Name: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model (Video) | JoVE Journal
Uploaded: 2018-01-21T13:00:00.000+00:00
Duration: 9 min 56 s
Description: Learn multimodal imaging techniques for tracking choroidal neovascularization in mice using SD-OCT and fluorescein angiography. This method enables longitudinal assessment of retinal pathology for ocular disease research.

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5 minutes

The authors would like to thank Yuliya Naumchuk (Loyola University Chicago) and Agne &#381;iniauskaite&#775; (Experimentica Ltd.) for excellent technical and videographic support. Dr. Kaja&#8217;s research program is supported by the Dr. John P. and Therese E. Mulcahy Endowed Professorship in Ophthalmology at Loyola University Chicago.

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the EC Directive 86/609/EEC for animal experiments, using protocols approved and monitored by the Animal Experiment Board of Finland.
1. Laser-induced mouse CNV model 5
<ol>
	<li>Inspect the eyes of the animal macroscopically for any abnormalities.</li>
	<li>Weigh the mouse.</li>
	<li>Calculate and prepare an appropriate amount of anesthetics to use, based on the weight of the animal, e.g. a mixture of medetomidine (1 mg/kg), ketamine (75 mg/kg), and distilled water (0.9% NaCl solution) at a ratio of 1:1.5:2.5, or ketamine (40-75 mg/kg), xylazine (5 mg/kg), and distilled water (0.9% NaCl solution) at a ratio of 1:2.5:1; for a 20 g mouse, inject 0.1 mL of mixture.</li>
	<li>Inject anesthetic intraperitoneally.</li>
	<li>Place mouse back into the cage and wait until animal is anesthetized. Confirm the mouse is properly anesthetized by lack of a pedal reflex.</li>
	<li>Ensure the use of laser safety personal protective equipment.</li>
	<li>Turn on a slit lamp and a 532 nm diode laser.</li>
	<li>Remove the mouse from the cage and place on the heating pad.</li>
	<li>Apply one drop of Tropicamide for pupillary dilation. Wait for 3-5 min for full (3 mm) pupillary dilation.</li>
	<li>Place the mouse on the stage of the slit lamp.</li>
	<li>Place one drop of opthalmic liquid gel on a coverslip to applanate the cornea.</li>
	<li>Orient the mouse eye with the optic nerve head in the center.</li>
	<li>Set the laser power to 100 mW, the duration to 100 ms, and the spot size to 50 &#181;m.</li>
	<li>Focus the laser beam on the retinal pigment epithelium (RPE).</li>
	<li>Make three laser shots into one eye by avoiding retinal blood vessels ideally at the 4, 8, and 12 o&#39;clock positions around the optic nerve, respectively. Inspect the fundus of the eye after all laser shots for absence of retinal bleeding. The contralateral eye serves as a non-lasered control.</li>
	<li>Discard the coverslip and place mouse back on the heating pad.</li>
	<li>Apply one drop of PEG gel drops on both eyes.</li>
</ol>
2. SD-OCT 6,7
<ol>
	<li>Place the mouse into the rodent alignment stage, and immobilize the head.</li>
	<li>Align the lens of the SD-OCT system (e.g., Bioptigen/Leica Envisu R2200) to face the eye for in vivo imaging using X- and Y-stage controllers.</li>
	<li>Perform SD-OCT scans to verify breaks of Bruch&#39;s membrane: once the SD-OCT scans the whole eye, manually move the reference line on the lasered sites. Breaks of Bruch&#39;s membrane should be clearly visible in lasered areas (see Figure 1).</li>
</ol>
3. Fluorescein angiography 7,8,9
<ol>
	<li>Remove mouse with the holder, and place it on the FA system (e.g., Heidelberg Spectralis HRA2).</li>
	<li>Focus at laser burn areas of the fundus of the eye using infrared reflectance mode with the head of the optic nerve in the middle of the viewing window.</li>
	<li>Inject 0.1 mL of 5% fluorescein sodium salt for a 20 g mouse subcutaneously or intraperitoneally.</li>
	<li>Focus on the choroidal level.</li>
	<li>Take an image from the choroidal focus level.</li>
	<li>Re-focus at the retinal level and take an image.</li>
	<li>Wait for 30 s and repeat steps 3.4-3.6.</li>
	<li>Remove the mouse from the holder and place it on the heating pad.</li>
	<li>Reverse anesthesia by &#945;2-antagonist for medetomidine, atipamezole (0.5 mg/kg, i.p.), or wait for animal recovery from anesthesia.</li>
	<li>Repeat in vivo SD-OCT and FA imaging in anesthetized animals on the follow-up days 5, 10, and 14.</li>
</ol>
4. CNV Grading
<ol>
	<li>Grade the damage of Bruch&#39;s membrane from OCT images and choroidal FA images taken immediately after the lasering on day 0 as following: 
	0 - Bruch&#39;s membrane was not damaged 
	1 - successful damage of Bruch&#39;s membrane</li>
	<li>Grade the presence of CNV from lasered spots that had leakage as observed by comparing dynamics of fluorescein signal in a series of retinal FA images as following: 
	0 - normal appearance of retina 
	0.5 - faint staining of leakage 
	1.0 - leaky CNV areas 
	Note: Use the OCT imaging for additional confirmation of CNV or in questionable FA where the presence of intraretinal fluid in OCT images would suggest CNV grading.</li>
</ol>
5. Retinal Thickness Measurements
<ol>
	<li>Use an automated segmentation software for retinal thickness measurements. Ensure that the total retinal thickness is considered, as the thickness of all layers from nerve fiber layer to RPE (healthy measurement sites), or to an imaginary line connecting RPE around the site of damage (lasered sites) (see also Figure 7).</li>
</ol>

<ol><li>Dobi, E. T., Puliafito, C. A., Destro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+model+of+experimental+choroidal+neovascularization+in+the+rat%5BTitle%5D)+AND+%22Arch.+Ophthalmol.+Chic.+Ill+1960%22%5BJournal%5D)">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/pubmed?term=((Evolution+of+neovascularization+in+mice+with+overexpression+of+vascular+endothelial+growth+factor+in+photoreceptors%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">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/pubmed?term=((Dramatic+inhibition+of+retinal+and+choroidal+neovascularization+by+oral+administration+of+a+kinase+inhibitor%5BTitle%5D)+AND+%22Am.+J.+Pathol%22%5BJournal%5D)">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/pubmed?term=((Animal+models+of+choroidal+and+retinal+neovascularization%5BTitle%5D)+AND+%22Prog.+Retin.+Eye+Res%22%5BJournal%5D)">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/pubmed?term=((A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((In+vivo+evaluation+of+laser-induced+choroidal+neovascularization+using+spectral-domain+optical+coherence+tomography%5BTitle%5D)+AND+%22Invest.+Ophthalmol.+Vis.+Sci%22%5BJournal%5D)">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/pubmed?term=((Optimization+of+an+Image-Guided+Laser-Induced+Choroidal+Neovascularization+Model+in+Mice%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((Neuroprotectin+D1+attenuates+laser-induced+choroidal+neovascularization+in+mouse%5BTitle%5D)+AND+%22Mol.+Vis%22%5BJournal%5D)">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/pubmed?term=((In-vivo+and+ex-vivo+characterization+of+laser-induced+choroidal+neovascularization+variability+in+mice%5BTitle%5D)+AND+%22Graefes+Arch.+Clin.+Exp.+Ophthalmol.+Albrecht+Von+Graefes+Arch.+Klin.+Exp.+Ophthalmol%22%5BJournal%5D)">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/pubmed?term=((A+Simple+Optical+Coherence+Tomography+Quantification+Method+for+Choroidal+Neovascularization%5BTitle%5D)+AND+%22J.+Ocul.+Pharmacol.+Ther.+Off.+J.+Assoc.+Ocul.+Pharmacol.+Ther.+Off.+J.+Assoc.+Ocul.+Pharmacol%22%5BJournal%5D)">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>

The author Symantas Ragauskas, Ph.D. is an employee (research scientist) and shareholder of Experimentica Ltd. that offers contract research services employing the preclinical CNV model used in this Article.
The author Eva Kielczewski is an employee (research applications engineer, OCT) of Leica Microsystems that produces SD-OCT systems used in this Article.
The author Joseph Vance is an employee (NA OCT Sales Director) of Leica Microsystems that produces SD-OCT systems used in this Article. Joseph Vance is also president and managing director of Spective, LLC.
The author Simon Kaja, Ph.D. is consultant Chief Scientific Officer and shareholder of Experimentica Ltd., a preclinical contract research organization that offers contract research services, incl. the preclinical CNV model used in this article. Simon Kaja, Ph.D. is also CEO of K&#38;P Scientific, LLC, a life sciences consulting firm, and serves as the Dr. John P. and Therese E. Mulcahy Endowed Professor in Ophthalmology at Loyola University Chicago, Stritch School of Medicine. The terms of this arrangement have been reviewed and approved by Loyola University Chicago in accordance with its conflict of interest policy.
The author Giedrius Kalesnykas, Ph.D. is an employee (CEO) and shareholder of Experimentica Ltd. that offers contract research services employing the preclinical CNV model used in this Article.

Multimodal imaging offers valuable tools for CNV pathology evaluation. Here we presented an imaging protocol consisting of FA, SD-OCT, and automatic segmentation for the quick, reproducible, and reliable evaluation of CNV pathology. A break of Bruch's membrane after laser administration was confirmed. In addition, the use of SD-OCT at this stage also allowed immediate visualization of possible intraretinal and subretinal hemorrhages, which may confound the interpretation of results. Retinal leaks were graded based on the...

The first successful attempt to mimic the pathology of human CNV in rodents was demonstrated almost three decades ago with a krypton laser in Long-Evans rats1. Thereafter, a krypton laser was used to break Bruch&#39;s membrane in the most popular mouse strain, C57BL/6J2,3,4. The success rate of CNV induction was verified with FA and histological stains. A rapid development of noninvasive imaging modalities, such as OCT, fostered the growth of the field of rodent preclinical models. The ability to monitor morphological changes in the retina at multiple time points in the same eye significantly contributes to the reduction of animal use, and increases efficiency in experimental studies. Histological evaluation of CNV lesions is rather straightforward, and requires labeling of abnormal vascular growth around the site of laser administration, image acquisition, and area/volume estimation using an image analysis software. In contrast, in vivo imaging modalities introduce more complex analyses of CNV pathology and its interpretation.
Here we present a simple and relatively fast method to grade induction, progression, and regression of CNV using FA, SD-OCT, and the automated segmentation method in the mouse laser-induced CNV model.

<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

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

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

A bubble or subretinal bleeding immediately after lasering is not always visible. Therefore, SD-OCT is particularly important to verify damage of Bruch's membrane. Figure 1 shows an example of OCT imaging at different time points after laser administration.
<img alt="Retinal imaging over 14 days; diagram shows lesion progression; ophthalmic research results." class="xfigimg" src="/files/ftp_upload/5...

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

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

Neovascular processes are a characteristic feature of several prevalent ocular pathologies. Most prominently, exudative age-related macular degeneration or Wet AMD for short, proliferative diabetic retinopathy, and retinopathy of prematurity. Together, these disorders account for most instances of legal blindness worldwide and are associated with additional ocular complications, such as vitreous hemorrhage and neovascular glaucoma.However, despite their prevalence, therapeutic options for ocular neovascular disorders are limited. The current standard of care for neovascularization associated with AMD and proliferative diabetic retinopathy is the intravitreal injection of humanized antibodies directed against a critical mediator of angiogenesis and neovascularization, vascular endothelial growth factor or VEGF. However, despite the efficacy in halting disease progression and improving functional vision, intravitreal injections are costly and far from being without risk.Complications can include infections, endophthalmitis, retinal detachment, and ocular hemorrhage. Therefore, there's an urgent need for the development of novel treatment options that are efficacious, safe, and less costly. In order to advance drug development for neovascular disorders, small animal models are of critical importance.Such models need to be reproducible, have established and validated readouts and endpoints, and ideally use a clinically-relevant reference compound to serve as positive control. In this protocol, we will present the mouse choroidal neovascularization, or CNV model, which is one of the most commonly employed models for both the study of pathophysiological mechanisms contributing to CNV and the development of novel anti-neovascular agents. In the CNV model, Bruch's membrane is ruptured using an argon laser.Thereby initiating neovascular processes originating from the choroid. Employing longitudinal in vivo imaging by spectral-domain optical coherence tomography, or SD-OCT for short, and fluorescein angiography, provides a means to follow the proliferation and regression of CNV. And thereby, to assess the efficacy and time course of novel pharmaceutical interventions.Recent advances in image processing furthermore allow for the automated segmentation for the measurement of retinal thickness, providing a methodology free of investigator bias to evaluate the presence of edema. Herein, we will discuss the usefulness of the new InVivoVue Diver software by Leica Microsystems for the automated segmentation of retinal layers in the mouse CNV model. Lastly, we will discuss how the histological analysis of retinal whole mounts can complement longitudinal in vivo imaging in this model.A single drop of tropicamide is applied onto each eye for pupillary dilation. Subsequently, the animal is placed gently into the rodent alignment stage. The mouse is adjusted to expose the eye and secured using the nose holder and a piece of laboratory tape placed gently over the back.Next, a drop of lubricant is applied onto the eye to moisturize, and any excess liquid is carefully removed using filter paper swabs. Lastly, the stage is aligned in front of the OCT device for baseline OCT imaging. in vivo spectral-domain optical coherence tomography imaging is performed at baseline, prior to laser application, in order to verify the absence of any retinal abnormalities.Gently remove the mouse from the holder. Place one drop of Viscotears Gel on a cover slip to applanate the cornea. Orient the mouse with the optic nerve head in the center, and focus the laser beam on the retinal pigment epithelium.Make three laser shots by avoiding retinal blood vessels at four, eight, and 12 o'clock positions around the optic nerve. Inspect the firmness of the eye after all laser shots for the absence of any retinal bleeding. As before, align the mouse in the holder and perform fluorescein angiography and OCT imaging to confirm damage of Bruch's membrane.For fluorescein angiography, first focus at laser burn areas using the infrared reflectance mode, which allows to visualize the sites laser was administered. Carefully inject 0.1 milliliters of 5%fluorescein sodium salt, for approximately 20 grams of mouse body weight without changing the mouse eye position. Start taking fluorescein angiography images at the level of the choroid and at the level of the retina, approximately every 30 seconds.Align the eye for OCT imaging as before and start imaging. Averaging the SD-OCT signal helps to better visualize the detailed retinal morphology, as shown in this sequence. Once the SD-OCT imaging has been performed, carefully remove the mouse from the holder.Apply lubricant for both eyes. At this point, you may choose to reverse anesthesia by subcutaneous injection of the alpha-2 antagonist for medetomidine, atipamezole, or wait for animal recovery from anesthesia. Repeat the in vivo SD-OCT and FA imaging in anesthetized animals on follow-up days five, 10, and 14.Use the automated segmentation feature by the InVivoVue Diver software for retinal thickness measurements. For the healthy retina, the total retinal thickness is considered as the thickness of all layers, from the nerve fiber layer to the RPE. In the healthy retina, automated segmentation can accurately detect individual retinal layers, as can be seen in this example.It is noteworthy to mention, that in places where CNV is present, manual measurement of retinal thickness should be performed for each CNV lesion site separately. From the nerve fiber layer to an imaginary line passing through the RPE layer. This sequence shows the workflow for manual thickness measurements at sites where CNV lesions are present.The nerve fiber layer is located at the top, the choroid at the bottom of the image. Using the mouse or touchpad, the borders of the nerve fiber layer and the choroid are manually determined, and the software automatically calculates the total retinal thickness based on these coordinates. This figure shows serial images taken every 20 seconds at the choroidal level.Image one was acquired in infrared reflectance mode. Whereas the others, after intraperitoneal fluorescein injection. White arrows in image one point to lasered sites, which show fluorescein leakage at later time points, highlighted by the white arrows in image 18.This image shows FA serial imaging acquired at the retinal level. The white arrow in image 18 points to a lasered site, which shows fluorescein leakage appearing faster than the other two, outlined in circles in image 18. This summary figure shows a time course of SD-OCT images.At baseline, immediately after lasering, and the follow-up time points on days five, 10, and 14. In summary, the protocol for the longitudinal in vivo imaging of CNV lesions using SD-OCT and FA imaging allows for the fast, multimodal, and reliable classification of CNV pathology and the presence of retinal edema. Thank you for your interest.

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Learn multimodal imaging techniques for tracking choroidal neovascularization in mice using SD-OCT and fluorescein angiography. This method enables longitudinal assessment of retinal pathology for ocular disease research.

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

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