagini SD-OCT, che deve essere eseguita manualmente.

Una bolla o subretinal sanguinamento immediatamente dopo lavorazione laser non è sempre visibile. Pertanto, SD-OCT è particolarmente importante per verificare il danno della membrana di Bruch. Figura 1 Mostra un esempio di formazione immagine OCT a tempi diversi dopo la somministrazione di laser.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> <strong class="xfi...

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

In Vivo Imaging multimodale e l'analisi di Mouse indotta da Laser modello di Neovascularization coroidico

Qui, presentiamo l'utilità di longitudinale in vivo imaging nel follow-up dei cambiamenti morfologici del neovascularization coroidico laser-indotta in topi.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

9.0K Views.  Experimentica Ltd.. Qui, presentiamo l'utilità di longitudinale in vivo imaging nel follow-up dei cambiamenti morfologici del neovascularization coroidico laser-indotta in topi.

Video: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography

The present protocol describes the implantation and evaluation of melanoma in the murine choroid utilizing optical coherence tomography.

Impianto e valutazione del melanoma nella coroide murina mediante tomografia a coerenza ottica

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, ...

Ricerca sul cancro

A Mouse Model for Laser-induced Choroidal Neovascularization

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

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

Un 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 Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Una piattaforma di microscopia multifoto personalizzata per l'imaging dal vivo di Mouse Cornea e Conjunctiva

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. ...

Bioingegneria

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging dei topi Reporter Cx3cr1gfp/gfp con tomografia a coerenza ottica Spectral-domain e scansione Laser oftalmoscopia

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

Digestione di interi occhi di topo per l'analisi citometrica a flusso multi-parametro dei fagociti mononucleari

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Immunologia e infezioni

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillare Due fotoni In vivo Imaging della retina del topo

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Neuroscienze

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.

Ottimizzazione del modello murino di occlusione venosa retinica per limitare la variabilità

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 ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

In vivo Letture di lesioni vascolari nella retina del topo per promuovere la riproducibilità

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Multiphoton Intravital Imaging per il monitoraggio del reclutamento dei leucociti durante l'arteriogenesi in un modello murino di arti posteriori

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...