Pathologies such as exudative age-related macular degeneration (AMD) are characterized by choroidal neovascularization (CNV) — abnormal growth of new blood vessels from the choroid into the subretinal space.1,2 The laser-induced CNV (L-CNV) animal model of exudative AMD relies on laser photocoagulation to disrupt Bruch's membrane, thus stimulating blood vessel growth from the choriocapillaris into the subretinal space.3 This model was originally developed in macaque4 and later adapted for use in mice and rats. L-CNV has become the gold standard for preclinical testing of current treatment modalities used for subretinal neovascularization.5–9 The model has proven invaluable for evaluating effects of drug therapies on CNV lesion progression in neovascular AMD research. 2,4,10,11 Significant benefits of L-CNV include its reproducibility and simplicity, which provide a straightforward methodology for studying subretinal neovascularization.4
The L-CNV model in non-human primates12 and in rodents8 undergoes three stages of development, mimicking the stages of development seen in human CNV.12 The first stage is an inflammatory response that initiates a growth phase.8 Soon after laser photocoagulation, a gas bubble forms under the retina in the area of laser application (indicating disruption of Bruch's membrane), and the retina becomes edematous and hazy.12 Three to 5 days after laser photocoagulation (second stage), choroidal fibrovascular tissue proliferates.8,12 Seven to 10 days after (third stage), choroidal neovascular tissue forms around the break in Bruch's membrane. At this time, almost no fluid is present between the newly formed subretinal membrane and the overlying retina. 8,12 Fourteen to 28 days following laser photocoagulation (still considered part of the third stage), fibrovascular tissue forms in the subretinal space around the break in Bruch's membrane. 8,12 This stage is also known as the reparative or involutional stage.12
Lesion development and progression is often tracked in vivo with fluorescein angiography (FA) to assess leakage from newly formed vessels.13 To obtain a precise measurement of lesion volume, choroidal flatmount preparations are typically stained with fluorescent dye that labels blood vessels, followed by confocal imaging to calculate three-dimensional (3-D) volumes of the CNV lesions. This technique has become widely accepted as a quantitative method to evaluate drug efficacy in ameliorating CNV lesion progression. 2,11,13,14 However, despite its accuracy in quantifying size of the neovascular complex, the technique is limited to ex vivo use.
Spectral-domain optical coherence tomography (SD-OCT) is a noninvasive, in vivo imaging method that allows collection of high-resolution, cross-sectional images of tissues.15 The technique has been used extensively in ophthalmology,15,16,2 providing detailed images for the anterior and posterior segments of the eye.17 SD-OCT has become an essential tool for the clinic in ocular pathologies such as exudative AMD,18–20 retinal tumors,21–24 macular edema,25 and retinal detachment.26–28
Recently, studies have demonstrated use of SD-OCT for in vivo evaluation of L-CNV in rodents, allowing quantification of lesion size and exudation parameters.2,8,29 These studies relied on two techniques for performing volume reconstruction from OCT images. In the first, L-CNV lesion size was estimated by fitting an ellipsoid to the lesion and calculating the volume of the ellipsoid. However, OCT volume measurements were found to be roughly 10-times greater than those obtained with ex vivo confocal reconstruction.2 As a result, the utility of this method may be limited to identifying gross effects of treatment conditions but not to performing precise in vivo volume measurements.
A more accurate technique for performing volume reconstruction of L-CNV lesions from OCT images relies on summing the area calculated from each horizontal cross-section passing through the lesion. Using this method in mice, Giani et al. found moderate correlation between CNV sizes measured using SD-OCT and confocal at days 5 and 7 after laser surgery. However, days 14 and 28 did not have a statistically significant correlation. Furthermore, direct comparisons between the volumes measured with OCT and those measured with confocal were not reported.8
In the present study, we describe a stereological method for quantifying lesion volumes from OCT images, which produces volume measurements that are statistically similar to those obtained from ex vivo confocal reconstruction. Application of this technique in vivo allows lesion volumes to be measured and tracked over time, without requiring animals to be euthanized.
Patients and Methods
A total of 18 male brown Norway rats (Envigo, Huntingdon, UK), aged P98 to P102, were used for this study. All procedures were performed under anesthesia, followed the guidelines of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Institutional Animal Care and Use Committee at the University of Southern California.
Rats were anesthetized with intraperitoneal injection of ketamine/xylazine (80 mg/kg to 90 mg/kg ketamine [Akorn, Lake Forest, IL] and 5 mg/kg to 10 mg/kg xylazine [Akorn, Lake Forest, IL]). Pharmacological mydriasis was induced with topical 1% tropicamide (Akorn, Lake Forest, IL) and 2.5% phenylephrine HCl (Paragon BioTeck, Portland, OR). Animals were euthanized on day 14 via intracardiac injection of sodium pentobarbital administered under deep anesthesia.
Laser Photocoagulation for L-CNV
At day 0, four laser burns were made in the posterior pole of each eye (36 eyes) using a green diode laser (IQ 532; IRIDEX, Mountain View, CA). A cover glass was used as a contact lens to provide visualization of the retina and optic nerve through a slit-lamp, as well as during laser application. Laser pulse parameters were 150-mW to 160-mW power, 50-ms duration, and 75-μm spot size. One burn was made per quadrant, between the retinal vessels in a peripapillary distribution. The production of a subretinal bubble at the time of laser application confirmed the rupture of Bruch's membrane.
Optical Coherence Tomography
OCT imaging was performed using an SD-OCT system (Bioptigen, Durham, NC) to assess in vivo progression of laser lesions after photocoagulation (at days 0, 3, 7, 10, and 14). OCT is a noncontact, noninvasive, nonionizing imaging technology that poses no additional risks to the animals. Imaging was performed under ketamine/xylazine anesthesia. Each lesion was imaged with OCT using 100 horizontal raster scans spaced 16-μm apart over an area of 1.6 mm x 1.6 mm.
FA was used to confirm Bruch's membrane rupture and to validate the presence of CNV in vivo. Following anesthesia, rats received dilation eye drops (1% tropicamide and 2.5% phenylephrine HCl) and 0.01 mL intraperitoneal injection of 10% sodium fluorescein dye. Sequential posterior pole images were taken using a RetCam 3 retinal camera (Natus Medical, Pleasanton, CA) with an 80° lens. Lesions that showed no leakage were excluded from analysis because those lesions likely represented laser impacts that did not result in CNV.
In Vivo Volume Analysis of L-CNV
The lesions were identified according to their position relative to the optic nerve: superotemporal, inferotemporal, superonasal, and inferonasal. We used a stereological method (3-D interpretation of two-dimensional cross-sections) to reconstruct the OCT images in 3-D and calculate lesion size. For this, we used the “Volumest” (“Volume Estimation”) plug-in30 of ImageJ (NIH, Bethesda, MD).31 Each image section passing through the lesion was delineated and measured by hand (Figure 1A). In each image, the CNV was identified as the subretinal hyperreflective material above the RPE layer. Before the measurement process, a single image in pixels was calibrated with known values from the OCT to establish the units in micrometers (1 pixel = 3.33 μm).
Laser-induced choroidal neovascularization (L-CNV) lesion volume calculated from (A) optical coherence tomography (OCT) images processed with ImageJ's “Volumest” plugin and from (B) confocal images of choroidal flatmounts that were stained with fluorescein labeled isolectin-B4 and processed with PerkinElmer's Volocity software. Arrows indicate the volume (in μm3) calculated by each software program.
Choroidal Flatmount Preparation and Ex Vivo CNV Volume Quantification
After OCT imaging on day 14, rats were euthanized, as described earlier. Before enucleating the eyes, a suture was placed at the 12 o'clock meridian to mark the orientation of the eye to facilitate direct comparison of each lesion between OCT (in vivo) and confocal (ex vivo). The anterior segment and retina were removed, and four relaxing radial incisions were made. The RPE-choroid-sclera complex was fixed overnight in 4% formalin at 4°C. Tissue was washed the next day and permeabilized by incubation with 0.5% Triton X-100 (Merck KGaA, Darmstadt, Germany) for 4 hours. The eyecups were blocked with 1% bovine serum albumin for 2 hours and placed in 1:50 fluorescein-labeled GSL I Isolectin B4 (endothelial cell marker; Vector Laboratories, Burlingame, CA) at 4°C overnight. The staining step was followed by two washes with Tris-buffered saline with 0.1% Tween-20 (TBST). Tissue was mounted on slides with mounting media (Vectashield; Vector Laboratories, Burlingame, CA) while making incisions in the eye cups to flatten them.
Flatmounts were visualized using the 10× objective of an UltraVIEW spinning disk confocal microscope (PerkinElmer, Waltham, MA). The image stacks were generated in the z-plane, with the microscope set to excite at 488 nm and to detect at 505 nm to 530 nm. Images were processed using the microscope's Volocity software by closely circumscribing and digitally extracting the fluorescent lesion areas throughout the entire image stack (Figure 1B). The extracted lesion was processed through the topography software to generate a digital topographic image representation of the lesion and an image volume. The topographic analysis program determines and displays the object's surface contours by detecting fluorescent signal from the top of the image stack and then measures everything under the surface to yield a final volume, which reflects the CNV fluorescence volume.
Pearson correlations were calculated to observe the association between lesion volumes measured by OCT and confocal microscopy. A modification of the Bland-Altman was performed to assess the gold standard (confocal) against the difference in volume between OCT and confocal. A paired t-test was used to determine if the mean difference between the two measurement techniques differed significantly from zero. Mean and standard deviation along with the 95% confidence intervals were reported. Analyses were run on matched paired data that had both OCT and confocal volumes available. All analyses were performed using SAS software version 9.4 (SAS Institute, Cary, NC).
Of the 144 laser burns that were made (18 rats x 8 burns each), 81% showed leakage on FA, consistent with L-CNV. Twenty-seven lesions did not show evidence of Bruch's membrane rupture and were thus excluded from analysis. The remaining 117 lesions were analyzed for comparison between volumes calculated from OCT (in vivo) and confocal (ex vivo) imaging. Figure 2 shows representative FA images of a leaky lesion on days 0, 3, 10, and 14. This same lesion, imaged by confocal microscopy, is shown in Figure 3.
Flourescein angiography (FA) imaging follow-up of a laser-induced choroidal neovascularization (L-CNV) lesion (arrows) based on angiographic patterns. Intraretinal edema is seen above and around the lesion on day 0 (A). On day 3, angiography late phases show hyperfluorescence due to leakage (B). Ten days after L-CNV, a staining pattern was observed with minimal leakage (C). On day 14, the angiographic (staining) pattern was similar to that of day 10 (D).
Representative confocal image of a laser-induced choroidal neovascularization (L-CNV) lesion after euthanasia on day 14. The lesion pictured is the same one followed by flourescein angiography (FA) in Figure 2.
Tracking L-CNV Progression With SD-OCT
SD-OCT images were acquired after laser treatment on days 0, 3, 7, 10, and 14. (In one rat, corneal opacity prevented imaging on day 7.) On day 0, OCT indicated signs of swelling, hyperreflectivity in the lasered area, and the disruption Bruch's membrane (Figures 4A and 4B). No volume measurement was performed on day 0 because of the lack of well-defined borders of the L-CNV. On day 3, the lesions presented a hyperreflective reaction originating from the RPE. As reported previously, day 3 lesions had a butterfly-like shape11 (Figure 4C). On day 7, lesions began to contract (choroidal fibrovascular tissue replaces the choroidal-RPE defect) and portrayed decreased swelling and increased RPE reflectivity (Figure 4D). On day 10 (Figure 4E), OCT showed less RPE hyperreflectivity as well as RPE and outer retina irregularity. On day 14 (Figure 4F), fibrovascular tissue was present in the OCT.
Representative spectral-domain optical coherence tomography (SD-OCT) images showing follow-up of a laser-induced choroidal neovascularization (L-CNV) lesion (arrowheads). The laser spot (A) and the disruption (arrow) of Bruch's membrane (B) are visible on day 0. By day 3, retinal hyperreflectivity exhibited a distinctive “butterfly-like” shape (C). Day 7 shows less swelling (D). On day 10, there is a decrease in retinal pigmented epithelium (RPE) hyperreflectivity and irregularity of the RPE and outer retina (E). Fibrovascular tissue is visible on day 14 (F).
Comparison Between Confocal and OCT Volume Measurements
Figure 5 shows CNV volume measurements calculated from SD-OCT images (days 3, 7, 10, and 14) and confocal microscopy (day 14). On day 14, correlations between OCT and confocal L-CNV volumes showed a positive association (Pearson's r = 0.50, P < .01; Figure 6). A Bland-Altman plot was generated to directly compare the lesion volumes measured by OCT and confocal (Figure 7). (Bland-Altman plots are a standard statistical method used to compare two measurement techniques.) For confocal volumes smaller than 0.004 mm3 (79% of the lesions), the difference between the OCT and confocal volume measurements was largely equally distributed around zero (Figure 7), signifying good agreement between the two techniques. For these smaller lesions, OCT and confocal measurements were not significantly different from each other (P = .90; Table). For larger volumes, however, OCT yielded smaller lesion sizes than confocal (Figure 7).
Choroidal neovascularization volume measurements calculated from spectral-domain optical coherence tomography (SD-OCT) images (days 3, 7, 10, and 14) and confocal microscopy (day 14). Error bars represent standard error of the mean (mean ± SEM).
Comparison of laser-induced choroidal neovascularization lesion volumes measured by optical coherence tomography (OCT) and confocal, 14 days after laser treatment. Pearson's r = 0.50, P < .01.
Bland-Altman plot of the difference in volume between the two measurement techniques (optical coherence tomography [OCT] – confocal) versus the mean confocal volume. Dotted horizontal lines represent the 95% limits for the differences: mean ± 1.96 × standard deviation. For confocal volumes smaller than 0.004 mm3 (dotted vertical line), the difference between the OCT and confocal volume measurements was largely equally distributed around zero.
Differences Between OCT and Confocal Volume Measurements
In this study, we describe a noninvasive method to quantify L-CNV volume in vivo using SD-OCT. Our technique relies on a stereological method similar to that used for 3-D reconstruction by confocal microscopy. The software we used (ImageJ and “Volumest” plug-in) is freely available to the public. With the aid of this program, we were able to accurately measure the volume of irregularly shaped lesions. Our approach can serve as a valuable tool to rapidly and quantitatively evaluate therapies for subretinal neovascularization in the L-CNV model. OCT provides in vivo information about exudation of the neovascular complex and morphologic features that are common to human pathology. Together, these details can aid in better understanding of CNV pathogenesis.8
Three other published studies have described use of OCT for evaluating L-CNV morphological features29 and measuring lesion volume in vivo in rodents.2,8 Our results agree with Giani et al.8 and Fukuchi et al.31 regarding the morphological progression of the lesions: Immediately after laser application, a subretinal bubble with a hyperreflective reaction and edema without well-defined borders forms. At day 3, lesions exhibit a butterfly-like shape,8 when the edema begins to retract. Around day 7, lesions have a more dome-like appearance with an increase in reflectivity, which represents the proliferative tissue, where fibrovascular tissue fills the defect generated by the laser-induced destruction of the RPE-choroidal complex.8 Despite not having the OCT-confocal volume correlations at more than one time point, we were still able to use OCT for monitoring L-CNV longitudinally in each animal.
Sulaiman et al.2 described OCT quantification of L-CNV lesion sizes by fitting an ellipsoid to the lesion and calculating its volume. They presented a statistically significant correlation (P < .05) between the OCT and confocal lesion volumes; however, lesion volumes measured by OCT were roughly 10 times the size of those measured by confocal. Presumably, this mismatch arises because CNV lesions are not perfect ellipsoids, but rather are arbitrarily shaped volumes with irregular borders.
Giani et al.8 applied L-CNV in mice and used a stereological method to calculate lesion volumes based on SD-OCT imaging (similar to the method we describe here). Giani et al. also reported the good correlation between CNV volume obtained with OCT and confocal. At days 5 and 7, there were Spearman correlations of Rho = 0.65 and Rho = 0.68, respectively (P < 0.05). However, at days 14 and 28, the correlation between the two methodologies was weaker and not significant, with Spearman coefficients of Rho = 0.183 and Rho = 0.571, respectively (P > .05).8 This differs from our results, where we observed a positive correlation on day 14 (Pearson's r = 0.50, P <.01). Furthermore, Giani et al. reported volumes in pixels, rather than in absolute measurement units. This prevented direct comparison of the lesion volumes measured by OCT and by the gold standard of confocal. Thus, we are the first to directly compare lesion volumes measured from stereological reconstruction of OCT images to those measured from 3-D confocal reconstruction.
To determine why our OCT volume measurements did not correlate well with confocal volume measurements for lesions with confocal volumes larger than 0.004 mm3, we examined the morphology of lesions measured by both techniques. For large lesions (>0.004 mm3), we found that the edges were more diffuse and not well defined, making it difficult to delineate the edges of the lesions in the confocal and OCT images. Because the confocal microscope software automatically detects the edges of each lesion, it is possible that the diffusivity caused the detection algorithm to overestimate the size of the large lesions. In contrast, lesion edges in the OCT images were manually traced with the “Volumest” ImageJ plug-in, which may have resulted in more precise measurements for lesions with diffuse edges.
OCT provides a rapid means to measure L-CNV lesion size during chronic experiments, without having to euthanize the animal. For each lesion, OCT image acquisition takes roughly 1 minute, followed by 1 to 2 minutes for calculating the lesion volume. In contrast, preparation of choroidal flatmounts for confocal imaging is a technically involved and time-consuming technique, requiring at least one overnight step as well as expensive reagents.13 The stereological technique that we used for OCT volume reconstruction relies on free software and is based on the same principle of volume quantification typically performed with confocal microscopy. Our quantification method may also be applied to the analysis of other focal intraocular lesions, such as choroidal nevus, metastasis, melanoma, or retinochoroiditis. Future work will focus on comparing OCT and confocal volume measurements at multiple time points. This will reveal whether OCT measurements are accurate at all stages of L-CNV development, or whether other factors may interfere with the accuracy of the technique.
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Differences Between OCT and Confocal Volume Measurements
|N||Mean||SD||95% CI||P Value|
|OCT Volume||92||0.00268||0.00103||(0.00247, 0.00289)||–|
|Confocal Volume||92||0.00267||0.00074||(0.00252, 0.00282)||–|
|Difference (OCT – Confocal)||92||0.00001||0.00084||(−0.00016, 0.00019)||.90|