Effect of vascular endothelial growth factor 165 on dopamine level in the retinas of guinea pigs with form-deprivation myopia

Experimental animals

A total of 90 healthy 3-week-old pigmented guinea pigs (male or female, 150–200 g) were purchased from Jinan Jinfeng Laboratory Animal Corporation randomly divided into six groups of 15 pigs in each group. The light environment of the animal was approximately a 12 h day/12 h night cycle, and the room temperature was maintained at 22–25 °C at 50–70% humidity. All animals had free access to food and water. The study was approved by the ethics committee of the Affiliated Hospital of Binzhou Medical University (approval number: 20220128-78), and the feeding and use of experimental animals strictly followed the relevant regulations of the animal management committee of Binzhou Medical University. All guinea pigs were euthanized with intraperitoneal injection of three times anesthetic dose (1% Pentobarbital sodium 120 mg/kg). During the experimental period in which disease signs (including weakness in the extremities, lethargy, lack of food intake, or unexpected infection) were detected in FDM models, 1% Pentobarbital sodium 120 mg/kg was given to guinea pigs for deep anesthesia, and then guinea pigs were euthanized.

Induction of form-deprivation myopia and injections

A white translucent nontoxic latex balloon was placed on the head of each guinea pig in a manner that it only covers the right eye of the guinea pig and fully exposes the left eye, mouth, nose, and ears. The balloons were washed once a day to keep the hood clean. An FDM guinea pig myopia model was established by continuous masking for 14 d.

Recombinant human protein VEGF165 (100-20; PEPROTECH, Cranbury, NJ, USA) was dissolved in sterile water for long-term storage at a concentration of 1 ng/µL in a −80 °C freezer. Stock solutions were diluted with phosphate buffer saline (PBS) solution immediately before use. Before FDM treatment, PBS solution (2 µL) and recombinant human protein VEGF165 (1, 5, and 10 ng VEGF165 were dissolved in 2 µL PBS solution) were injected into the vitreous of the right eye of each guinea pig. Tropicamide phenylephrine eye drops were used 20 min before surgery to open the pupils. After the intraperitoneal injection of anesthetics (1% Pentobarbital sodium 40 mg/kg), the eye areas and ocular surfaces of the guinea pigs were fully disinfected. The eyeball was fixed with microscopic toothed forceps under the microscope. The microsyringe was injected vertically downward 1 mm behind the temporal limbus, and the needle was tilted 20° toward the eyeball wall to avoid the lens. The microsyringe was further advanced about 3 mm, and the drug was injected into the vitreous. The needle hole was gently clamped with forceps for 30 s after injection. Ofloxacin eye ointment and sterile gauze dressing were applied after the operation. All experiments were conducted by experienced investigators.

Ocular measurements

Ocular refraction and AL were recorded in both eyes of each animal before and 2 weeks after treatment. All measurements were carried out under minimal lighting after facemask removal. Before a measurement, tropicamide phenylephrine eye drops were applied to the right eye, and refraction was measured by a streak retinoscope (YZ24; 66 Vision-Tech, Suzhou, China) after the pupil was fully dilated. The equivalent spherical lens (spherical degree + 1/2 cylindrical lens degree) was used for data analysis, and the average value of five measurements was used as the refractive value.

Propmecaine hydrochloride eye drops were applied to the right eye before the length of the eye axis was measured and after anesthesia was applied on the surface of the cornea. Ultrasound (AVISO; Quantel Medical, Cournon-d’Auvergne, France) was used to measure the length of the eye axis when the probe was aimed at the center of the cornea and perpendicular to the plane of the cornea. The length of the eye axis of the right eye was continuously measured five times in manual mode, and the average value was calculated to an accuracy of 0.01 mm.

HPLC analysis

All guinea pigs were sacrificed via euthanasia. The removed eyeball was placed on ice, and retinas were harvested within 30 s under a dissecting microscope, immediately frozen in liquid nitrogen, and stored at −80 °C until all the tissue samples were collected. Each milligram of frozen sample was added to 20 µL of freshly prepared homogenate (0.1 M H₃ClO₄, 0.1 mM EDTA Na₂, and internal standard DHBA) and frozen at −40 °C. After low-temperature centrifugation (20,000 rpm, 30 min, 4 °C), the supernatant was collected and tested on the machine. An HPLC system (Thermo UniMate 3000 Pump, flow rate 0.2 ml/min, ESA Coulochem III. Electrochemical Detector, 5041 Cell voltage 350 mV, Guard Cell voltage 360 mV, Rang 100 nA, Filter 10S; Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the levels of DA and DOPAC. The column was Thermo Acclaim rapid separation liquid chromatography (2.1 mm × 100 mm; C18, 2.2 µm; column temperature, 40 °C). The mobile phase contained NaH₂PO₄ (90 mM), citric acid (50 mM), OSA (1.7 mM), EDTA (50 µM), and acetonitrile (4.5%). Data were acquired and analyzed using a Chromeleon 6.9 chromatography workstation, and target concentrations were calculated using internal standards.

Immunofluorescence analysis

The right eyeball was removed on ice, and the excess connective tissue and extraocular muscles were removed under the microscope to preserve the optic nerve and maintained the integrity of the eyeball. For immunofluorescence, the eyes were fixed in Bouin’s fixative solution (PH0976; Scientific Phygene, Fuzhou, China) for 24 h and then transferred to a 20% sucrose solution. After 12 h, 30% sucrose solution was added. The solution was left to stand for 12 h at 4 °C. The anterior segments of the eyes were removed, and the eyecups were embedded in optimum cutting temperature media (Order Number 4583; SAKURA, Torrance, CA, USA) and then frozen at −80 °C for long-term storage. The frozen eyecups were sectioned vertically on a freezing microtome (CM1950; Leica, Wetzlar, Germany) at a thickness of 8 µm. The tissue sections were adsorbed on viscous slides, treated with acetone at 4 °C, washed three times with PBS solution, and rinsed three times with 0.3% Trition X-100. Retinal sections were blocked with 5% albumin bovine (Biosharp, Anhuim, China) in a 37 °C incubator for 30 min. The sections were then incubated with primary antibodies (TH, 1:100; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by appropriate fluorophore-conjugated secondary antibodies (1:500; Abcam, Cambridge, UK) for 1 h at 37 °C in the protected state. After thorough washes, each sample was gently mounted with a cover glass, observed the number of TH-positive cells under an upright fluorescence microscope (BX51+DP72; Olympus, Tokyo, Japan), and photographed. The number of TH-positive cells were counted using a fluorescence microscope under a ×20 objective. To assess the number of TH-positive cells under immunofluorescence microscopy, positive cells of the photomicrographs obtained from retinal sections of different experimental groups were measured. Data obtained in six cross sections were averaged for one sample, and six samples were collected for each differently manipulated group.

Western blot analysis

All guinea pig eyeballs were removed, and the retinas were harvested and weighed. RIPA lysis buffer (Biosharp, Anhui, China) and protease inhibitor cocktail (MedChem Express, Monmouth Junction, NJ, USA) was added to the tissues, and an ultrasonic crusher was used to further fragment and homogenize the tissues. The samples were centrifuged at 12,000 r/min for 30 min in a 4 °C centrifuge (TGL-20R; Shan Dong BaiO Medical technology CO., LTD., Shandong, China), and the supernatant was collected. The concentration was quantified using a BCA protein assay kit (Biosharp, Anhui, China). All the samples were boiled with 5× loading buffer at 95 °C for 10 min. From each group of retinal tissues, 20 µg of protein homogenate was extracted and added to 10% sodium dodecyl sulfate polyacrylamide gel for electrophoresis and transferred to 0.45 mm-thick polyvinylidene fluoride membrane. Nonspecific membrane binding was blocked with 5% BSA for 2 h at room temperature. The membranes were incubated with the primary antibodies anti-TH (1:1,000; Cell Signaling Technology, Danvers, MA, USA), anti-CD31 (1:1,000; Abcam, Cambridge, UK), and GAPDH (1:10,000) overnight at 4 °C. After the membranes were washed with Tris-buffered saline containing 0.1% Tween-20, they were incubated against the secondary antibody (goat anti-rabbit 1:10,000) for 2 h at room temperature. TH and CD31Protein bands were visualized using a ChemiScope Capture Image acquisition software (ChemiScope 6200 Touch; Clinx Science Instruments Co., Ltd., Shanghai, China), and ImageJ was used to analyze the optical density. Each experiment was repeated three times.

Hematoxylin and eosin (HE) staining

Frozen retinal tissues were cut into 8 µm sections. The retinal sections were fixed by acetone at 4 °C and then washed with 1% PBS for 5 min three times. All the samples were stained with hematoxylin and eosin (HE), dehydrated with ethanol and xylene, and sealed with neutral glue. The number of vascular endothelial nuclei distributed within the retinal boundary membrane were counted using a microscope under ×20 objectives. Six samples were randomly selected from each differently manipulated group and statistically analyzed after calculating the mean number of vascular endothelial cell nuclei.

Statistical analysis

The statistical software SPSS 25.0 was used for statistical analysis. All data were confirmed by Shapiro–Wilk test to be normally distributed and expressed as mean ± SD. The between-group data were homogeneous with variance according to the Levene’s test. One-way ANOVA was used for the overall comparison of the data in each group. LSD-t test was performed to compare the groups. P < 0.05 indicated statistical significance.

Refraction error and ocular parameters

Before the experiment, all guinea pigs in each group showed hyperopia in both eyes. No significant difference in refraction or AL was found between the right and left eyes of each animal. After 2 weeks of FDM treatment, the FDM eyes showed significant ocular AL elongation and myopic shift (refraction error, blank eye vs. FDM eye, +2.417D vs. −3.083D, AL, blank eye vs. FDM eye, 7.710 mm vs. 8.120 mm, P < 0.05) (Figs. 1A and 1B).

To evaluate the efficacy of the three concentrations of recombinant human VEGF165 in attenuating progressing myopia, PBS buffer and 1, 5, and 10 ng of recombinant human protein VEGF165 were injected intravitreal into the eyes of the FDM guinea pigs. The groups with intravitreal injection of recombinant human VEGF165 had significantly shorter AL than the groups with intravitreal injection of PBS buffer and FDM (Fig. 1D). Moreover the refractive error of the three VEGF groups were significantly hyperopic compared with that of the PBS buffer and FDM groups (Fig. 1C). Moreover, 1 ng of recombinant human VEGF165 was the most effective in attenuating myopia progression in terms of AL elongation and refractive error.

Expression of DA and DOPAC levels in the retina

We first explored whether the retinal DA levels in the eyes form deprived for 2 weeks were reduced in guinea pigs. The levels of DA and DOPAC in the retina of the guinea pigs in the FDM group were significantly lower than those in the normal eyes according to the HPLC assay, and the differences were statistically significant (P < 0.05). Three different concentrations of VEGF165 were injected into the vitreous of the guinea pigs. The most significant increases in the levels of DA and DOPAC were observed in the retinas of the guinea pigs in the 1 ng group, followed by those in the 5 ng groups, and those of the 10 ng groups had the least significant increases (Figs. 2A and 2B). No significant difference in DOPAC/DA (DA metabolic rate) in the retina was found among all groups (P > 0.05) (Fig. 2C).

Number of TH⁺ cells in immunofluorescence

Given that TH is a rate-limiting enzyme for DA synthesis, we counted the number of TH⁺ cells in the retina to reflect the amount of DA. The immunofluorescence results showed that the expression of TH⁺ cells in the retina was green punctate fluorescence. TH⁺ cells were located in the plane of the inner nuclear layer, where the somata of DA amacrine cells are located (Fig. 3A). The results of pairwise comparison showed that the number of TH⁺ cells in the FDM group was significantly lower than that in the blank group. The number of TH⁺ cells in the FDM group was not significantly different from that in the PBS group (P > 0.05), and the number of TH⁺ cells increased in the 1, 5, and 10 ng groups. The 1 ng group had the highest number of TH⁺ cells. Therefore, the highest expression of TH⁺ was achieved through treatment with a low concentration of VEGF165 (Fig. 3B).

TH/CD31 protein expression level

Western blot analysis was performed to assess retinal TH protein and CD31 protein expression levels after treatment with three groups of VEGF165 at different concentrations. Band of approximately 55 and 130 kDa, corresponding to the molecular weights of TH and CD31, were detected in the retinal protein extracts. The results of Image J gray value analysis showed that TH protein expression was significantly reduced in the FDM group compared with the blank group. DA secretion was reduced in FDM. The protein detection results of VEGF165 at different concentrations showed that TH expression increased in the three groups, consistent with the above experimental results, and demonstrated the inhibitory effect of VEGF165 on FDM. The highest efficacy was observed in the 1 ng group, and VEGF165 had the most significant effect at low concentrations (Figs. 4A and 4C).

CD31, as a specific marker on vascular endothelial cells, was used in detecting the promoting effect of the intravitreal injection of VEGF165 on neovascularization in the retina. Densitometric analysis did not reveal significant difference in CD31 protein expression level among the three experimental groups (blank, FDM, and PBS). By contrast, in the three groups with different concentrations of VEGF165, the gray value analysis results showed that the expression of CD31 protein increased. The 10 ng group had the highest expression level, whereas and 1 ng group had the lowest. The results indicated that the expression of CD31 protein is positively correlated with the amount of VEGF165 (Figs. 4B and 4D).

HE staining result of the retina

The results of HE staining of retinal sections showed that the retinas of the guinea pigs in the blank, FDM, and PBS group were neatly arranged, and no or few vascular endothelial cells were distributed in the inner boundary membrane (Fig. 5A). The results of HE staining in the three groups with different concentrations of VEGF165 showed increase in number of vascular endothelial cell nuclei in the inner boundary membrane and the number of vascular endothelial cell nuclei increased with the VEGF165 dose. Compared with the blank group, the FDM and PBS groups showed no significant difference in the number of vascular endothelial cell nuclei in the retina (P > 0.05), and the number of vascular endothelial cell nuclei in the retina in the 1, 5, and 10 ng groups increased significantly compared with that in the blank group. The differences were statistically significant (all P < 0.05). The number of vascular endothelial nuclei in the 10 ng group was the highest, and the number of vascular endothelial nuclei in the 5 ng group was higher than that in the 1 ng group (Fig. 5B).