Pathologies such as exudative age-related macular decline (AMD) are characterized by choroidal neovascularization (CNV) — aberrant advance of new claret argosy from the choroid into the subretinal space.1,2 The laser-induced CNV (L-CNV) beastly archetypal of exudative AMD relies on laser photocoagulation to agitate Bruch’s membrane, appropriately aesthetic claret barge advance from the choriocapillaris into the subretinal space.3 This archetypal was originally developed in macaque4 and afterwards acclimatized for use in mice and rats. L-CNV has become the gold accustomed for preclinical testing of accustomed assay modalities acclimated for subretinal neovascularization.5–9 The archetypal has authentic invaluable for evaluating furnishings of biologic therapies on CNV bane progression in neovascular AMD research. 2,4,10,11 Cogent allowances of L-CNV accommodate its reproducibility and simplicity, which accommodate a aboveboard alignment for belief subretinal neovascularization.4
The L-CNV archetypal in non-human primates12 and in rodents8 undergoes three stages of development, artful the stages of development apparent in beastly CNV.12 The aboriginal date is an anarchic acknowledgment that initiates a advance phase.8 Soon afterwards laser photocoagulation, a gas balloon forms beneath the retina in the breadth of laser appliance (indicating disruption of Bruch’s membrane), and the retina becomes edematous and hazy.12 Three to 5 canicule afterwards laser photocoagulation (second stage), choroidal fibrovascular tissue proliferates.8,12 Seven to 10 canicule afterwards (third stage), choroidal neovascular tissue forms about the breach in Bruch’s membrane. At this time, about no aqueous is present amid the anew formed subretinal film and the aloft retina. 8,12 Fourteen to 28 canicule afterward laser photocoagulation (still advised allotment of the third stage), fibrovascular tissue forms in the subretinal amplitude about the breach in Bruch’s membrane. 8,12 This date is additionally accustomed as the reparative or involutional stage.12
Lesion development and progression is generally tracked in vivo with fluorescein angiography (FA) to appraise arising from anew formed vessels.13 To access a complete altitude of bane volume, choroidal flatmount affairs are about decrepit with beaming dye that labels claret vessels, followed by confocal imaging to account three-dimensional (3-D) volumes of the CNV lesions. This address has become broadly accustomed as a quantitative adjustment to appraise biologic ability in ameliorating CNV bane progression. 2,11,13,14 However, admitting its accurateness in quantifying admeasurement of the neovascular complex, the address is bound to ex vivo use.
Spectral-domain optical adherence tomography (SD-OCT) is a noninvasive, in vivo imaging adjustment that allows accumulating of high-resolution, cross-sectional images of tissues.15 The address has been acclimated abundantly in ophthalmology,15,16,2 accouterment abundant images for the antecedent and afterwards segments of the eye.17 SD-OCT has become an capital apparatus for the dispensary in ocular pathologies such as exudative AMD,18–20 retinal tumors,21–24 macular edema,25 and retinal detachment.26–28
Recently, studies accept accustomed use of SD-OCT for in vivo appraisal of L-CNV in rodents, acceptance altitude of bane admeasurement and elimination parameters.2,8,29 These studies relied on two techniques for assuming aggregate about-face from OCT images. In the first, L-CNV bane admeasurement was estimated by applicable an ellipsoid to the bane and artful the aggregate of the ellipsoid. However, OCT aggregate abstracts were begin to be almost 10-times greater than those acquired with ex vivo confocal reconstruction.2 As a result, the account of this adjustment may be bound to anecdotic gross furnishings of assay altitude but not to assuming complete in vivo aggregate measurements.
A added authentic address for assuming aggregate about-face of L-CNV lesions from OCT images relies on accretion the breadth affected from anniversary accumbent array casual through the lesion. Appliance this adjustment in mice, Giani et al. begin abstinent alternation amid CNV sizes abstinent appliance SD-OCT and confocal at canicule 5 and 7 afterwards laser surgery. However, canicule 14 and 28 did not accept a statistically cogent correlation. Furthermore, complete comparisons amid the volumes abstinent with OCT and those abstinent with confocal were not reported.8
In the present study, we call a stereological adjustment for quantifying bane volumes from OCT images, which produces aggregate abstracts that are statistically agnate to those acquired from ex vivo confocal reconstruction. Appliance of this address in vivo allows bane volumes to be abstinent and tracked over time, afterwards acute animals to be euthanized.
A complete of 18 macho amber Norway rats (Envigo, Huntingdon, UK), age-old P98 to P102, were acclimated for this study. All procedures were performed beneath anesthesia, followed the guidelines of the Affiliation for Assay in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and were accustomed by the Institutional Beastly Care and Use Committee at the University of Southern California.
Rats were anesthetized with intraperitoneal bang 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 contemporary 1% tropicamide (Akorn, Lake Forest, IL) and 2.5% phenylephrine HCl (Paragon BioTeck, Portland, OR). Animals were euthanized on day 14 via intracardiac bang of sodium pentobarbital administered beneath abysmal anesthesia.
At day 0, four laser burns were fabricated in the afterwards pole of anniversary eye (36 eyes) appliance a blooming diode laser (IQ 532; IRIDEX, Mountain View, CA). A awning bottle was acclimated as a acquaintance lens to accommodate decision of the retina and optic assumption through a slit-lamp, as able-bodied as during laser application. Laser beating ambit were 150-mW to 160-mW power, 50-ms duration, and 75-μm atom size. One bake was fabricated per quadrant, amid the retinal argosy in a peripapillary distribution. The assembly of a subretinal balloon at the time of laser appliance accepted the breach of Bruch’s membrane.
OCT imaging was performed appliance an SD-OCT arrangement (Bioptigen, Durham, NC) to appraise in vivo progression of laser lesions afterwards photocoagulation (at canicule 0, 3, 7, 10, and 14). OCT is a noncontact, noninvasive, nonionizing imaging technology that poses no added risks to the animals. Imaging was performed beneath ketamine/xylazine anesthesia. Anniversary bane was beheld with OCT appliance 100 accumbent raster scans spaced 16-μm afar over an breadth of 1.6 mm x 1.6 mm.
FA was acclimated to affirm Bruch’s film breach and to validate the attendance of CNV in vivo. Afterward anesthesia, rats accustomed aborticide eye drops (1% tropicamide and 2.5% phenylephrine HCl) and 0.01 mL intraperitoneal bang of 10% sodium fluorescein dye. Sequential afterwards pole images were taken appliance a RetCam 3 retinal camera (Natus Medical, Pleasanton, CA) with an 80° lens. Lesions that showed no arising were afar from assay because those lesions acceptable represented laser impacts that did not aftereffect in CNV.
The lesions were articular according to their position about to the optic nerve: superotemporal, inferotemporal, superonasal, and inferonasal. We acclimated a stereological adjustment (3-D estimation of two-dimensional cross-sections) to reconstruct the OCT images in 3-D and account bane size. For this, we acclimated the “Volumest” (“Volume Estimation”) plug-in30 of ImageJ (NIH, Bethesda, MD).31 Anniversary angel breadth casual through the bane was delineated and abstinent by duke (Figure 1A). In anniversary image, the CNV was articular as the subretinal hyperreflective actual aloft the RPE layer. Before the altitude process, a distinct angel in pixels was calibrated with accustomed ethics from the OCT to authorize the units in micrometers (1 pixel = 3.33 μm).
Laser-induced choroidal neovascularization (L-CNV) bane aggregate affected from (A) optical adherence tomography (OCT) images candy with ImageJ’s “Volumest” plugin and from (B) confocal images of choroidal flatmounts that were decrepit with fluorescein labeled isolectin-B4 and candy with PerkinElmer’s Volocity software. Arrows announce the aggregate (in μm3) affected by anniversary software program.
After OCT imaging on day 14, rats were euthanized, as declared earlier. Before enucleating the eyes, a abutment was placed at the 12 o’clock acme to mark the acclimatization of the eye to facilitate complete allegory of anniversary bane amid OCT (in vivo) and confocal (ex vivo). The antecedent articulation and retina were removed, and four adequate adorable incisions were made. The RPE-choroid-sclera circuitous was anchored brief in 4% formalin at 4°C. Tissue was done the aing day and permeabilized by evolution 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 corpuscle marker; Vector Laboratories, Burlingame, CA) at 4°C overnight. The staining footfall was followed by two washes with Tris-buffered acrid with 0.1% Tween-20 (TBST). Tissue was army on slides with ascent media (Vectashield; Vector Laboratories, Burlingame, CA) while authoritative incisions in the eye cups to abrade them.
Flatmounts were visualized appliance the 10× cold of an UltraVIEW spinning deejay confocal microscope (PerkinElmer, Waltham, MA). The angel endless were generated in the z-plane, with the microscope set to accelerate at 488 nm and to ascertain at 505 nm to 530 nm. Images were candy appliance the microscope’s Volocity software by carefully circumscribing and digitally extracting the beaming bane areas throughout the complete angel assemblage (Figure 1B). The extracted bane was candy through the cartography software to accomplish a agenda topographic angel representation of the bane and an angel volume. The topographic assay affairs determines and displays the object’s apparent contours by audition beaming arresting from the top of the angel assemblage and again measures aggregate beneath the apparent to crop a final volume, which reflects the CNV fluorescence volume.
Pearson correlations were affected to beam the affiliation amid bane volumes abstinent by OCT and confocal microscopy. A modification of the Bland-Altman was performed to appraise the gold accustomed (confocal) adjoin the aberration in aggregate amid OCT and confocal. A commutual t-test was acclimated to actuate if the beggarly aberration amid the two altitude techniques differed decidedly from zero. Beggarly and accustomed aberration forth with the 95% aplomb intervals were reported. Analyses were run on akin commutual abstracts that had both OCT and confocal volumes available. All analyses were performed appliance SAS software adaptation 9.4 (SAS Institute, Cary, NC).
Of the 144 laser burns that were fabricated (18 rats x 8 burns each), 81% showed arising on FA, constant with L-CNV. Twenty-seven lesions did not actualization affirmation of Bruch’s film breach and were appropriately afar from analysis. The actual 117 lesions were analyzed for allegory amid volumes affected from OCT (in vivo) and confocal (ex vivo) imaging. Figure 2 shows adumbrative FA images of a adulterated bane on canicule 0, 3, 10, and 14. This aforementioned lesion, beheld by confocal microscopy, is apparent in Figure 3.
Flourescein angiography (FA) imaging aftereffect of a laser-induced choroidal neovascularization (L-CNV) bane (arrows) based on angiographic patterns. Intraretinal edema is apparent aloft and about the bane on day 0 (A). On day 3, angiography backward phases actualization hyperfluorescence due to arising (B). Ten canicule afterwards L-CNV, a staining arrangement was empiric with basal arising (C). On day 14, the angiographic (staining) arrangement was agnate to that of day 10 (D).
Representative confocal angel of a laser-induced choroidal neovascularization (L-CNV) bane afterwards afterlife on day 14. The bane pictured is the aforementioned one followed by flourescein angiography (FA) in Figure 2.
SD-OCT images were acquired afterwards laser assay on canicule 0, 3, 7, 10, and 14. (In one rat, corneal caliginosity prevented imaging on day 7.) On day 0, OCT adumbrated signs of swelling, hyperreflectivity in the lasered area, and the disruption Bruch’s film (Figures 4A and 4B). No aggregate altitude was performed on day 0 because of the abridgement of categorical borders of the L-CNV. On day 3, the lesions presented a hyperreflective acknowledgment basic from the RPE. As appear previously, day 3 lesions had a erfly-like shape11 (Figure 4C). On day 7, lesions began to arrangement (choroidal fibrovascular tissue replaces the choroidal-RPE defect) and portrayed decreased abscess and added RPE reflectivity (Figure 4D). On day 10 (Figure 4E), OCT showed beneath RPE hyperreflectivity as able-bodied as RPE and alien retina irregularity. On day 14 (Figure 4F), fibrovascular tissue was present in the OCT.
Representative spectral-domain optical adherence tomography (SD-OCT) images assuming aftereffect of a laser-induced choroidal neovascularization (L-CNV) bane (arrowheads). The laser atom (A) and the disruption (arrow) of Bruch’s film (B) are arresting on day 0. By day 3, retinal hyperreflectivity apparent a characteristic “erfly-like” actualization (C). Day 7 shows beneath abscess (D). On day 10, there is a abatement in retinal bistered epithelium (RPE) hyperreflectivity and abnormality of the RPE and alien retina (E). Fibrovascular tissue is arresting on day 14 (F).
Figure 5 shows CNV aggregate abstracts affected from SD-OCT images (days 3, 7, 10, and 14) and confocal microscopy (day 14). On day 14, correlations amid OCT and confocal L-CNV volumes showed a complete affiliation (Pearson’s r = 0.50, P < .01; Figure 6). A Bland-Altman artifice was generated to anon analyze the bane volumes abstinent by OCT and confocal (Figure 7). (Bland-Altman plots are a accustomed statistical adjustment acclimated to analyze two altitude techniques.) For confocal volumes abate than 0.004 mm3 (79% of the lesions), the aberration amid the OCT and confocal aggregate abstracts was abundantly appropriately broadcast about aught (Figure 7), blame acceptable acceding amid the two techniques. For these abate lesions, OCT and confocal abstracts were not decidedly altered from anniversary added (P = .90; Table). For beyond volumes, however, OCT yielded abate bane sizes than confocal (Figure 7).
Choroidal neovascularization aggregate abstracts affected from spectral-domain optical adherence tomography (SD-OCT) images (days 3, 7, 10, and 14) and confocal microscopy (day 14). Absurdity confined represent accustomed absurdity of the beggarly (mean ± SEM).
Comparison of laser-induced choroidal neovascularization bane volumes abstinent by optical adherence tomography (OCT) and confocal, 14 canicule afterwards laser treatment. Pearson’s r = 0.50, P < .01.
Bland-Altman artifice of the aberration in aggregate amid the two altitude techniques (optical adherence tomography [OCT] – confocal) against the beggarly confocal volume. Dotted accumbent curve represent the 95% banned for the differences: beggarly ± 1.96 × accustomed deviation. For confocal volumes abate than 0.004 mm3 (dotted vertical line), the aberration amid the OCT and confocal aggregate abstracts was abundantly appropriately broadcast about zero.
Differences Amid OCT and Confocal Aggregate Measurements
In this study, we call a noninvasive adjustment to quantify L-CNV aggregate in vivo appliance SD-OCT. Our address relies on a stereological adjustment agnate to that acclimated for 3-D about-face by confocal microscopy. The software we acclimated (ImageJ and “Volumest” plug-in) is advisedly accessible to the public. With the aid of this program, we were able to accurately admeasurement the aggregate of anyhow shaped lesions. Our access can serve as a admired apparatus to rapidly and quantitatively appraise therapies for subretinal neovascularization in the L-CNV model. OCT provides in vivo advice about elimination of the neovascular circuitous and morphologic actualization that are accepted to beastly pathology. Together, these capacity can aid in bigger compassionate of CNV pathogenesis.8
Three added appear studies accept declared use of OCT for evaluating L-CNV morphological features29 and barometer bane aggregate in vivo in rodents.2,8 Our after-effects accede with Giani et al.8 and Fukuchi et al.31 apropos the morphological progression of the lesions: Immediately afterwards laser application, a subretinal balloon with a hyperreflective acknowledgment and edema afterwards categorical borders forms. At day 3, lesions display a erfly-like shape,8 back the edema begins to retract. About day 7, lesions accept a added dome-like actualization with an access in reflectivity, which represents the proliferative tissue, area fibrovascular tissue fills the birthmark generated by the laser-induced aition of the RPE-choroidal complex.8 Admitting not accepting the OCT-confocal aggregate correlations at added than one time point, we were still able to use OCT for ecology L-CNV longitudinally in anniversary animal.
Sulaiman et al.2 declared OCT altitude of L-CNV bane sizes by applicable an ellipsoid to the bane and artful its volume. They presented a statistically cogent alternation (P < .05) amid the OCT and confocal bane volumes; however, bane volumes abstinent by OCT were almost 10 times the admeasurement of those abstinent by confocal. Presumably, this conflict arises because CNV lesions are not complete ellipsoids, but rather are arbitrarily shaped volumes with aberrant borders.
Giani et al.8 activated L-CNV in mice and acclimated a stereological adjustment to account bane volumes based on SD-OCT imaging (similar to the adjustment we call here). Giani et al. additionally appear the acceptable alternation amid CNV aggregate acquired with OCT and confocal. At canicule 5 and 7, there were Spearman correlations of Rho = 0.65 and Rho = 0.68, appropriately (P < 0.05). However, at canicule 14 and 28, the alternation amid the two methodologies was weaker and not significant, with Spearman coefficients of Rho = 0.183 and Rho = 0.571, appropriately (P > .05).8 This differs from our results, area we empiric a complete alternation on day 14 (Pearson’s r = 0.50, P <.01). Furthermore, Giani et al. appear volumes in pixels, rather than in complete altitude units. This prevented complete allegory of the bane volumes abstinent by OCT and by the gold accustomed of confocal. Thus, we are the aboriginal to anon analyze bane volumes abstinent from stereological about-face of OCT images to those abstinent from 3-D confocal reconstruction.
To actuate why our OCT aggregate abstracts did not associate able-bodied with confocal aggregate abstracts for lesions with confocal volumes beyond than 0.004 mm3, we advised the assay of lesions abstinent by both techniques. For ample lesions (>0.004 mm3), we begin that the edges were added broadcast and not able-bodied defined, authoritative it difficult to abut the edges of the lesions in the confocal and OCT images. Because the confocal microscope software automatically detects the edges of anniversary lesion, it is accessible that the diffusivity acquired the apprehension algorithm to aggrandize the admeasurement of the ample lesions. In contrast, bane edges in the OCT images were manually traced with the “Volumest” ImageJ plug-in, which may accept resulted in added complete abstracts for lesions with broadcast edges.
OCT provides a accelerated agency to admeasurement L-CNV bane admeasurement during abiding experiments, afterwards accepting to euthanize the animal. For anniversary lesion, OCT angel accretion takes almost 1 minute, followed by 1 to 2 account for artful the bane volume. In contrast, alertness of choroidal flatmounts for confocal imaging is a technically complex and time-consuming technique, acute at atomic one brief footfall as able-bodied as big-ticket reagents.13 The stereological address that we acclimated for OCT aggregate about-face relies on chargeless software and is based on the aforementioned assumption of aggregate altitude about performed with confocal microscopy. Our altitude adjustment may additionally be activated to the assay of added focal intraocular lesions, such as choroidal nevus, metastasis, melanoma, or retinochoroiditis. Future assignment will focus on comparing OCT and confocal aggregate abstracts at assorted time points. This will acknowledge whether OCT abstracts are authentic at all stages of L-CNV development, or whether added factors may baffle with the accurateness of the technique.
Differences Amid OCT and Confocal Aggregate Measurements
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