This work was supported by grants from BrightFocus Foundation, Retina&#160;Research Foundation, Mullen Foundation, and the Sarah Campbell Blaffer Endowment in Ophthalmology to YF, NIH core grant 2P30EY002520 to Baylor College of Medicine, and an unrestricted grant to the Department of Ophthalmology at Baylor College of Medicine from Research to Prevent Blindness.

The animal experiments conducted in this study received approval from the Institutional Animal Care and Use Committees (IACUC) at Baylor College of Medicine. All procedures were carried out in compliance with the guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Young (7-9 weeks) and old (12-16 months) C57BL/6J male and female mice were used for the present study. The animals were obtained from a commercial source (see Table of Materials).
1. Preparation of the imaging system
<ol>
	<li>Position a heating pad on the imaging platform (see Table of Materials) to ensure the mouse&#39;s body temperature is maintained during imaging. Activate the heating pad and adjust the temperature to 35 &#176;C.</li>
	<li>Remove the dust cover and turn on the laser scanning ophthalmoscope. Place a 55&#176; lens on the machine.</li>
	<li>Set up the imaging software (see Table of Materials) and input essential information, including genotype, gender, age, etc. When the imaging session commences, select the IR (Infrared channel) for capturing ICGA images.</li>
</ol>
2. Animal preparation prior to ICGA and FA
<ol>
	<li>Weigh the mouse to determine the amount of anesthesia (Ketamine/Xylazine 70-100/2.5-10 mg/kg, see Table of Materials) needed. 
	NOTE: Optimizing the dosage is crucial as the metabolic profile varies among different mouse strains. It is essential to determine the appropriate dosage to avoid the mouse waking up before completing the imaging or the risk of an overdose and potential animal mortality.</li>
	<li>Use a 1 mL sterile syringe with a 30-32 G needle to deliver anesthesia by intraperitoneal injection. Gently pinch one of the mouse&#39;s paws to check whether the mouse is adequately anesthetized. If the animal exhibits any response or movement, it is advisable to wait for additional time before proceeding to the next step. This allows the animal to settle and ensures optimal conditions for the subsequent procedures.</li>
	<li>Administer 1% tropicamide ophthalmic solution drops to dilate both eyes of the mouse. Wait at least 30 s before using 0.5% proparacaine hydrochloride drops (see Table of Materials) on both eyes to reduce eye movement and blinking. Follow this up with lubricant eye gel drops. 
	NOTE: Throughout the entire duration of anesthesia, it is important to address the issue of extreme dryness in the mouse&#39;s eyes, which can lead to corneal opacity. This opacity makes subsequent imaging challenging due to hindered visibility. To prevent this, it is crucial to apply lubricant eye gel drops continuously to keep the mouse&#39;s eyes moisturized.</li>
	<li>Place the mouse on a heating water pad. 
	NOTE: Mice possess a high surface area to volume ratio, resulting in increased heat loss to the environment. When combined with the temperature drop induced by anesthesia, this can pose a significant risk to the mouse, potentially leading to death due to hypothermia. It is crucial to take necessary precautions to prevent hypothermia and ensure the mouse&#39;s well-being during the procedure.</li>
	<li>Prepare a 1:1 volume mixture of 2 mg/mL ICG and 20 mg/mL Fluorescein dye (see Table of Materials). Administer 250 &#181;L of the mixture through intraperitoneal injection using a 1 mL syringe and a 32 G needle.
	<ol>
		<li>Insert the needle in the bottom left quadrant of the mouse abdomen near the hind legs at an angle parallel to the mouse&#39;s skin to avoid perforating any organs.</li>
		<li>Carefully withdraw the plunger and verify that no blood has entered the syringe cap. Proceed to inject the dye slowly and steadily, maintaining a consistent pace. 
		NOTE: The ICG dye must be filtered with a 0.22 &#181;m syringe filter before use.</li>
	</ol>
	</li>
</ol>
3. ICGA and FA
<ol>
	<li>Place the mouse on the heating pad of the imaging platform to start imaging.</li>
	<li>Position the mouse&#39;s body at a 45-degree angle to the camera and rotate its head slightly downwards. This allows the optic nerve to be in the center of the camera&#39;s focus.</li>
	<li>Using a cotton swab, delicately wipe the eye to remove the layer of lubricant eye drops or gels on the eye being imaged first. Ensure to apply the lubricant gel drops immediately after completing the imaging procedure. 
	NOTE: Leaving the eye without lubrication for more than 1 min while under anesthesia is not recommended.</li>
	<li>Move the camera toward the mouse&#39;s eye. Select the FA channel from the acquisition module. The luminescence emitted from the FA channel can be used to position in the center of the mouse cornea for faster placement.</li>
	<li>Position the mouse&#39;s head in a way that the optic nerve is centered on the screen, avoiding the need to tilt the laser scanning ophthalmoscope at an angle. Make minor adjustments to the mouse&#39;s head position to achieve the desired alignment.</li>
	<li>On the acquisition module, select the ICGA channel. Ensure that the laser intensity is set to 100% and select the 55&#176; option to match the appropriate lens. This ensures optimal settings for the laser scanning ophthalmoscope. 
	NOTE: To prevent oversaturation, it may be necessary to use a lower laser intensity, typically around 25%-50%, when imaging the early stage. Adjusting the laser intensity in this range can help capture clear and accurate images without causing oversaturation.</li>
	<li>When the eye occupies the entire screen on the imaging software, make adjustments to the sensitivity and focus settings to obtain the clearest image of the CNV membrane.
	<ol>
		<li>Rotate the round black button on the acquisition module to adjust the sensitivity of the image.</li>
		<li>Rotate the knob on the ophthalmoscope to adjust the focus. The optimal focus for visualizing the retinal vasculature using FA is typically within the range of 35-45 D (diopters). On the other hand, the ideal focus for visualizing the choroid using ICGA is generally between 10-15 D. 
		NOTE: Due to the different sizes of CNV membrane, it may be necessary to adjust the focus in 10-30 D to obtain optimal imaging of the vascular morphology.</li>
	</ol>
	</li>
	<li>Once the focus and the sensitivity have been adjusted to achieve the best possible image, press the round black button on the acquisition module to normalize the image. Once normalization is complete (all frames captured), click on the acquire button on the touchscreen panel to save the image. Moving the camera around to image CNV from different angles may be necessary. The mouse can also be oriented in different positions to provide the best image of the CNV lesion. 
	NOTE: One may have to readjust the focus or positioning of the laser scanning ophthalmoscope when viewing vasculature farther from the optic nerve.</li>
	<li>Switch back to the FA channel using the acquisition module. Complete the same steps listed in steps 3.6-3.7 to adjust the sensitivity and focus of each image to capture the leakage of the CNV lesion. 
	NOTE: Ensure the correct sensitivity is selected to avoid oversaturation of the CNV leakage and artificially increase the leakage area.</li>
	<li>Image the &#34;early phase&#34; of ICGA and FA 3-4 min post-injection. 
	NOTE: The &#34;early phase&#34; is the time when the choroidal vasculature can be clearly and distinctly seen. During the middle phase, which typically occurs between 4-8 min, the retinal and choroidal vessels are much more faded and diffused. As the late phase (&#62;8-10 min) sets in, both the choroidal and retinal vessels become indiscernible. Hyperfluorescent CNV lesions, however, exhibit maximal contrast against the diminished background. These mentioned timings are variable and depend on the concentration and amount of ICG dye injected. A greater amount of ICG dye tends to increase the timeline of each phase and provide more distinct vessels. A phase should be defined based on the key features listed above rather than an absolute time.</li>
	<li>Once all images have been acquired, apply a gel lubricant or ointment to the mouse&#39;s eye and carefully monitor the mouse on the heating pad for recovery. It usually takes 1.5 h for the mouse to recover fully.</li>
	<li>Place the mice back into their cages and the designated holding area once they have fully recovered from the anesthesia and are awake.</li>
	<li>Before shutting down the imaging system and laser, export the images as either TIFF or JPEG files for subsequent analysis.</li>
</ol>
4. RPE/choroid flat-mount and staining
<ol>
	<li>Fix the eyes in 4% paraformaldehyde overnight. Wash the eyes with PBS three times. Remove the lens and cornea.</li>
	<li>Incubate the eyes in blocking solution (10% bovine serum albumin, 0.6% Triton X-100 in PBS) for 1 h at room temperature. Repeat PBS wash three times.</li>
	<li>Incubate the eyes with isolectin GS-IB4 Alexa-flour 568 conjugate and anti-&#945;-smooth muscle actin antibody (see Table of Materials) overnight in the blocking solution.</li>
	<li>Wash three times with PBS. Incubate the samples with an Alexa Fluor 488 goat anti-rabbit secondary antibody (see Table of Materials) for 2 h at room temperature.</li>
	<li>Perform 4 radial cuts from the edge to the equator. Carefully remove the retina17. 
	NOTE: Care must be taken to ensure the neovascular membrane is not accidentally detached.</li>
	<li>Mount the flat-mounted choroid on a glass slide. Visualize the CNV using confocal microscopy.</li>
</ol>

Characterizing Different Lesion Types of Laser-Induced CNV in Mouse

<ol>
	<li>Fleckenstein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration.">Age-related macular degeneration.</a> Nature Reviews Disease Primers. 7 (1), 1-25 (2021).</li><li>Wong, W. L., 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=Global+prevalence+of+age-related+macular+degeneration+and+disease+burden+projection+for+2020+and+2040:+a+systematic+review+and+meta-analysis.">Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: a systematic review and meta-analysis.</a> The Lancet Global Health. 2 (2), e106-e116 (2014).</li><li>Maguire, M. 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=Five-Year+outcomes+with+anti-vascular+endothelial+growth+factor+treatment+of+neovascular+age-related+macular+degeneration:+the+comparison+of+age-related+macular+degeneration+treatments+trials.">Five-Year outcomes with anti-vascular endothelial growth factor treatment of neovascular age-related macular degeneration: the comparison of age-related macular degeneration treatments trials.</a> Ophthalmology. 123 (8), 1751-1761 (2016).</li><li>Yang, S., Zhao, J., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+anti-VEGF+therapy+in+neovascular+age-related+macular+degeneration:+a+comprehensive+review.">Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: a comprehensive review.</a> Drug Design, Development and Therapy. 10, 1857-1867 (2016).</li><li>Ehlken, C., Jungmann, S., B&#246;hringer, D., Agostini, H. T., Junker, B., Pielen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+anti-VEGF+agents+is+an+option+for+nonresponders+in+the+treatment+of+AMD.">Switch of anti-VEGF agents is an option for nonresponders in the treatment of AMD.</a> Eye. 28 (5), 538-545 (2014).</li><li>Heier, J. 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=Intravitreal+aflibercept+(VEGF+trap-eye)+in+wet+age-related+macular+degeneration.">Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration.</a> Ophthalmology. 119 (12), 2537-2548 (2012).</li><li>Rofagha, S., Bhisitkul, R. B., Boyer, D. S., Sadda, S. R., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEVEN-UP+Study+Group+Seven-year+outcomes+in+ranibizumab-treated+patients+in+ANCHOR,+MARINA,+and+HORIZON:+a+multicenter+cohort+study+(SEVEN-UP).">SEVEN-UP Study Group Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: a multicenter cohort study (SEVEN-UP).</a> Ophthalmology. 120 (11), 2292-2299 (2013).</li><li>Krebs, I., Glittenberg, C., Ansari-Shahrezaei, S., Hagen, S., Steiner, I., Binder, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-responders+to+treatment+with+antagonists+of+vascular+endothelial+growth+factor+in+age-related+macular+degeneration.">Non-responders to treatment with antagonists of vascular endothelial growth factor in age-related macular degeneration.</a> British Journal of Ophthalmology. 97 (11), 1443-1446 (2013).</li><li>Mettu, P. S., Allingham, M. J., Cousins, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incomplete+response+to+Anti-VEGF+therapy+in+neovascular+AMD:+Exploring+disease+mechanisms+and+therapeutic+opportunities.">Incomplete response to Anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities.</a> Progress in Retinal and Eye Research. 82, 100906 (2021).</li><li>Otsuji, 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=Initial+non-responders+to+ranibizumab+in+the+treatment+of+age-related+macular+degeneration+(AMD).">Initial non-responders to ranibizumab in the treatment of age-related macular degeneration (AMD).</a> Clinical Ophthalmology (Auckland, N.Z). 7, 1487-1490 (2013).</li><li>Cobos, E., 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=Association+between+CFH,+CFB,+ARMS2,+SERPINF1,+VEGFR1+and+VEGF+polymorphisms+and+anatomical+and+functional+response+to+ranibizumab+treatment+in+neovascular+age-related+macular+degeneration.">Association between CFH, CFB, ARMS2, SERPINF1, VEGFR1 and VEGF polymorphisms and anatomical and functional response to ranibizumab treatment in neovascular age-related macular degeneration.</a> Acta Ophthalmologica. 96 (2), e201-e212 (2018).</li><li>Kitchens, J. W., 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+pharmacogenetics+study+to+predict+outcome+in+patients+receiving+anti-VEGF+therapy+in+age+related+macular+degeneration.">A pharmacogenetics study to predict outcome in patients receiving anti-VEGF therapy in age related macular degeneration.</a> Clinical Ophthalmology (Auckland, N.Z.). 7, 1987-1993 (2013).</li><li>Rosenfeld, P. J., Shapiro, H., Tuomi, L., Webster, M., Elledge, J., Blodi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+patients+losing+vision+after+2+Years+of+monthly+dosing+in+the+phase+III+Ranibizumab+clinical+trials.">Characteristics of patients losing vision after 2 Years of monthly dosing in the phase III Ranibizumab clinical trials.</a> Ophthalmology. 118 (3), 523-530 (2011).</li><li>Spaide, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+angiography+signs+of+vascular+abnormalization+with+antiangiogenic+therapy+for+choroidal+neovascularization.">Optical coherence tomography angiography signs of vascular abnormalization with antiangiogenic therapy for choroidal neovascularization.</a> American Journal of Ophthalmology. 160 (1), 6-16 (2015).</li><li>Lumbroso, B., Rispoli, M., Savastano, M. C., Jia, Y., Tan, O., Huang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography+angiography+study+of+choroidal+neovascularization+early+response+after+treatment.">Optical coherence tomography angiography study of choroidal neovascularization early response after treatment.</a> Developments in Ophthalmology. 56, 77-85 (2016).</li><li>Zhu, L., 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=Combination+of+apolipoprotein-A-I/apolipoprotein-A-I+binding+protein+and+anti-VEGF+treatment+overcomes+anti-VEGF+resistance+in+choroidal+neovascularization+in+mice.">Combination of apolipoprotein-A-I/apolipoprotein-A-I binding protein and anti-VEGF treatment overcomes anti-VEGF resistance in choroidal neovascularization in mice.</a> Communications Biology. 3 (1), 386 (2020).</li><li>Zhang, Z., Shen, M. M., Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+AIBP,+apoA-I,+and+Aflibercept+overcomes+anti-VEGF+resistance+in+neovascular+AMD+by+inhibiting+arteriolar+choroidal+neovascularization.">Combination of AIBP, apoA-I, and Aflibercept overcomes anti-VEGF resistance in neovascular AMD by inhibiting arteriolar choroidal neovascularization.</a> Investigative Ophthalmology &amp; Visual Science. 63 (12), 2 (2022).</li><li>Koehn, D., Meyer, K. J., Syed, N. A., Anderson, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine/Xylazine-induced+corneal+damage+in+mice.">Ketamine/Xylazine-induced corneal damage in mice.</a> PloS One. 10 (7), e0132804 (2015).</li><li>Li, X. -. T., Qin, Y., Zhao, J. -. Y., Zhang, J. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lens+opacity+induced+by+different+kinds+of+anesthetic+drugs+in+mice.">Acute lens opacity induced by different kinds of anesthetic drugs in mice.</a> International Journal of Ophthalmology. 12 (6), 904-908 (2019).</li><li>Zhou, T. E., 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=Preventing+corneal+calcification+associated+with+xylazine+for+longitudinal+optical+coherence+tomography+in+young+rodents.">Preventing corneal calcification associated with xylazine for longitudinal optical coherence tomography in young rodents.</a> Investigative Ophthalmology &amp; Visual Science. 58 (1), 461-469 (2017).</li><li>Ikeda, W., Nakatani, T., Uemura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataract-preventing+contact+lens+for+in+vivo+imaging+of+mouse+retina.">Cataract-preventing contact lens for in vivo imaging of mouse retina.</a> BioTechniques. 65 (2), 101-104 (2018).</li><li>Kumar, S., Berriochoa, Z., Jones, A. D., Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+abnormalities+in+choroidal+vasculature+in+a+mouse+model+of+age-related+macular+degeneration+by+time-course+indocyanine+green+angiography.">Detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography.</a> Journal of Visualized Experiments. 84, e51061 (2014).</li></ol>

The authors have nothing to disclose.

This study demonstrated the use of indocyanine green angiography (ICGA) to identify the vascular morphology of arteriolar and capillary choroidal neovascularization (CNV) in mouse models with laser-induced CNV. The hemoglobin-bound and infrared light properties of indocyanine green (ICG) dye enabled the detection of CNV morphology, which is challenging to achieve using fluorescein angiography (FA), the current method employed by the research community.
The first critical step in the protocol i...

Age-related macular degeneration (AMD) is a prevalent condition that leads to severe vision loss in older individuals1. In the United States alone, the number of AMD patients is projected to double, reaching nearly 22 million by 2050, compared to the current 11 million. Globally, the estimated number of AMD cases is expected to reach a staggering 288 million by 20402.
Choroidal neovascularization (CNV), also known as &#34;wet&#34; or neovascular AMD, can have devastating effects on vision due to the formation of abnormal blood vessels beneath the central retina. This leads to hemorrhaging, retinal exudation, and significant vision loss. The introduction of anti-vascular endothelial growth factor (VEGF) therapies, which target extracellular VEGF, has revolutionized CNV treatment. However, despite these advancements, up to 50% of patients exhibit suboptimal responses to these therapies, with ongoing disease activity such as fluid accumulation and unresolved or new hemorrhages3,4,5,6,7,8,9,10,11,12,13,14.
Clinical studies have indicated that anti-VEGF resistance in CNV patients often corresponds to the presence of arteriolar CNV, characterized by large-caliber branching arterioles, vascular loops, and anastomotic connections9. Repeated anti-VEGF treatment can contribute to vessel abnormalization, the development of arteriolar CNV, and ultimately, resistance to anti-VEGF therapies14,15. In cases of arteriolar CNV, persistent fluid leakage is likely due to heightened exudation caused by inadequately formed tight junctions at arteriovenous anastomotic loops, particularly under conditions of high blood flow9. Conversely, individuals who respond well to anti-VEGF treatment tend to exhibit capillary CNV.
In our studies using animal models, we have demonstrated that laser-induced CNV in older mice develops arteriolar CNV and shows resistance to anti-VEGF treatment16,17. Conversely, laser-induced CNV in younger mice leads to the development of capillary CNV and high responsiveness to anti-VEGF treatment. Thus, it is crucial to differentiate between CNV vascular types for both mechanistic and therapeutic investigations.
In clinical settings, CNV is commonly classified based on fluorescein angiography (FA) leakage patterns (e.g., Type 1, Type 2), which use fluorescein dye to track exudation and identify areas of pathological leakage. In AMD research, CNV is predominantly studied using FA in animal models. However, FA fails to reveal the vascular morphology of CNV. Moreover, FA only captures images in the visible light spectrum and cannot visualize the choroidal vasculature beneath the retinal pigment epithelium (RPE). In contrast, indocyanine green (ICG), which exhibits strong affinity for plasma proteins, facilitates predominant intravascular retention and enables visualization of vascular structure and blood flow9. By utilizing the near-infrared fluorescence property of ICG, it becomes feasible to image the retinal and choroidal pigment using ICG angiography (ICGA). In this context, a protocol is presented that combines FA and ICGA to investigate the leakage and vascular morphology of laser-induced choroidal neovascularization (CNV) in young and old mice, where capillary and arteriolar CNV are observed.

<table><tbody><tr><td>32-G Insulin Syringe</td><td>MHC Medical Products</td><td>NDC 08496-3015-01</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-rabbit secondary antibody</td><td>Invitrogen</td><td>&amp;nbsp;A11008</td><td></td></tr><tr><td>Anti-&amp;alpha; smooth muscle Actin antibody</td><td>Abcam</td><td>ab5694</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Santa Cruz Biotechnology, Inc.</td><td>sc-2323&amp;nbsp;</td><td></td></tr><tr><td>C57BL/6J mice (7-9 weeks)</td><td>The Jackson Laboratory</td><td>Strain #:000664</td><td></td></tr><tr><td>Fluorescein Sodium Salt</td><td>Sigma-Aldrich</td><td>MFCD00167039</td><td></td></tr><tr><td>Gaymar T Pump Heat Therapy System</td><td>Gaymar</td><td>TP-500</td><td>Water circulation heat pump for mouse recovery after imaging</td></tr><tr><td>GenTeal Gel</td><td>Genteal</td><td>NDC 58768-791-15</td><td>Clear lubricant eye gel</td></tr><tr><td>GS-IB4 Alexa-Flour 568 conjugate</td><td>Invitrogen</td><td>&amp;nbsp;I21412</td><td></td></tr><tr><td>Heidelberg Eye Explorerer</td><td>Heidelberg Engineering, Germany</td><td>HEYEX2</td><td></td></tr><tr><td>Indocyanine Green</td><td>Pfaultz &amp;amp; Bauer</td><td>I01250</td><td></td></tr><tr><td>Ketamine</td><td>Vedco Inc.</td><td>NDC 50989-996-06</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Acros Organics&amp;nbsp;</td><td>416785000</td><td></td></tr><tr><td>Proparacaine Hydrochloride Ophthalmic Solution (0.5%)</td><td>Sandoz</td><td>NDC 61314-016-01</td><td></td></tr><tr><td>Spectralis Multi-Modality Imaging System</td><td>Heidelberg Engineering, Germany</td><td>SPECTRALIS HRA+OCT</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X100-1L</td><td></td></tr><tr><td>Tropicamide ophthalmic solution (1%)</td><td>Bausch &amp;amp; Lomb</td><td>NDC 24208-585-64</td><td>For dilation of pupils</td></tr><tr><td>Xylazine</td><td>Lloyd Laboratories</td><td>NADA 139-236</td><td></td></tr></tbody></table>

characterization of vascular morphology of neovascular age-related macular degeneration by indocyanine green angiography

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging population. Choroidal neovascularization (CNV) or wet AMD, which accounts for 10%-20% of all AMD cases, is responsible for an alarming 80%-90% of AMD-related blindness. Current anti-VEGF therapies show suboptimal responses in approximately 50% of patients. Resistance to anti-VEGF treatment in CNV patients is often associated with arteriolar CNV, while responders tend to have capillary CNV. While fluorescein angiography (FA) is commonly used to assess leakage patterns in wet AMD patients and animal models, it does not provide information about CNV vascular morphology (arteriolar CNV vs. capillary CNV). This protocol introduces the use of indocyanine green angiography (ICGA) to characterize lesion types in laser-induced CNV mouse models. This method is crucial for investigating the mechanisms and treatment strategies for anti-VEGF resistance in wet AMD. It is recommended to incorporate ICGA alongside FA for comprehensive assessment of both leakage and vascular features of CNV in mechanistic and therapeutic studies.

Following the protocol, ICGA and FA were performed on laser-induced CNV in young (7-9 weeks) and old (12-16 months) C57BL/6J mice. FA provides information about the location and leakage of the CNV lesions (Figure 1, left panels), while ICGA reveals the vascular morphology of the CNV lesions (Figure 1, right panels). In young mice, capillary CNV dominates the CNV lesions. In contrast, old mice exhibit arteriolar CNV characterized by large caliber vessels, vascula...

Watch this Scientific Journal Video about Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography at JoVE.com

Author Spotlight: Overcoming Anti-VEGF Resistance Through Advanced Vascular Morphology Assessment in Choroidal Neovascularization

Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

Age-related macular degeneration (AMD) is a leading cause of blindness among older individuals, and its prevalence is rapidly increasing due to the aging ...

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JoVE Journal

Medicine

1.0K Views.  Baylor College of Medicine. Currently, fluorescein angiography (FA) is the preferred method for identifying leakage patterns in animal models of choroidal neovascularization (CNV). However, FA does not provide information about vascular morphology. This protocol outlines the use of indocyanine green angiography (ICGA) to characterize different lesion types of laser-induced CNV in mouse models.

Video: Author Spotlight: Overcoming Anti-VEGF Resistance Through Advanced Vascular Morphology Assessment in Choroidal Neovascularization

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

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

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

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

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

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.

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.

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

Neuroscience

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

OCT-Based Multimodal Imaging of Human Donor Eyes for AMD Research

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Author Spotlight: Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Characterization of Vascular Morphology of Neovascular Age-Related Macular Degeneration by Indocyanine Green Angiography

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Przeglądaj więcej filmów

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

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

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

Using Retinal Imaging to Study Dementia

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model