**Background:** Myopia is a highly prevalent ophthalmic disease projected to affect nearly 50% of the global population by 2050, with high myopia being a major cause of blindness. Atropine is the most effective evidence-based pharmacological agent for slowing myopia progression, but its mechanism remains debated. Vasoactive intestinal peptide (VIP) is a neuropeptide expressed in the retina and visual cortex, involved in visual development and potentially in myopia. VIP is linked to dopamine signaling, which is known to regulate axial elongation. However, the effect of atropine on retinal VIP expression had not been previously reported. This study hypothesized that VIP plays a role in myopia formation and that atropine may control myopia by modulating retinal VIP levels.
**Methods:** Thirty 3-week-old pigmented guinea pigs were randomly divided into three groups (n=10 each): normal control (NC, no intervention), monocularly form-deprived (FDM, right eye covered with a semi-transparent latex balloon), and FDM treated with 1% atropine sulfate eye gel daily (FDM+AT). Refractive error (by streak retinoscopy after cycloplegia with 0.5% tropicamide) and axial length (by A-scan ultrasonography) were measured at 0, 2, and 4 weeks. After 4 weeks, eyeballs were enucleated, fixed, paraffin-embedded, and sectioned. Hematoxylin and eosin (H&E) staining was used to assess histopathological changes in the retina, choroid, and sclera. Immunohistochemistry (IHC) and in situ hybridization (ISH) were performed to evaluate VIP protein and mRNA expression, respectively, with mean optical density quantified using Image-Pro Plus. Statistical analysis used one-way ANOVA with LSD post-hoc test and independent t-test; p<0.05 was considered significant.
**Key Results:** At baseline, there were no significant differences in refractive error (F=0.015, P>0.05) or axial length (F=0.054, P>0.05) among groups. After 4 weeks, the NC group showed a shift from hyperopia to emmetropia (mean refractive error: 0.850±1.385 D; axial length: 7.890±0.084 mm). The FDM group developed significant myopia (-2.450±0.956 D) and axial elongation (8.272±0.087 mm) compared to NC (both P<0.001). The FDM+AT group had significantly less myopia (-0.025±1.502 D) and shorter axial length (7.921±0.107 mm) than FDM (both P<0.01), and did not differ significantly from NC (P>0.05). H&E staining revealed that in the FDM group, retinal ganglion cells and inner/outer nuclear layers were disorganized; choroidal thickness (ChT) was reduced (50.614680±3.497180 μm vs. 75.584831±4.061680 μm in NC, P<0.001); scleral thickness was thinner (127.918865±9.967032 μm vs. 205.891324±9.525299 μm in NC, P<0.001); and choroidal blood vessel lumen and area were decreased. In the FDM+AT group, ChT was significantly increased (98.436654±4.388368 μm) compared to both NC and FDM (P<0.001), and scleral thickness (201.452932±9.200460 μm) was similar to NC (P>0.05). IHC and ISH showed that VIP protein and mRNA expression were significantly upregulated in the FDM group (mean optical density: IHC 0.157880±0.042568, ISH 0.032904±0.009690) compared to NC (IHC 0.038271±0.012142, ISH 0.013041±0.002360; both P<0.01). In the FDM+AT group, VIP expression (IHC 0.039503±0.009082, ISH 0.013807±0.001666) was significantly lower than FDM (P<0.001) and not significantly different from NC (P>0.05). VIP signals were mainly localized in the nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segment.
**Clinical Implications:** This study provides the first evidence that atropine treatment reduces retinal VIP expression in a form-deprivation myopia model, suggesting that the VIP signaling pathway may be a key molecular mechanism for atropine's anti-myopia effect. The findings support the involvement of VIP in myopia pathogenesis, as its upregulation during form deprivation correlates with myopic shifts and structural changes. The ability of atropine to normalize VIP levels, along with its effects on choroidal and scleral morphology, highlights VIP as a potential therapeutic target for myopia control. However, the study has limitations: it used a single high dose (1%) of atropine, did not include an internal control (unaffected eye), and relied on histopathological processing that may affect retinal thickness measurements. Future research should explore dose-response relationships, use in vivo imaging (e.g., OCT angiography) to monitor choroidal blood flow, and investigate the interaction between atropine, VIP, and dopamine pathways in both the eye and visual cortex.