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Abstract
Chicks are an excellent model for studying myopia. To study the change of the ocular structures in chicks, ultrasound is mostly used. However, it suffers from limited spatial resolution. In this study, we investigated the axial length (AL) and the thickness of different ocular structures in chicks’ eye undergoing visually induced changes using a swept-source optical coherence tomography (SS-OCT) system in vivo. Two groups of chicks wore a translucent plastic goggle (n = 6) over the right eye to induce form-deprivation myopia. Following 12 days of form deprivation, goggles were removed in one group of chicks (n = 3), and they were allowed to experience 5 days of unrestricted vision (recovery). Goggles remained in place for a total of 17 days for the remaining 3 chicks. A separate group of 3 chicks were untreated and served as normal control. Ocular dimensions were measured in control, myopic, and recovered eyes using an SS-OCT system. We found myopic chick eyes had significantly thicker AL, lens thickness (LT), anterior chamber depth (ACD), and vitreous chamber depth (VCD), but significantly thinner retina thickness (RT) and choroid thickness (ChT) compared to the control eyes. Following 5 days of recovery, the cornea thickness (CT), retina pigment epithelium thickness (RPET), and ChT were significantly thicker, while the ACD and LT became significantly thinner compared to that of myopic eyes. SS-OCT can serve as a promising tool to provide measurements of the entire ocular structures, for evaluating the change of thickness and depth of different ocular structures in chicks in vivo. The change of AL in the myopic and recovered chick eyes can be attributed to the thickness alterations of different ocular structures. Altogether, this work demonstrated the feasibility of SS-OCT in chick myopic research and exhibited new insights into the changes of ocular structures in chicks experiencing myopia after unrestricted vision recovery.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Myopia is one of the most common ocular disorders that causes the irreversible low vision of approximate 1.6 billion people worldwide [1–3]. Myopic eyes are characterized by excessive axial elongation compared to normal eyes. In high myopia, retinal thinning, posterior staphyloma, and chorioretinal atrophy, can occur, putting individuals at risk of several blinding eye diseases including retina degeneration, glaucoma, and retinal detachment [1,2]. There is strong and compelling evidence suggesting that myopia can be induced by alterations of the visual environment [4]. Although the cause of myopia is complex, many clinical and experimental studies suggest that environmental factors such as education, reading, time spent outdoor, near work, and light exposure, are risk factors for myopia development [5]. If myopia cannot be controlled and corrected in time, myopia can progress leading to high myopia and the blinding eye conditions associated with high myopia [6].
Animal models play a critical role in vision research for a large number of ocular diseases and conditions including refractive errors, cataracts, retinal degeneration, retinal detachment, diabetic retinopathy, glaucoma, amblyopia, and corneal injuries [7–12]. Compared with other animal models, chick is regarded as an underutilized animal model with many advantages in cost, size, and ease of handling due to relatively large eyes, rapid eye growth, highly sensitive control of refractive state, excellent optics, active accommodation, high visual acuity, and easy drug delivery by intravitreal injection [13,14]. Although primates are the most ideal animal model for myopia research since they have similar ocular anatomy and physiology to humans, it takes a long time to induce myopia in primates and costs of housing and breeding can be prohibitive. A few investigators have attempted to use mice for studies of myopia, however, the small size of mouse eyes complicates ocular measurements using conventional ocular equipment [15–18] and it is unclear whether the mouse eye responds to changes in the visual environment, as has been clearly demonstrated in humans, primates, guinea pigs and chicks. Therefore, chicks are becoming the most commonly used animal model for the study of emmetropization [19–21], ocular aberration [22,23], myopia [24–31], and hyperopia [32,33].
Currently, the thickness/depth change of different ocular dimensions in chicks’ eyes with myopia and vision correction had been measured by several techniques. J. Wallman et al. first introduced form-deprivation myopia in chicks and measured vitreous chamber depth (VCD) by histologic section staining [17,27,34]. R. Ashby et al. utilized infrared photoretinoscopy, ultrasonography, and infrared photokeratometry to monitor the change of ocular elongation [35,36]. J.R. Phillips et al. observed the change of VCD and lens thickness (LT) before and after myopia via retinoscopy and ultrasonography [37,38]. The laser Doppler interferometry (LDI) was also used to measure the change of retina thickness in chick myopia [39]. Additionally, X. Y. Zhu et al. imaged and measured the choroid thickness change (ChT) by ultrasonography in myopic and recovered chick eyes [31,40–43]. Histology can achieve high-resolution imaging but it is terminal. Retinoscopy and ultrasonography can provide in vivo measurements but the spatial resolution is limited to resolve the ocular structures. Therefore, an imaging method that can provide high-resolution and in vivo measurement for systematic dimension changes of different structures in myopic chick eyes is highly needed.
Optical coherence tomography (OCT) is a rapid, high resolution, noninvasive, and noncontact imaging modality [34–36], playing an important role in the measurement of human retinal thickness (RL) [37–40], choroidal thickness (ChT) [41–44], and scleral thickness (ST) [45–47]. OCT has become essential to detect the length or thickness change of RNFL [48,49], peripapillary RNFL [50], and macular retina [51] in human myopic eyes. Compared to typical ophthalmic B-scanner and ultrasound biomicroscopy (UBM) that owned 30 to 150 μm axial resolution [52], OCT is able to provide a 1-15 μm axial resolution [53]. Conventional ophthalmic laser interferometer can offer <10 μm axial resolution [54], while it cannot provide images of different ocular structures and is difficult to distinguish the border between different ocular layers, e.g., RPE, choroid and sclera. The previous studies have demonstrated that OCT is able to observe the retinal degeneration [55], choroidal thickness distribution [56], and accommodation of iris [57] in chicks. Although OCT has been widely used to characterize morphological structures and understand physiological changes in myopia, using OCT to study the change of various structural thicknesses of unrestricted-vision recovered myopic eyes has not been reported in avian species. Swept-source optical coherence tomography (SS-OCT) that uses a wavelength-tunable laser and a dual-balanced photodetector, presents great sensitivity and high signal-to-noise ratio (SNR) and has been widely used in different applications [58]. In the recent ophthalmology study, SS-OCT has been demonstrated to better distinctly differentiate the true border between two structural layers such as sclera and choroid [59–61], thereby could serve as a promising tool to observe the change of various structural thickness and ocular length in myopic and unrestricted-vision recovered eyes in chicks.
In this study, we demonstrated the feasibility of SS-OCT system in measuring the structural thickness and ocular length in chick eyes in vivo and investigated the change of ocular length and various structural thickness undergoing accelerated growth induced by form-deprivation (goggle-wearing) and in chick eyes undergoing decelerated growth during recovery from form deprivation (goggle-removing).
2. Materials and methods
2.1 Ethics and animals
Animals were managed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research, with the Animal Welfare Act, and with the National Institutes of Health Guidelines. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center. White Leghorn male chicks (Gallus gallus) were obtained as 2-day-old hatchlings from Ideal Breeding Poultry Farms (Cameron, TX). Chicks were housed in temperature-controlled brooders with a 12-hour light/dark cycle and were given food and water ad libitum. At the end of experiments, chicks were euthanized by overdose of isoflurane inhalant anesthetic (IsoThesia; Vetus Animal Health, Rockville Center, NY), followed by decapitation.
2.2. Induction of myopia and recovery from myopia
Form deprivation myopia (FDM) was induced in 3 to 4 day-old chicks (n=6) by applying translucent plastic goggles to one eye (right eyes) for 12 days, as previously described [62]. The contralateral eyes (left eyes) of all chicks remained untreated. Chicks were checked twice a day to ensure that their googles were still intact. Following 12 days of form deprivation, goggles were removed from a subset of form-deprived chicks and chicks were allowed to experience unrestricted vision (recover) for 5 days (n=3). A separate group of chicks were untreated and served as controls (n=3). All chicks were housed together in brooders and were maintained on a 12/12 hour light/dark cycle.
2.3 Optical coherence tomography (OCT)
In this study, a SS-OCT imaging system was used for chick eye imaging. The OCT system utilized a wavelength-swept laser as a light source and the laser was centered at 1310 nm with 100 nm full-width at half-maximum (FWHM) bandwidth providing an imaging depth of 8 mm in the air [63,64]. This system schematic was illustrated in Fig. 1. The system had a wavelength-swept frequency of 200 kHz with the output power of ∼10 mW incident onto the chick eyes [65–68]. This system provided 10.6 μm axial resolution in tissue. A commercial objective (OCT-LK3, Thorlabs, New Jersey, USA) was used to provide 13 μm lateral resolution. The objective provided a 36 mm focal length and 10 mm x 10 mm field of view. The swept source laser was connected to a fiber coupler with 97:3 ratio that split 97% of the laser power towards the fiber-based Michelson interferometer and 3% of the laser power towards the Mach-Zehnder interferometer (MZI), which generated a frequency-clock signal for triggering the OCT sampling. The 97% of the laser power was further split to the reference arm and sample arm with a 50:50 fiber coupler. In the sample arm, the laser beam was collimated through a collimator and reflected by a mirror to a pair of two-dimensional (2D) galvanometer scanners. The beam was further focused by the objective to scan the sample laterally in the (x, y) plane. The reflected signal from the reference and sample arms was combined in the 50:50 ratio fiber coupler to form the interference fringes [69–68]. The interference fringe signals from different depths received by the balanced photodetector were encoded with different frequencies. The polarization controllers in both arms were used to maximize the interference fringe signal and decrease the background noise [66,67]. Finally, the resulting signal was collected and processed on the computer to generate a depth-resolved intensity profile of the chick eyes. The sensitivity of the system was ∼98 dB.
Fig. 1. Imaging schematic of SS-OCT for ocular structure measurement. SSL, swept-source laser. FC, fiber coupler. PC, polarization controller. C, collimator. BD, balance photodetector. CL, circulator. MZI, Mach-Zehnder interferometer (frequency clocks). DAQ, data acquisition board. M, mirror. O, objective lens. GSM, galvanometer scanning mirror. CP, computer.
2.4 Measurement
The internal ocular structures were imaged during anesthesia using SS-OCT. 1.5% isoflurane in oxygen without cycloplegic agents were utilized to anesthetize chicks before SS-OCT imaging. Chicks were positioned on a custom-made stage with a soft stand for fixing chick head. An elastic metal double arm clip was used to expand chick eyelid to provide the scanning window and keep the chick eye still while imaging. Each right eye was imaged after the topical instillation of two drops of Genteal Severe gel and dilating drops. The scanning light beam was located into the chick eye through the objective with 36 mm focal length and 10 mm x 10 mm field of view. The focal point of scanning laser beam was adjusted by the optical intensity and reflected intensity on the imaging screen. The distance between the chick eye and objective depended on location of the focal point. Even when the focus was on the lower part of the eye (e.g., choroid), the objective did not reach the chick eye and the distance between the chick eye and objective was around 20 mm. Each scanning region was fixed to approach the pecten following the chick beak direction to keep the image of each chick eye from the same imaging position, as shown in Fig. S1. The scanning region was selected to cover the whole iris of each chick eye as shown in Fig. S2. The horizontal orientation of the iris plane (perpendicular to the laser beam) can help to assure nearly the same area/angle from each chick eye being used for thickness analysis. To further eliminate the effect of locations on thickness measurement from each chick, we picked the 2D cross-sectional imaging plane which passed the center (blue dashed line in Fig. S2) of the scanning window (iris) for thickness calculation. Eight 2D SS-OCT structural images were obtained at the same imaging plane. Each B-scan contained 960 × 6000 pixels on X and Z axes (Z presents the depth direction) with a pixel size of 6.25 × 1.33 μm2. All measurements of various structure thickness of chick eyes were completed via ImageJ FIJI software (1.53f, NIH, USA). Each B-scan frame was processed by Despeckle for noise reduction and Median Blur Filter for image optimization. The measurement was calculated by averaging each targeted structure in the eight 2D SS-OCT image. All layers were segmented manually for measurements. The thickness/depth measurement was corrected by applying the refractive index of 1.369 for cornea, 1.335 for anterior chamber, 1.437 for lens, 1.335 for vitreous chamber, and 1.351 for retina, RPE, choroid, and sclera [71]. The internal ocular dimensions were composed of the cornea thickness (CT), anterior chamber depth (ACD), lens thickness (LT), vitreous chamber depth (VCD), retina thickness (RT), retina pigment epithelium thickness (RPET), choroid thickness (ChT), and sclera thickness (ST). Axial length (AL) of chick eyes was defined as the sum of all ocular dimensions.
2.5 Tissue preparation
To prepare ocular tissues for analysis, chicks were euthanized by an overdose of isoflurane inhalant anesthetic (IsoThesia; Vetus Animal Health, Rockville Center, NY). Eyes were enucleated and cut along the equator to separate the anterior segment and posterior eye cup. Anterior tissues were discarded, and the vitreous body was removed from the posterior eye cups. An 8 mm punch was taken from the posterior pole of the chick eye using a dermal biopsy punch (Miltex Inc., York, PA). Punches were located nasal to the exit of the optic nerve, with care to exclude the optic nerve and pecten oculi. Tissue punches were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer [pH 7.3], overnight at 4°C. Tissues were then dehydrated in a graded series of ethanol in PBS and embedded in JB-4 resin (Polysciences, Inc., Warrington, PA) as previously described [72]. Then, 4-µm-thick sections were cut using glass knives on an Ultracut E ultramicrotome (Reighert-Jung) and stained with 1% Toluidine blue O in dH2O. The stained sections were imaged with a Nikon Eclipse Ts2-FL/Ts2 inverted microscope equipped with a Nikon color 5.9 Mp DS-Fi3 camera, operated with Nikon NIS-Elements software, version 4.00.
2.6 Data analysis
In the data statistics, the Mean ± Standard Error (M ± Str) of the mean was used for comparing the interocular structure difference between right eyes of three groups (i.e. control, myopic, and recovered). Two-sample Student’s t-test were used to compare various ocular structure parameters between any two groups. P-values < 0.05 was taken to be significant, P-values > 0.05 was nonsignificant. The repeatability of the OCT cross-sectional map averages was assessed by pooled standard deviation obtained from the eight measurements taken from each eye. The reproducibility of measurements was demonstrated by the coefficient of variation (CoV) [73]. CoV < 100% was considered high-reproducibility (low-variance) and CoV > 100% was considered as low-reproducibility (high-variance).
3. Results
In this study, 18 eyes (9 left eyes & 9 right eyes) from 9 chicks were imaged. The 9 chicks included 3 normal untreated chicks, 3 chicks undergoing form deprivation-induced myopia (left eyes are untreated, right eyes are myopic), and 3 chicks recovering from form-deprivation-induced myopia (left eyes are contralateral control, right eyes are unrestricted-vision recovered). We first focused the laser beam on the upper side of the chick eye, Fig. 2 shows the structural images of cornea, anterior chamber and lens, CT, ACD, and LT can be easily identified and measured. We then lowered the beam focus to reach retina and previously observed structures (cornea, anterior chamber, and lens) began to flip over the upper side of the zero-delay plane (there was 8 mm at each side of the zero-delay plane of the OCT system and the zero-delay plane was set at the top of the image) as shown in Fig. 3. With retina front surface and lens back surface in the same image, we can calculate the vitreous chamber depth (VCD) as indicated by the blue line in Fig. 3. Figure 4 shows the focused structural images of retina, RPE, choroid, and sclera, and the thickness measurements corresponding to ocular structures from SS-OCT. Different ocular structures can be clearly resolved. There were obvious changes in choroid thickness with a reduced thickness in myopic eyes and increased thickness in recovering eyes. The chorio-scleral-interface within the choroid was clearly observed in chick eyes. Particularly, both the fibrous layer and cartilaginous layer of the sclera were observed and the statistics about the sclera included the fibrous and cartilaginous layer in this study. Table 1 shows the distribution of the repeatability and coefficient of variation in the measurement of each chick eye structure. The pooled standard deviation of the SS-OCT measurements was 7.8 µm to 28.6 µm and the CoV value of measurements was 0.2% to 7.2% in the chick eye (cornea, anterior chamber, lens, vitreous chamber, retina, RPE, choroid, and sclera) demonstrating that SS-OCT was a stable and reliable imaging modality for the repeatable measurement of chick eyes. The segmentation of the RPE by our OCT intensity images in this study included the boundary between inner and outer photoreceptor segments and the end tips of the photoreceptors [74]. Figure 5 shows the histology of the myopic and recovering eyes with retina, RPE, choroid, and sclera. The changes in choroid thickness observed in tissue sections of fixed and embedded chick eyes are consistent with what we observed using SS-OCT in vivo. The retinas came off the control tissue more readily than they did from recovered eyes. The phenomenon was not clearly understood, but the RPE (and retina) adhered more tightly to the myopic and recovered choroids than it did in the control eyes. It was thought to be due to differences in fluid flow across the RPE. A summary and comparison of ocular tissue thicknesses and chamber depths were provided in Figs. 6, Fig. S3, Table 2, and Table 3. In the following sections, we analyzed the measurements on different ocular structures, respectively.
Fig. 2. OCT images of the upper chick eye in normal (A), myopic (B), and recovered (C) chick right eyes. Cornea, lens, and iris can be clearly resolved on the images. Cornea thickness (CT), anterior chamber depth (ACD), and lens thickness (LT) were indicated in red, cyan and while lines, respectively. Lens front and back surfaces were labeled using yellow dashed lines in (A). C, Cornea. I, Iris. LFS, lens front surface. LBS, lens back surface. Scale-bar: 500μm.
Fig. 3. The vitreous chamber depth (VCD) measurement from OCT images in normal (A), myopic (B), and recovered (C) chick experimental eyes. VCD was indicated by the blue line. Retina front surface, lens front and back surfaces were labeled using yellow dashed lines in (A). I, Iris. LRS, lens rear surface. LFS, lens front surface. RFS, retina front surface. VCD is the length between LRS and RFS. Scale-bar: 500μm.
Fig. 4. OCT images of the retina, retina pigment epithelium (RPE), choroid, and sclera layers in normal (A), myopic (B), and recovered (C) chick right eyes. (A1)-(C1), Enlarged images of the areas indicated by blue, green, and yellow dashed squares in (A)-(C) respectively. RT, RPET, ChT, and ST were indicated in red, pink, yellow and cyan lines, respectively. Scale-bar in (A)-(C): 200μm. Scale-bar in (A1)-(C1): 100μm.
Fig. 5. Histology of ocular structures in myopic and recovered chick eyes. (A) recovered chick’s contralateral control eye (left), (B) recovered chick’s experimental eye (right), (C) myopic chick’s contralateral control eye (left), (D) myopic chick’s experimental eye (right).
Fig. 6. Data analysis of CT, ACD, LT, VCD, RT, RPET, ChT, ST and AL in normal, myopic, and recovered chick eyes. (A)-(I) Comparison of CT, ACD, LT, VCD, RT, RPET, ChT, ST, and AL in control, myopic, and recovered chick eyes. N = 3. NS: P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001. CT, cornea thickness. ACD, anterior chamber depth. LT, lens thickness. VCD, vitreous chamber depth. RT, retina thickness. RPET, retinal pigment epithelium thickness. ChT, choroid thickness. ST, sclera thickness. AL, axial length.
Table 1. Distribution of the repeatability and coefficient of variation of the measurement for CT, ACD, LT, VCD, RT, RPET, ChT, and ST in normal left chick eye. Rep, repeatability.
Table 2. Summary and comparison of different ocular structure thickness and AL between the right chick eyes in control, myopic, and recovered groups (M ± Str). N = 3. CT, cornea thickness. ACD, anterior chamber depth. LT, lens thickness. VCD, vitreous chamber depth. RT, retina thickness. RPET, retinal pigment epithelium thickness. ChT, choroid thickness. ST, sclera thickness. AL, axial length. C-M, Control versus Myopic eyes (ocular structure thickness in Myopic subtract that of in Control). C-R, Control versus Recovered eyes (ocular structure thickness in Recovered subtract that of in Control). M-R, Myopic versus Recovered eyes (ocular structure thickness in Recovered subtract that of in Myopic). Diff, difference value in thickness/depth.
Table 3. Summary of different ocular structure thickness/depth and AL of the right chick eyes in control, myopic, and recovered groups (M ± Str). N = 3. C, control. M, myopic. R, recovered.
3.1 Cornea thickness
Among the comparison of CT between the right eyes for the control, myopic, and recovered groups, there was no significant difference in control versus myopic (C-M) and control versus recovered (C-R) comparisons. The difference of CT in right eyes for C-M (myopic – control) is -8.88 ± 5.58 µm, 3.31 ± 4.89 µm for C-R (recovered – control), as shown in Table 2 and Figs. S3-A. However, the difference of CT in myopic versus recovered (M-R, recovered – myopia) comparison was 12.19 ± 3.26 µm, and CT in the recovered group was significantly larger than that of the myopic group (P < 0.05, Fig. 6(A) and Fig. S3-A).
3.2 Anterior chamber depth
In Fig. 6(B), there was a significant difference between the right eyes for all the three groups (P < 0.001 for C-M and C-R, P < 0.05 for M-R). The differences of ACD in C-M, C-R, and M-R were 400.51 ± 80.56 µm, 200.41 ± 25.47 µm, and -200.10 ± 62.59 µm (Table 2 and Fig. S3-B), respectively. ACD in the myopic eyes was significantly larger than both the control and recovered eyes.
3.3 Lens thickness
In Fig. 6(C), the difference in LT between the right eyes was significant for the control, myopic, and recovered groups (P < 0.001 for C-M and M-R). The LT in the myopic eyes was significantly larger (495.68 ± 108.91 µm) than that in the control eyes and was significantly larger (421.90 ± 57.56 µm) than that in the recovered eyes. The LT was larger (73.77 ± 51.50 µm) in the recovered eyes than that in control eyes (Fig. S3-C).
3.4 Vitreous chamber depth
In Fig. 6(D), the VCD of the right eyes was significantly bigger in both myopic and recovered groups than the control group (P < 0.001 for C-M and C-R). There was no significant difference in VCD of the right eyes in M-R group (P > 0.05). The differences of VCD between the right eyes in C-M, C-R, and M-R were 1360.52 ± 42.03 µm, 1148.79 ± 73.44 µm, and -211.73 ± 64.80 µm (Table 2 and Fig. S3-D), respectively.
3.5 Retina thickness
Compared with the control group, the RT of the right eyes was significantly smaller in both myopic and recovered groups (P < 0.001 for C-M and C-R; Fig. 6(E)). There was no significant difference in RT of the right eyes in M-R group (P > 0.05), which was consistent with histology in Fig. 5. The difference of RT between right eyes were -34.08 ± 9.85 µm, -22.06 ± 6.61 µm, and 12.02 ± 5.73 µm in the C-M, C-R, and M-R (Fig. S3-E), respectively.
3.6 Retinal pigment epithelium thickness
In Fig. 6(F) and Fig. S3-F, RPET of the right eyes in the recovered group was significantly larger than that of the control (P < 0.01) and myopic groups (P < 0.01). However, the difference of RPET between the control and myopic eyes was not significant.
3.7 Choroid thickness
Figure 6(G) shows that ChT had a significant difference among the rights eyes for the control, myopic, and recovered groups (P < 0.001 for all). The difference between the right eyes in C-M, C-R, and M-R groups were -73.17 ± 13.92 µm, 427.49 ± 19.36 µm, and 500.67 ± 6.72 µm (Fig. S3-G), respectively.
3.8 Sclera thickness
The difference in ST of right eyes in the myopic group was larger than that of the control group (10.68 ± 12.66 µm) and that of the recovered group (2.20 ± 13.65 µm, Fig. 6(H) and Fig. S2-H). The ST in recovered groups was also larger than that of the control group (8.48 ± 7.16 µm). However, the difference in ST of right eyes among the control, myopic, and recovered groups was not significant. In the cartilaginous scleral layer, as shown in Fig. 7, the thickness in the myopic group (75.50 ± 8.09 µm) was thicker than that of the control (61.94 ± 4.52 µm) and recovered (58.39 ± 3.87 µm) groups, but the recovered group was thinner compared to the control group. The difference in cartilaginous scleral layer of right eyes among the control, myopic, and recovered groups was not significant. Interestingly, the thickness of the fibrous scleral layer in the recovered group (123.92 ± 4.72 µm) was significantly thicker than that of the control (111.89 ± 2.26 µm, P < 0.05) and myopic (109.00 ± 1.79 µm, P < 0.01) groups.
Fig. 7. OCT image of the retina, RPE, choroid, chorio-sclera-interface, cartilaginous layer, and fibrous layer and data analysis of cartilaginous and fibrous layers in normal, myopic, and recovering chick right eyes. (A) OCT intensity image of the retina, RPE, choroid, chorio-sclera-interface, cartilaginous layer, and fibrous layer. (B) comparison of the thickness of the cartilaginous scleral layer in control, myopic, and recovered chick eyes. (C) comparison of the thickness of the fibrous scleral layer in control, myopic, and recovered chick eyes. N = 3. NS: P > 0.05, *: P < 0.05, **: P < 0.01, ***: P < 0.001. RPE, retinal pigment endothelium. Ch, choroid. C-S-I, chorio-sclera-interface. CS, cartilaginous layer. FS, fibrous layer.
3.9 Axial length
AL was the sum of all ocular tissue thickness and chamber depths. In Fig. 6(I) and Fig. S1-I, AL in the control eyes was significantly smaller (1764.66 ± 163.53 µm) compared to the myopic eyes, and also significantly smaller (1663.58 ± 86.57 µm) compared to that in the recovered eyes (P < 0.001 for both). The difference in AL between right eyes in myopic and recovered eyes was not significant.
4. Discussion
In this study, SS-OCT was used to evaluate changes in ocular size and ocular tissue thicknesses in chicks in vivo non-invasively reared under visual conditions. To our knowledge, this is the first study to report the change of various ocular structures between the myopic and recovered eyes (unrestricted vision correction) in chicks using SS-OCT measurements. Our results showed that the CT of the right eyes (experimental eye) in the myopic group was thinner than in the control group. The similar conclusion has been demonstrated in human eyes that the CT became progressively thinner in the myopic and highly myopic eyes [75,76]. Because of the limited number of samples in this study, more samples are needed in future studies to further demonstrate if the thinning is significant. Interestingly, the CT of recovering chick eyes was significantly thicker than that of the myopic chick eyes. There were no relevant reports about the change of the CT after unrestricted vision correction. There was no significant difference for CT between the control and recovered eyes. This result suggested that CT returned to normal thickness during recovery from induced myopia.
We also demonstrated that ACD was significantly larger in the myopic eyes. This finding is in agreement with several studies on human eyes that demonstrated increased ACD in the myopic eyes as compared with the control eyes, and ACD tended to be larger with more myopic refractive error [77–79]. Our results also demonstrated that ACD in the recovered eyes somewhat reduced as compared with that of the myopic eyes, however, the ACD was still significantly larger compared to the control eyes. These results suggested that the increased ACD associated with form deprivation myopia began to return to a normal size during recovering period.
In the myopic group, the experimental eyes showed that the LT in the myopic eyes was larger than that in the control eyes. This result aligns with previously published data stating that LT in human eyes was larger in the myopic eyes [80]. Moreover, this result was similar to the data published by Y Guo et al. that the thicker lens and deeper anterior chamber existed in severer myopic eyes [81]. Additionally, our result demonstrated that the thickness of the lens altered to significantly smaller after recovery from myopia and the lens of the recovered eyes showed no significant thickness difference with that of the control eyes.
As expected, VCD was significantly increased in the myopic chick eyes as compared with the controls. This is in agreement with previously observed in human eyes [31,80,82,83] and in previous studies on form deprivation myopia in chicks and other animal models of myopia [84,85]. The VCD was also longer in the recovered eyes as compared with the controls, but shorter than in the myopic eyes. It has been previously demonstrated that upon removal of the occluder, the vitreous chamber growth rapidly decelerates to compensate for the imposed myopic defocus [86] and also as a result of choroidal thickening during recovery [87]. Since chicks in the recovered group experienced myopic defocus for five days, while eyes of chicks in the form-deprivation myopia group continued to elongate at an accelerated rate, this result is entirely expected.
In our results, the RT in the myopic eyes was significantly thinner than in the control eyes. This result was consistent with previous studies in human eyes that reported that the RT was significantly thinner in the myopic eyes at the inner and outer zones of macula than in non-myopic eyes [40,88,89]. Additionally, the previous study showed that the middle to the inner retina was the primary cause to result in retina thinning in the myopic eyes [90]. RT was also significantly thinner in the recovered eyes compared to the control eyes, similar to that of contralateral control eyes as indicated by histological analysis (Fig. 5(B) and Fig. 5(D)). These results suggested that the thinner retina of the myopic eyes may begin to increase somewhat during the recovery from induced myopia.
Our ocular measurements determined that the RPET of the myopic eyes was larger than that of the control eyes. A similar result was also demonstrated in a previous study on human myopic eyes where increased AL of myopic eyes was associated with a significant increase of superior RPET [91]. Additionally, studies on pig and human eyes demonstrated that the RPE cell density decreased with longer AL [90,92]. Thus, there might be a negative correlation between the RPE thickness and RPE cell density. Moreover, we found that the RPET of the recovered eyes was significantly larger than that of the myopic eyes and control eyes. This result was also supported by the histology in Fig. 5(B) and Fig. 5(D). There have been no related reports on RPET in the myopic and recovered eyes in chicks, possibly due to limitations in resolution of standard imaging systems currently used, such as high frequency A-scan ultrasonography.
ChT was significantly decreased in the myopic eyes as compared to that of the control eyes. This result is consistent with previous studies on human eyes and experimental animal models. It has been shown that ChT has a negative correlation with the degree of myopia and becomes markedly thinner in the myopic eyes [41,43,44,93,94]. This choroidal thinning can lead to myopic macular degeneration [47]. Importantly, after removal of the occluder, ChT of the recovered eyes was 2-3 times thicker than that of the control and myopic eyes which was also evident upon histological examination (Fig. 5(B) and 5(D)). Choroidal thickening during recovery from myopia has been previously shown to be robust in chicks and significant thickening can be presented within 3-7 recovering days after removing goggles [31,95]. It has been speculated that choroidal thickening in the recovered eyes is a rapid mechanism for moving the retina closer to the focal point as compensation of the imposed myopic defocus (choroidal accommodation) [87]. Additionally, the chorio-scleral-interface was showed within the choroid as reported by the previous study [96] and the relative location kept unchanged and closed to the lower surface of the choroid. This result demonstrated that the unrestricted vision did not change the position of chorio-scleral-interface within the choroid.
In the study, the ST of the myopic eyes was thicker than that of the control eyes, but the difference was not significant. Although scleral thinning is well-documented in human myopia and in mammalian models of myopia [84], the chick myopia showed a reversed condition and the thickening has not been reported in the chick sclera of the myopic eyes. We need to notice that the structural differences between the chick sclera and mammalian sclera, which consists of a cartilaginous layer and fibrous layer, while the mammalian sclera is exclusively cartilaginous [96,97]. In our results, the cartilaginous sclera layer became thicker in myopia and became thinner in the recovered group. However, the fibrous scleral layer in the recovered group became significantly thicker than that of the control and myopic groups. Therefore, the potentially internal balance of the cartilaginous and fibrous layers in the chick sclera may be responsible for maintaining nonsignificant change in the chick sclera after myopia and unrestricted vision recovery.
In this study, our results exhibited that a significant increase of AL in the myopic eyes compared to the control eyes, which was consistent with the previous reports that there was a significant thickening of the AL in myopic eyes [3,31,80,81,91,98], and the difference became greater with increased periods of form deprivation [78,98,99]. Furthermore, the AL of myopic eyes was positively correlated with the thickness of the ACD and VCD, but negatively correlated with the thickness of the retina and choroid. These results were in agreement with related reports that the AL was significantly correlated with the thicker central cornea, deeper anterior chamber, thicker lens, thinner retina, thinner sclera, and thinner choroid in myopia [3,24,77,81,85,89,99,100]. Additionally, the AL of recovered eyes demonstrated a similar trend of thickening with the thickness of RPE, choroid, and VCD, but an inverse change trend with the thickness of retina as compared to the control chick eyes.
In summary, we applied SS-OCT to non-invasively evaluate changes in ocular size and ocular tissue thickness in eight layers in chick eyes in vivo after myopia and unrestricted vision recovery. To our knowledge, the changes in ocular size and ocular tissue thickness of CT, RPE, and two scleral layers were measured for the first time, providing new insights for myopia studies. Particularly, we found that the CT of recovered chick eyes was significantly thicker than that of the myopic chick eyes. There were no relevant reports about the change of the CT after unrestricted vision recovery. We demonstrated that the RPET of the recovered eyes was significantly larger than that of the myopic eyes and control eyes. There were no previous reports on RPET in the myopic and recovered eyes in chicks. Although the entire chick sclera showed no significant difference between chick myopic and recovered eyes, the fibrous sclera layer was significantly thicker than that of the control and myopic chick eyes. There were no relevant reports demonstrated the internal change of the cartilaginous and fibrous layers after myopia and unrestricted vision recovery.
There are several limitations for this current study. Firstly, the chick number in the current study was limited. The limited sample size might cause relatively bigger statistical errors. To obtain more sufficient and significant statistical results, a larger size of animals will be used in future study. Secondly, raw data of OCT contained a large amount of speckle causing them to be grainy and limited contrast. To enhance the image quality of the chick ocular tissues from SS-OCT, the Despeckle [101,102] and Median filters [103,104] that were confirmed to restore speckled OCT images and remove background noises, were applied to adjust the intensity contrast of images to better exhibit the layer borders of different structures. Recently, the automatic layer segmentation and learnable despeckling/denoising have been reported to effectively distinguish retinal layers automatically and significantly improve the image quality [105,106]. Due to the limitation of the sample size for the training data, the automatic layer segmentation and learnable despeckling were not applied for the current study. Thirdly, the FOV in this study was limited and could be improved for deriving more effective data of the wider field of the entire eye in future studies. The wide-field OCT has been demonstrated to provide wide-field structure [107] and angiography for predicting eye diseases [108] and offer imaging of large-volume samples for other biomedical applications [109]. Besides, one limitation of this study is that the extent of the induced myopia was not measured, which could be used to correlate with the ocular structural changes in the next study.
5. Conclusion
In summary, for the first time, the present study demonstrates the application of high-resolution SS-OCT for in vivo measurements of ocular tissues and ocular chamber depths in chicks induced by form-deprivation or recovery from form deprivation. Our work proves that SS-OCT has the potential to be applied for diagnosing and monitoring of myopia development and drug evaluation in the future studies.
Funding
University of Oklahoma; National Institutes of Health (EY09391); National Cancer Institute (P30CA225520).
Acknowledgement
This work was funded by 2020 Junior Faculty Fellowship from University of Oklahoma (QG Tang), 2021 Faculty Investment Program from University of Oklahoma (QG Tang), and Startup Fund from University of Oklahoma (QG Tang). The authors thank Dr. Hong Liu, Xi Sun, Majood Haddad, and Meredith Jones for the help with the language and grammar polish. Research reported in this publication was supported in part by a Stephenson Cancer Center Trainee Research Award funded by the National Cancer Institute Cancer Center Support Grant P30CA225520 awarded to the University of Oklahoma Stephenson Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Disclosures
The authors declare no conflicts of interest.
Data Availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
Supplemental document
See Supplement 1 for supporting content.
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