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 (se...

Characterizing Different Lesion Types of Laser-Induced CNV in Mouse

<ol>
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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. 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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<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>&#160;A11008</td>
			<td></td>
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			<td>Anti-&#945; smooth muscle Actin antibody</td>
			<td>Abcam</td>
			<td>ab5694</td>
			<td></td>
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			<td>Bovine Serum Albumin</td>
			<td>Santa Cruz Biotechnology, Inc.</td>
			<td>sc-2323&#160;</td>
			<td></td>
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			<td>C57BL/6J mice (7-9 weeks)</td>
			<td>The Jackson Laboratory</td>
			<td>Strain #:000664</td>
			<td></td>
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			<td>Fluorescein Sodium Salt</td>
			<td>Sigma-Aldrich</td>
			<td>MFCD00167039</td>
			<td></td>
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			<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>
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			<td>GenTeal Gel</td>
			<td>Genteal</td>
			<td>NDC 58768-791-15</td>
			<td>Clear lubricant eye gel</td>
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			<td>GS-IB4 Alexa-Flour 568 conjugate</td>
			<td>Invitrogen</td>
			<td>&#160;I21412</td>
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			<td>Heidelberg Eye Explorerer</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>HEYEX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pfaultz &#38; Bauer</td>
			<td>I01250</td>
			<td></td>
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			<td>Ketamine</td>
			<td>Vedco Inc.</td>
			<td>NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics&#160;</td>
			<td>416785000</td>
			<td></td>
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			<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 Heidelberg Engineering, Germany SPECTRALIS HRA+OCT Tropicamide ophthalmic solution (1%) Bausch &#38; Lomb NDC 24208-585-64 for dilation of pupils GenTeal Gel Genteal NDC 58768-791-15 clear lubricant eye gel Ketamine Vedco Inc NDC 50989-996-06 Xylazine Lloyd Laboratories NADA 139-236 Acepromazine Vedco Inc NDC 50989-160-11 32-G Needle Steriject PRE-32013 1-ml syringe BD 309659 Indocyanine Green Pfaltz &#38; Bauer I01250</td>
			<td>Heidelberg Engineering, Germany</td>
			<td>SPECTRALIS HRA+OCT</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>X100-1L</td>
			<td></td>
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			<td>Tropicamide ophthalmic solution (1%)</td>
			<td>Bausch &#38; Lomb</td>
			<td>NDC 24208-585-64</td>
			<td>For dilation of pupils</td>
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		<tr>
			<td>Xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>NADA 139-236</td>
			<td></td>
		</tr>
	</tbody>
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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.

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

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

We are working on the mechanism and treatment strategies of age-related macular degeneration, a prevalent condition causing severe vision loss in older individuals. Particularly, we're developing new treatments to tackle the issue of drug resistance to the current treatment of corneal neovascularization, which is the wet type of AMD. Our lab recently developed new treatment to ameliorate anti-VEGF resistance in CNV.This is achieved by using mouse CNV model. Our research has shown that old mice with laser-induced CNV develop arteriolar CNV, which is resistant to anti-VEGF treatment. In contrast, young mice with capillary CNV are responsive to the treatment.In AMD research, CNV is almost exclusively studied by fluorescein angiography to reveal leakage patterns in animal models. However, FA does not show CNV vascular morphology, and thus it is not suitable to study anti-VEGF resistance in AMD. The current FA method used for CNV imaging lacks vital information regarding the vascular morphology of CNV lesions, such as capillary or arteriolar CNV.However, by combining ICGA and FA, this protocol can evaluate both CNV leakage and vascular morphology. By assessing both leakage and CNV lesion morphology, it is possible to study the underlying molecule mechanism relating to arteriolar CNV, and therefore provide opportunities to find new targets to treat anti-VEGF resistance for AMD patients.

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

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.

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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Medicine

Preparation of CNV Mouse Model for Indocyanine Green Angiography (IGCA) and Fluorescein Angiography (FA)

After anesthetizing the mouse, gently pinch one of the mouse's paws to check whether the mouse is adequately anesthetized. Administer 1%tropicamide ophthalmic solution drops to dilate the mouse's eyes and wait for 30 seconds. Then to reduce eye movement and blinking, apply 0.5%proparacaine hydrochloride drops to both eyes, followed by lubricant eye gel drops and position the mouse on a heating water pad.Prepare a dye mix containing an equal volume of indocyanine green, or ICG, and fluorescein dye. Inject 250 microliters of the mixture through an intraperitoneal injection into the lower left quadrant near the hind legs of the mouse, positioning it parallel to the skin to prevent organ perforation. Carefully retract the plunger, ensuring no blood has entered the syringe cap.Proceed by gradually injecting the dye at a steady pace. Next, place the mouse on the heating pad of the imaging platform. Adjust the positioning of the mouse's body at a 45 degree angle relative to the camera and gently angle the head slightly downwards.Delicately wipe the eye to be imaged using a cotton swab to remove the layer of lubricant eyedrops or gels. Transition the camera towards the mouse's eye and select the FA channel from the acquisition module. Arrange the mouse's head so the optic nerve is centered on the screen, avoiding the need to tilt the laser scanning ophthalmoscope.Now, switch to the ICGA channel on the acquisition module. Once the eye occupies the entire screen on the imaging software, rotate the round black button on the acquisition module for image sensitivity adjustments. Use the ophthalmoscope's knob to fine tune the focus.Next, press the round black button on the acquisition module to normalize the image. After normalization, click the ACQUIRE button on the touchscreen panel to save the image. Switch to the FA channel using the acquisition module and adjust both the sensitivity and focus of each image as shown earlier to capture the leakage of the choroidal neovascularization or CNV lesion.Then capture images for the early phase of ICGA and FA three to four minutes after injection. Once all the necessary images are captured, apply a gel lubricant or ointment to the eye of the mouse. Capillary CNV dominates the CNV lesions in young mice.In contrast, old mice exhibit arteriolar CNV, characterized by large caliber vessels, vascular loops, and anastomotic connections. Both young and old mice show clear visibility of the retinal vasculature in FA.In the ICGA images of young mice, the retinal vasculature is not visible and the choroidal vessels appear faded. In the ICGA images of old mice, partial retinal vasculature can be observed while the choroidal vessels appear faded.Arteriolar CNV in old mice exhibits a larger CNV size and significantly more leakage compared to capillary CNV in young mice.

725 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.

Characterizing Different Lesion Types of Laser-Induced CNV in Mouse

Summary