3.1 SE and ocular biometric parameters of EM, non-HM and HM groups
As is shown in Figure 1, the SE of the HM group was significantly lower than those of the EM and non-HM groups (P<0.001) (Figure 1A). Since axial myopia accounts for most cases of myopia, AL was measured in our study. The AL of the non-HM group was longer than that of the EM group, whereas the AL of the HM group was the longest among the three groups (P<0.001) (Figure 1B). However, only the ACD of the HM group was significantly higher than those of the EM and non-HM groups (P<0.05) (Figure 1C). As an important parameter for myopia, VCD was detected and displayed similar results: HM>non-HM>EM (P<0.001) (Figure 1D). Meanwhile, AL/CR (Figure 1E) and VCD/AL (Figure 1F) also showed similar tendencies, i.e., HM>non-HM>EM (P<0.001).
3.2 Alterations in choroid thickness and choriocapillaris vessel density in the EM, non-HM and HM groups
Figure 2A was the choroidal thickness map detected by OCTA. The results obtained via OCTA (Figure 2B) showed that pCTs and pCTi of HM were significantly less than those of the non-HM and EM groups (P < 0.01). Moreover, the pCTt of the three groups displayed an HM<non-HM<EM pattern (P<0.01). The pCTn of the HM group was also less than that of the EM group (P< 0.05). The SFCT of the HM group was significantly less than those of the non-HM and EM groups (P < 0.01).
Figure 3A,3B was the choriocapillaris vessel density map detected by OCTA. Similar results were discovered in all macular measurements; that is, macular choriocapillaris vessel density in the HM group was significantly less than those in the non-HM and EM groups (P<0.01) (Figure 3C). On the other hand, as Figure 3D shown: PCVDc in the HM group was lower than that in the EM group (P < 0.05). There was no significant difference in PCVDs among the three groups. PCVDi in the HM and non-HM groups was significantly lower than that in the EM group (P<0.05). PCVDt and PCVDn in the HM group were lower than those in the non-HM and EM groups (P < 0.001).
3.3Alterations in the levels of VEGFA, VEGFB, VEGFC and PlGF in the tears of the EM, non-HM and HM groups
The levels of VEGFA, VEGFB, VEGFC and PlGF were measured via ELISA. As shown in Figure 4A, VEGFA level demonstrated significant differences among the groups: HM<non-HM<EM (P<0.001). There was no significant difference in the level of VEGFB among the three groups. The level of VEGFC in the HM group was lower than those in the EM and HM groups (P<0.05). The level of PlGF in the EM group was higher than those in the HM and non-HM groups (P< 0.01). In addition, analysis of the correlation revealed a negative correlation between VEGFA levels and AL (Figure 4B), whereas VEGFA levels were positively correlated with SE (Figure 4C). Therefore, VEGFA levels may be related to the degree of myopia.
3.4 Alterations in SE and AL in the FDM guinea pig group
There were no significant differences in SE and AL between the control and FDM groups before form deprivation (0 w) (P>0.05). However, 4 weeks after modeling, SE in the FDM group was significantly lower than that in the control group (P<0.001) (Figure 5A), whereas AL in the FDM group was much longer than that in the control group (P<0.001) (Figure 5B). Moreover, the alterations in the SE and AL of the FDM group were greater than those of the control group (P < 0.001) (Figure 5C, 5D).
3.5 Change in the choroidal vessel in the FDM group
Choroidal sections dissected from the eyeballs of guinea pigs were labeled with α-SMA before being observed with a confocal microscope (Figure 6A-6F). Then, the vascular area fraction score was obtained via ImageJ, which was used to reflect the vascular density in the choroid. As shown in Figure 6G, the vascular area fraction score in the FDM group was significantly less than that in the control group (p < 0.001), which suggested less vascular density in the choroid after onset of myopia in the FDM group.
3.6 Alterations in VEGFA expression in the FDM group
The expression of VEGFA in the choroid was assessed by immunohistochemistry (n = 3 per group) and Western blotting (n = 6 per group). As shown in Figure 7A-7D, VEGFA was expressed in the cytoplasm of endothelial cells in the choroidal vessels. Meanwhile, Western blotting was applied to detect alterations in the expression of VEGFA in the choroid, which revealed lower expression of VEGFA in FDM guinea pigs than in the control group (P<0.01) (Figure 7E, 7F).
3.7 The effects of conbercept on SE, AL and choroidal vessels in the FDM group
After form deprivation for 4 w and 6 w, there were significant differences in SE, AL, and the change in SE and AL between the FDM and control groups (P < 0.001). After form deprivation for 4 weeks, conbercept was injected intravitreally into the guinea pigs. Two weeks after the injection, it was observed that SE was significantly lower in the FDM + conbercept group than those in the control and FDM + vehicle groups (P<0.001) (Figure 8A), and AL was significantly longer in the FDM + conbercept group than those in the control and FDM + vehicle groups (P<0.001) (Figure 8B). Alterations in the changes in SE and AL in the FDM + conbercept group compared with the FDM + vehicle and control groups were also observed (P<0.001) (Figure 8C,8D). On the other hand, the vascular area fraction score was shown in Figure 8E, which represented the choroidal vascular density of the FDM + conbercept group was less than those of the other two groups, and the FDM + vehicle group showed less choroidal vascular density than the control group (P<0.001) (Figure 8F-8N). Therefore, intravitreal injection of conbercept may aggravate myopia and reduce vascular density in the choroid.