Animals
A total of 136 pigmented guinea pigs (3 weeks old) from the Beijing Keyu Animal Centre (Beijing, China) were raised on a daily cycle of 12-h illumination and 12-h darkness during the experimental period (on at 8:00 AM, off at 8:00 PM), with free access to water and food supplemented with vitamin C. Form-deprivation myopia was induced by the animals wearing a latex face mask (Suzhou, China) on the right eye. The animals were monitored 3 times during the 12-hour light period to ensure that the face masks were in place and were promptly changed to a suitable size when necessary. These face masks were made by latices, which were opaque and soft with light transmission of 60%. The face masks did not contact the cornea, which allowed the right eyes to blink freely. The animal research in this study was approved by the Animal Care and Ethics Committee at Tian Jin Eye Hospital, Tianjin, China. The treatment and care of the animals complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Experimental Design and Groups
The animals wore the face mask for 21 days to induce myopia. Then, the face masks were removed, and the guinea pigs were re-exposed to the normal visual environment. Ninety-six guinea pigs were divided into four groups: the recovery group (REC, n=24), the REC plus SP600125 group (REC-SP, n=24), the REC plus anisomycin group (REC-AN, n=24) and the REC plus DMSO group (vehicle) (REC-DM, n=24). SP600125 (Abcam, Cambridge, MA, USA) and anisomycin (Abcam, Cambridge, MA, USA) were injected into the REC-SP group and the REC-AN group, respectively, at the same concentration and volume (0.2 nmol, 0.01 ml). SP600125, which is an inhibitor of JNK, and anisomycin, which is an agonist of JNK, were both dissolved in DMSO and diluted with PBS to their final concentrations. In the REC-DM group, the form-deprived eyes were injected with sterilized DMSO solution (0.01 ml, diluted with PBS), which is the carrier. In the REC group, the guinea pigs were raised without any injection after the face mask was removed. Moreover, the normal control group (NC) included 32 guinea pigs that were free of form deprivation and were left untreated.
All intravitreal injections were performed once using a microinjector (Shanghai Meter Glass Factory, Shanghai, China) with a 30-gauge needle at 1.5 mm posterior to the temporal limbus under anesthesia with 2.5 g/kg chloral hydrate (i.p.) when the face masks were removed. The animals in the four experimental groups were divided into the following three subgroups (n=8, each), based on the length of exposure to the normal environment: 1) the 3d group (3 days of exposure) the 7d group (7 days of exposure), and 3) the 10d group (10 days of exposure). The normal control group was also divided into three subgroups (n=8, each) corresponding to the time of the experimental group. We also added two groups in this experiment: first, the form deprivation for 21 days (FD 21d) group (n=8); second, a normal control for 21 days (NC 21d) group (n=8) corresponding to the time of the FD 21d group. The randomly assigned experimental animal groups are shown in Table 1.
Table 1. Experimental animal group classification (n=8 in each group)
Groups
|
Time after removal of deprivation (or the corresponding time in the NC group)
|
Time of deprivation
|
Intravitreal injection
|
3 days
|
7 days
|
10 days
|
21 days
|
|
REC
|
REC 3d
|
REC 7d
|
REC 10d
|
|
|
REC-SP
|
REC-SP 3d
|
REC-SP 7d
|
REC-SP 10d
|
|
SP600125
|
REC-AN
|
REC-AN 3d
|
REC-AN 7d
|
REC-AN 10d
|
|
anisomycin
|
REC-DM
|
REC-DM 3d
|
REC-DM 7d
|
REC-DM 10d
|
|
DMSO
|
FD
|
|
|
|
FD 21d
|
|
NC
|
NC 3d
|
NC 7d
|
NC 10d
|
NC 21d
|
|
REC: recovery; REC-SP: REC plus SP600125; REC-AN: REC plus anisomycin; REC-DM: REC plus DMSO; NC: normal control.
Refractive Error and Axial Length (AL) Measurements
All animals underwent biometric measurements (refractive error and AL) at the end of each FD and recovery period on the experimental days. Biometric measurement was performed by a research optometrist who was masked to the treatment group assignment with the help of an animal care assistant.
Refractive errors were measured by hand-held streak retinoscopy on awake, behaving animals. The measurement was performed one hour after instillation of 1 or 2 drops of tropicamide phenylephrine eye drops (Santen Pharmaceutical Co., Ltd., Osaka, Japan) to achieve cycloplegia in a dark room on the experimental days. All refractive error data presented refer to the spherical equivalent (averages of results for the two principal meridians) refraction. Three replicate measures of the same eye were recorded with the mean used for statistical analysis.
Ocular axial length was measured by high frequency A-scan ultrasonography (10-MHz, Suoer, Tianjin, China) on the same day following refraction measurements. Topical cornea anesthesia was carried out by administering a drop of 0.5% proparacaine hydrochloride (Alcon, Puurs, Belgium) 2 min prior to the ultrasound measurement. The ultrasound probe had direct contact with the cornea during the measurement. The average of 8 measurements was adopted.
Measurement of choroidal thickness
With the SD RTVue OCT (Optovue XR-100-2, Fremont, CA, USA), the scan patterns were the retina cross line mode, which consists of 2 orthogonally oriented 9-mm lines consisting of 1024 A-scans after the animals were anaesthetized with 2.5 g/kg chloral hydrate (i.p.). CT was defined as the distance from the outermost layer of the retinal pigment epithelium (RPE) to the inner scleral border, presumed to be the chorioscleral interface [13], using the caliper system provided by the software. The choroidal thicknesses at 3 mm from the optic disc of 18 radial lines were measured manually by two independent researchers. The mean overall CT was obtained by calculating the average values of the CT measurements at all eccentricities. If the line was blurred, especially for the chorioscleral interface and RPE interface, the center of the line was traced and measured. Measurements by the first examiner were performed three times to evaluate the intraobserver variability. All measurements were performed at the same time of the day (between 5:00 pm and 9:00 pm) in all guinea pigs to avoid the effect of diurnal CT variation on the results [14]
Collection of Choroid Samples
Tissue Isolation
At the end of form deprivation and each recovery period, the guinea pigs were terminally euthanized with 2.5 g/kg chloral hydrate (i.p.), and the right eyes in all animals were enucleated. Using a surgical microscope (Zeiss, American) and razor blade, we cut the eyes of the guinea pigs perpendicular to the anteroposterior axis and approximately 1 mm posterior to the ora serrata on the ice plate. The anterior segment of the eye was discarded. For isolation of the choroid, the retina and RPE were removed as a sheet with a small spatula, and then, the choroid was carefully removed from the underlying sclera. Occasionally, small bits of RPE adhered to the choroid, and these pieces were removed by gentle brushing with a brush and rinsing with PBS. The tissues were stored in liquid nitrogen at −80°C until use.
Western Blot Analysis
Total protein was extracted from the frozen choroids of the eye with ice-cold extraction buffer as well as protease inhibitors. Protein was separated on a 10% resolving gel and transferred to cellulose acetate membrane (Millipore). Then, these membranes were blocked with 5% nonfat dry milk in 0.1% Tween 20 for 2 hours at room temperature. Afterwards, these membranes were cultured overnight with a primary antibody against p-JNK 1 (Abcam, Cambridge, MA, USA) and AQP-1 (Abcam, Cambridge, MA, USA) at a 1:500 dilution at 4°C in the blocking buffer. Furthermore, these membranes were washed with 0.1% Tween 20 and then treated with goat anti-rabbit IgG conjugated to alkaline phosphatase (1: 2000) for 1 hour at 37°C. The filters were stripped and reprobed for GAPDH normalization. The controls for nonspecific binding lacked primary antibodies. A film scanner (Image Master VDS; Amersham Biosciences, Inc., Piscataway, NJ) was used to scan the films.
Statistical Analysis
The statistical analyses were performed using version 17.0 SPSS software (SPSS, Inc., Chicago, IL, USA). All data are reported as the mean ± standard deviation (SD) with a 95% confidence interval. Paired sample t-tests were used to compare the biometric data, which had a normal distribution,
between the experimental and normal control eyes, while data without a normal distribution were assessed by the Mann–Whitney U test. One-way analyses of variance (ANOVA) with a Bonferroni correction was performed for comparisons among the different groups. A p<0.05 was considered significant.