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/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>

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 fluorescein signal from FA images. The use of SD-OCT provided a more detailed description of CNV pathology. Moreover, longitudinal SD-OCT analysis at various time points throughout the follow-up period highlighted temporal differences in the pathology that would remain elusive if relying on FA alone. 
Total retinal thickness was measured using automated segmentation. Retinal thickness at sites CNV was induced was manually measured. The histological evaluation of choroidal flatmount is verified, and the area of neovascularization is measured using image analysis software Image J. 
The proper transparency of the visual axis is critical for successful performance of the presented protocol. Dryness of the cornea and formation of cataracts are the main factors involved in troubleshooting. Therefore, once the mouse gets anesthetized, the eyes should be constantly hydrated with artificial tears or gel to maintain proper hydration of the cornea. The proposed protocol should be performed preferentially within 10 min from the induction of anesthesia. Extended anesthesia time may cause the formation of cataracts and prevent in vivo imaging. 
The described protocol is limited to observational grading of CNV progression based on vascular leaks at the level of the retina. Quantitative assessment of retinal leak can be added using Heidelberg Spectralis software, which allows the delineation of the area of the leak, and provides quantitative data on the region of interest. In addition, Sulaiman and colleagues (2015) recently proposed a calculation of CNV volume from in vivo acquired OCT images using the ellipsoid method10. The ellipsoid model most likely introduces bias toward overestimation of volume of lesions as CNV in most cases has an irregular shape. However, high correlation between volume measurements from confocal analysis of histological samples and proposed ellipsoid quantification from OCT images provides evidence that the method is a valuable tool for the quantitative evaluation of CNV volume10. 
To conclude, we believe the presented combination of different in vivo imaging modalities, together with automated segmentation and histological analysis, provides reproducible and reliable evaluation of CNV pathology in preclinical studies. The method could be particularly useful for proof of concept therapeutic intervention studies.

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

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&#39;s membrane. Figure 1 shows an example of OCT imaging at different time points after laser administration.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg" /> 
Figure 1: OCT en face view of eye fundus (VIP image) shows three lasered areas outlined in white, green and red circles. OCT B-scan images were taken prior the lasering (baseline), immediately after the lasering to verify a break of Bruch&#39;s membrane (0 days, arrows point to the site of damage), and 5, 10 and 14 days after laser administration. As can be seen from the area outlined in white (first row of images), CNV did not develop at later timepoints. The area outlined in green and red developed CNV, which was detected on follow-up day 5. However, at the 10 and 14 day timepoints, these CNV lesions regressed. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figures 2 and 3 show serial imaging using FA, which confirmed successful damage of Bruch&#39;s membrane in all three spots on day 0 in a male 10-week-old C57BL/6jRj mouse.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56173/56173fig2.jpg" /> 
Figure 2: Serial FA imaging taken every 20 s (images 1 through 18) at the choroidal level immediately after laser administration. White arrows in image 1 point to lasered sites, which show fluorescein leakage at later timepoints (white arrows in image 18). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56173/56173fig3.jpg" /> 
Figure 3: Serial FA imaging taken every 20 s (images 1 through 18) at the retinal level immediately after laser administration. CNV area that has fluorescein leakage and a grading of 1 (leaky CNV) is pointed out by the white arrow in image 18. Note an increasing intensity, as well as fluorescein positive area, during the timecourse of FA imaging (white arrow in images 8 and 11). Two areas outlined in white in the image 18 were graded as having a faint FA signal (grading 0.5). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition to the grading of CNV pathology, SD-OCT is also useful to reveal additional information in the lesion site, e.g., presence of subretinal fluid, edema, and CNV regression. Figure 4 shows the main pathological hallmarks of laser-induced CNV in mice.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56173/56173fig4.jpg" /> 
Figure 4: Spectral Domain Optical Coherence Tomography Imaging of CNV pathology. SD-OCT provides a detailed CNV pathology within retinal tissue, as can be seen from these representative images on scarring tissue, CNV formation, and liquid accumulation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Macular edema is one of the main pathological hallmarks of wet form AMD in humans. In the laser-induced CNV model, retinal thickness can be evaluated using automated segmentation. Manual measurement of selected lasered sites is required to measure retinal thickness at the site of CNV. Figure 5 shows an example of a report generated after automated segmentation.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56173/56173fig5.jpg" /> 
Figure 5: Quantification of retinal thickness. Retinal thickness as measured by automated segmentation <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The use of automated segmentation is a fast way to provide an overview of retinal thickness (Table 1). Figures 6A and 6B show representative examples of automated segmentation from a healthy retinal area and from the retinal area with CNV pathology, respectively. Despite minor inaccuracies found in distinguishing individual retinal layers, overall, the software reliably recognizes the total retinal thickness in pigmented mice.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56173/56173fig6.jpg" /> 
Figure 6: Automated segmentation of retinal layers. Automated segmentation of healthy retinal area (A) and retinal area containing CNV (asterisk in image B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In order to evaluate the retinal thickness at lasered areas, each lasered area was measured manually as following: the total retinal thickness was considered as the thickness of all layers from nerve fiber layer to the imaginary line connecting RPE around the site of damage (Figure 7 and Table 1).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Area</td>
			<td>Day 5</td>
			<td>Day 10</td>
			<td>Day 14</td>
		</tr>
		<tr>
			<td>Total retinal thickness, &#956;m</td>
			<td>218&#177;7.8</td>
			<td>220&#177;7.2</td>
			<td>221&#177;9.8</td>
		</tr>
		<tr>
			<td>Lasered area 1</td>
			<td>200</td>
			<td>204</td>
			<td>214</td>
		</tr>
		<tr>
			<td>Lasered area 2</td>
			<td>226</td>
			<td>217</td>
			<td>220</td>
		</tr>
		<tr>
			<td>Lasered area 3</td>
			<td>222</td>
			<td>223</td>
			<td>227</td>
		</tr>
		<tr>
			<td colspan="4">Data are presented as mean&#177;SD</td>
		</tr>
	</tbody>
</table>
Table 1. Total retinal thickness and retinal thickness at CNV sites during a 14-day follow-up as determined by automated segmentation using inVivoDiver software (v. 3.0.8).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56173/56173fig7.jpg" /> 
Figure 7: Manual measurement of retinal thickness at lasered area with CNV pathology. Yellow line indicates the total retinal thickness from nerve fiber layer to an imaginary RPE layer (black line) at the site of laser administration. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Histologically, the CNV lesions were confirmed using isolectin GS-IB4 labeling (Figure 8A). Image analysis software Image J was used to calculate the area of CNV lesion (Figure 8B).
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56173/56173fig8.jpg" /> 
Figure 8: Histological analysis. Histological stain of CNV lesion from choroidal flatmount (in green, A) can be quantified using thresholding in Image J (B). The scale bar for A is 50 &#956;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
&#160;

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

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

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 ...

in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.1K Views.  Experimentica Ltd.. Hier presenteren we het nut van longitudinale in vivo imaging in de follow-up van de morfologische veranderingen van laser-geïnduceerde choroidal neovascularization in muizen.

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.

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

Medicine

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.

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

Cancer Research

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.

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.

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

Bioengineering

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.

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.

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

Immunology and Infection

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.

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

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).

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.

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

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

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

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