Morphologic and functional changes in the retina and sclera induced by form deprivation high myopia in guinea pigs

Our aim was to study the morphologic and functional changes in the retina and sclera induced by form deprivation high myopia (FDHM) in guinea pigs and explore the possible mechanisms FDHM formation. Forty 3-weeks-old guinea pigs were randomized into the blank control (Group I, 20 cases) and model groups (20 cases). In the model group, the right eyes of the guinea pigs were sutured 8 weeks to induce FDHM (Group II) and the left eyes were considered a self-control group (Group III). The diopters were measured with retinoscopy. The anterior chamber depth (AC), lens thickness (L), vitreous depth (V) and axial length (AL) were measured using ultrasonometry A. Retinal and scleral morphology and ultrastructural features were observed with light and electron microscopy. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the retina and sclera were detected with a chemical colorimetric assay. deviation (`x±s). Comparisons between Group I and Group II were performed using variance analysis. Comparisons between Group II and Group III were performed using paired sample t tests. A value of  < 0.05 was considered statistically significant between the groups.

2 Abstract Background Our aim was to study the morphologic and functional changes in the retina and sclera induced by form deprivation high myopia (FDHM) in guinea pigs and explore the possible mechanisms FDHM formation.

Methods
Forty 3-weeks-old guinea pigs were randomized into the blank control (Group I, 20 cases) and model groups (20 cases). In the model group, the right eyes of the guinea pigs were sutured 8 weeks to induce FDHM (Group II) and the left eyes were considered a self-control group (Group III). The diopters were measured with retinoscopy. The anterior chamber depth (AC), lens thickness (L), vitreous depth (V) and axial length (AL) were measured using ultrasonometry A. Retinal and scleral morphology and ultrastructural features were observed with light and electron microscopy. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the retina and sclera were detected with a chemical colorimetric assay.

Results
After remaining stitched for eight weeks, the diopters of Group II changed from (+3.59±0.33) D to (-7.96±0.55) D, and these values were significantly higher than those of Group I (+0.89±0.32) D and Group III (-0.55±0.49) D. The vitreous chamber depth (4.12±0.13) mm and axial length (8.93±0.22) mm of Group II were significantly longer than that of Group I (3.71±0.23) mm and (7.95±0.37) mm, respectively and Group III (3.93±0.04) mm and (8.01±0.15) mm, respectively (P < 0.05). With the prolongation of form deprivation, the retina and scleral tissue became thinner, the ganglion cell and inner and outer nuclear layers of the retina became decreased, and the arrangement was disordered.
In Group II, the activity of SOD was significantly lower than that in Group I and Group III, and the content of MDA was significantly higher in Group II than in Group I and Group III. The differences were statistically significant (P < 0.05).

Conclusions
These findings suggested that in the FDHM guinea pigs model, the diopter, the vitreous chamber 3 depth, and axial length increased significantly with prolongation of monocular FD time, and morphological structural changes in the retina and sclera were observed. Oxygen free radicals might participate in the formation of FDHM.

Background
Myopia is a global epidemic ametropia caused by a combination of genetic and environmental factors [1]. Worldwide, the number of people with high myopia (HM) is about 163 million, accounting for 2.7% of the total population. It is estimated that by 2050, the number of people with HM will increase to 938 million, accounting for 9.8% of the total population. The number of people with HM in China is 87 million, accounting for 6.3% of the total population. It is estimated that by 2050, the number of people with HM will exceed 175 million, accounting for 13% of the total population [2]. Surveys have shown that the prevalence of HM in Chinese adolescents is 6.69-38.4%; that is, China is a typical country with a high HM incidence [3,4]. With myopic diopters increasing, especially up to HM, the incidence of fundus lesions, risk of blindness and medical costs also increase [5]. It has been reported that pathological myopia (PM) is an important cause of global vision loss, and the prevalence of PM is 0.9 to 3.1%. The prevalence of PM associated with visual impairment in European countries is 0.1% to 0.5%, and the value in Asian countries is high, ranging from 0.2% to 1.4%. Studies in China have shown that PM is the most common cause of blindness, accounting for 26.1% of the cases [6]. At present, the occurrence of myopia tends to be higher in younger people and is in a more advanced stage. HM fundus lesions, especially macular degeneration, are the main causes of blindness in East Asian countries [7].
The form deprivation myopia (FDM) theory was founded in 1977. After Wiesel et al. [8] sutured the eyelids of newborn rhesus monkeys, the sutured eyes caused obvious axial myopia. According to the research needs, some scholars have carried out myopia research on the eyes of animals such as tree shrews [9,10], chickens [11], mice [12], rats [13], and guinea pigs [14]. Studies have confirmed that form deprivation (FD) in young animals leads to abnormal growth of the eye axis and formation of obvious myopia [15,16]. Guinea pigs are widely used in the current research on myopia because the eyeball structure and emmetropization mechanism is similar to that of human myopia. The mechanism of HM formation is very complicated, and the local retinal regulation mechanism is a focus of research. This study intended to establish a guinea pig FDM model to simulate HM, observe changes in retinal and scleral morphology, determine the content of malondialdehyde (MDA) in the retina and sclera, and assess the activity of superoxide dismutase (SOD). The relationship between oxygen free radicals and the formation of form deprivation high myopia (FDHM) provides new ideas and a basis for further research on myopia, especially HM.

Animal Model and Grouping
Forty pigmented guinea pigs at two weeks of age weighing 100 g to 140 g were obtained from Beijing

Main equipment and reagents
A strip light inspection mirror was purchased from Suzhou Liuliu Vision Technology Co. Ltd. A lens box was purchased from Danyang Medical Instrument Factory. A type A ophthalmic ultrasound instrument (ODM-1000) was purchased from Tianjin Maida Medical Technology Co. Ltd. SOD (KGT001100) and an MDA (KGT004) test kit were purchased from Beijing Benuowei Biotechnology Co. Ltd.

Biological measurements
All guinea pigs were treated with mydriatic optometry before and after FD for eight weeks. Before FD, tropicamide eye drops were used to fully enlarge the pupil and the ciliary muscle was paralyzed. The optometry was performed in a dark room with a band-shaped photoreceptor. Each eye was measured three times to obtain an average value. The astigmatism degree was transformed into half of the count and this was included in the spherical mirror. Surface anesthesia was performed with 0.4% oxybuprocaine hydrochloride eye drops. The length of each component of the eyeball was measured with ultrasonometry A. The probe of ultrasonometry A was perpendicular to the cornea and located in the center of the pupil. Meanwhile, the cornea was not pressed. When the pattern was stable and clear, the posterior capsule of the lens and the double peak of the retina were higher than the baseline, and the image and results were recorded. Each eye was repeatedly measured 8 times to obtain an average value. Recorded data included the anterior chamber depth (AC), lens thickness (L), vitreous chamber depth (V) and axial length (AL).

Light microscopy Specimen Preparation
After FD for eight weeks, guinea pigs were anesthetized with an overdose intraperitoneal injection of 1% pentobarbital sodium (100 mg/kg). Five eyeballs of each group were removed with the optic nerve 1 to 2 mm long and fixed in 4% paraformaldehyde (Solarbio, Beijing, China). The eyecups were made by cutting the eyeball along the limbus and removing the anterior segment, and then the eyecups were fixed again in 4% paraformaldehyde at 4 ℃ for 24 hours. After gradient alcohol dehydration and xylene transparent and paraffin machine embedding, the sclera and retina were continuously sectioned along the longitudinal axis. Sections had a thickness of 4 μm and were stained with haematoxylin-eosin (HE). A microphotographic system (OLYMPUS BH-2, Japan) was used to observe the histology and morphology of the sclera and retina.

Electron microscopy Specimen Preparation
Fives eyeballs in each group were fixed in 2.5% glutaraldehyde solution (Solarbio, Beijing) at 4 ℃ for 72 hours. First, the sclera and retina were sliced to a 2mm × 2 mm size and washed three times with phosphate buffer (PBS) for 10 minutes each time. Second, the eyeball samples were fixed in 1% citric 6 acid for two hours and rinsed twice with double distilled water for 10 minutes each time. Third, ethanol acetone was used to dehydrate stepwise. Fourth, the samples were infiltrated and embedded with Epon812 epoxy resin. Then, the slice was cut into a 10 nm sample and double stained with uranyl acetate and lead citrate. Finally, a Hitachi H-600 transmission electron microscope was used to observe the sclera and retina ultrastructure and take photos.

Colorimetric detection of the MDA content and SOD activity
After FD for eight weeks, ten guinea pigs in each group were selected and then the eyeballs were removed after the same administration of anesthesia as described above. After removing the anterior segment of the eye on an ice cube, the sclera and retina were peeled off with a microscopic tweezer and put into a cryotube. The wet weight was weighed with a precision analytical balance, and doubledistilled water was added to make a homogenate. The homogenate was centrifuged at 3000 r/min for 5 minutes, and the supernatant was taken for testing. According to the SOD vitality test kit instructions, we used a 96-well plate to set the sample well and the blank control well. We then added the sample to be tested and other various solutions in turn, and we added the reaction start working solution, mixed it well, and incubated it at 37 °C for 30 minutes. We measured the absorbance at 450 nm, and calculated the SOD vitality according to the formula in the instruction manual. In reference to the MDA kit standard tube absorbance, we took 0.1 ml as the standard. The standard tube absorbance minus the standard blank tube absorbance was 0.103-0.112. We calculated the MDA content according to the formula in the manual. After the experiment, the guinea pigs were euthanized by increasing an anesthetic dose pentobarbital sodium (50mg/kg ip). We have concerned for the welfare of animals and our method of euthanasia followed the AVMA Guidelines for the Euthanasia of Animals.

Statistical Analysis
SPSS statistics 22.0 software was used to analyze the results. Data were recorded as the mean ± standard deviation (`x±s). Comparisons between Group I and Group II were performed using variance analysis. Comparisons between Group II and Group III were performed using paired sample t tests. A value of < 0.05 was considered statistically significant between the groups. 7

Changes in the refractive status of guinea pig eyeballs
The guinea pigs were born with hyperopia. The diopters of the 3-week-old guinea pigs were about +3.50D. Table 1 shows that there was no significant difference in the diopters among the groups before FD (P > 0.05). After receiving FD for eight weeks in the right eyes, the myopic diopters of Group II were significantly increased compared with Group I (P < 0.05). The diopter of Group II was higher than that of Group III and the difference was statistically significant (P < 0.05).

Ultrasonometry A measurement results and statistics
After FD, the vitreous cavity of group II was deepened and the axial length of the eye was longer.
Compared with group I, the difference was statistically significant (P < 0.01). The depth of the vitreous cavity and the axis of the right eye in the guinea pigs in group III were compared. The length was less than in group II, and the difference was also statistically significant (P = 0.000). There was no significant difference in the depth of the anterior chamber and the thickness of the lens among the groups before and after FD (P > 0.05; Table 2). The retinal structure of group I was clear, the thickness of the ganglion cell layer was normal, the monolayer was distributed, the nucleus was round, the nucleolus was clear and neatly arranged, there was no cell loss or deformation, and the retinal pigment epithelium (RPE) included the visual cells. The outer segment protruded as small protrusions, arranged in a brush-like shape and it was neatly dense (Fig. 1A). After FD for eight weeks, the retinas of group II became thinner, the ganglion cell and inner and outer nuclear layers were all reduced, the nucleus was small and uneven, and the cells were arranged in a disorderly manner. The microvilli protruding from the RPE layer were shorter and even fused to break (Fig. 1B). The retinas of group III showed only a decrease in ganglion cells, a small nucleus, and clear structures in the remaining layers (Fig. 1C).

Observation of guinea pig retinas under an electron microscope
Under an electron microscope, the retinal disks of group I were arranged neatly and tightly. The nuclear layer of cell membranes was smooth and completed with uniform chromatin. Compared with group I, the retinal disks of group II showed swelling and deformation, irregular contraction of the inner and outer nuclear layers and RPE cell membrane, irregular accumulation of the nuclear chromatin, mitochondrial swelling, deformation and vacuolization (Fig. 2, Fig. 3).

Observation of guinea pig sclera under a light microscope
The scleral thickness of group I was normal. The fibroblasts were stained blue-purple and were fusiform or oblong, the extracellular matrix was pink, the collagen fibers were neatly arranged, and the diameters were uniform (Fig. 4A). The sclera of the guinea pigs in the group II had obviously become thin, the fibroblast cell nuclei were distorted, the collagen fibers were sparsely distributed, the diameter was obviously reduced, the arrangement was disordered, twisted, broken, and separated, the interfiber space was increased, and the extracellular matrix was increased (Fig. 4B).
The scleral thickness of the group III was slightly thinner, but the morphology was not significantly abnormal (Fig. 4C).

Observation of guinea pig sclera under an electron microscope
The scleral tissue was composed of fibroblasts and collagen bundles parallel to the wall of the eyeball.
The collagen fibers constitute the framework of the scleral tissue. The cross-sectional diameter of the 9 collagen fibers was observed. There was no significant difference between the sclera of group I and group III. Compared with the two groups, the diameter of the FDHM was significantly reduced and the fiber density was decreased (Fig. 5).

MDA content and SOD activity in the retinas and sclera of guinea pigs
The statistical results showed that the activity of SOD and MDA, the main products of lipid peroxide, had an opposite trend. That is to say, under normal circumstances, the SOD of the retina and sclera remained high in the normal group, while MDA was at a low level. However, after FD, compared with Group I, the content of SOD in the retinas and sclera of guinea pigs in Group II decreased, while the content of MDA increased (P < 0.01). There was no significant difference compared with Group III (P > 0.05; Table 3, 4).

Discussion
Since the refractive status and orthotopic mechanism of guinea pigs are similar to that of humans, guinea pigs are born with hyperopia. In three weeks, the axial length of the guinea pigs grows rapidly and the diopter decreases. The refractive state of 11-week-old guinea pigs is stabilized in the mild hyperopic eye. The FDM model has been widely used in experimental research on myopia [17,18].
Studies have shown that, after FD, the cornea is flattened, the lens is thickened, the anterior chamber is deepened, the axial length is increased, and the diopter is decreased [19]. In this study, 3-week-old guinea pigs were selected as a myopic animal model. At this time, guinea pigs were in a critical period of visual development and were sensitive to the model manipulations. By prolonging the time of FD, the aim was to cause high myopia [20]. The retina is a tissue with a high oxygen demand, strong metabolism and active redox reaction. Under physiological conditions, the intraocular tissues, such as the retina, are continuously stimulated by external light to cause photooxidation, which promotes the formation of oxygen free radicals. At the same time, effective antioxidants, such as SOD, in the cells can promptly scavenge oxygen free radicals. The formation and degradation are in a dynamic equilibrium, which provides protection for tissues such as the retina and sclera. Under pathological conditions, excessive generation of oxygen free radicals or insufficient antioxidant capacity of the body can trigger lipid peroxidation, and its main metabolite, MDA, causes damage to cells and tissues. The content of MDA reflects the degree of attack on oxygen by oxygen cells. SOD is an important antioxidant enzyme in the body, and its activity reflects the body's ability to scavenge oxygen free radicals [21,22].
The results of this study showed that, after eight weeks of FD due to guinea pig eyelid suture, the myopic diopter decreased, with the highest at -8.00D refraction, and the biometric data of Ultrasonometry A increased, in which the vitreous cavity was elongated and the axial length became longer. Compared with Group I, the difference was statistically significant (P < 0.01), which indicated that the high myopia model was successful. There was no significant difference in all parameters between Group II and the Group III (both P > 0.05). Deprivation in Group II did not interfere with the natural development of the contralateral eye, which is consistent with the findings of other researchers such as Zhao [23].

Conclusions
In summary, this experiment produced a HM status in the guinea pigs FD for eight weeks. We

Consent for publication
Not applicable.

Availability of data and material
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request at any time.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by the National Natural Science Foundation of China (No. 81574029). The funders had no role in the design or conduct of this study or in writing the manuscript.

Authors' contributions
YX Z and MJ designed the study, analyzed the data and wrote the manuscript. YX Z, YD, JR Z, and MQ J performed the animal study. YL Q and TT D revised and made suggestions to the manuscript. All authors read and approved the final manuscript.
14 Figure 1 Observation of light microscopic observation of guinea pig retina (HE×400) Figure 2 Observation of electron microscopic observation of guinea pig retina membrane (×4000) Figure 3 Observation of electron microscopic observation of guinea pig retinal inner nuclear layer (×4000) Figure 4 Sclera morphological structure changes in guinea pigs of each group.

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.