Subretinal deposits induce choroidal new blood vessels to penetrate Bruch’s membrane

Abstract

Abstract: : Purpose: CNV in AMD is strongly associated with the existence of abnormal deposits of ECM (extracellular matrix) located between Bruch’s membrane and the RPE. At issue is whether the sub–RPE deposits and CNV are simply two independent manifestations of the same disease, or there is a causal relation between the two. The present work attempts to answer this question. Methods: An artificial ECM deposit was created in an adult Sprague–Dawley rat by injecting Matrigel or collagen gel to the subretinal space on the temporal side of an eye. The sclera was exposed and an incision made between the limbus and equator using a sharp #33 needle to reach the subretinal space. The injection needle (blunt #33) was introduced to the incision at a 5–10° angle toward the posterior pole and inserted 0.1–0.2 mm into the subretinal space. Matrigel (growth factor depleted) or collagen (rat tail collagen I) (1.2 µl) was injected slowly so that the material pushed its way into the subretinal space to create a bleb (1.5–2 mm in diameter). The needle site was on the anterior edge of the bleb. Animals were sacrificed 60–70 days after injection and processed with Vessel Paint to stain blood vessels. The anterior portion of an eye was removed and the eyecup was embedded in 5% agarose. Serial sections (100 µm) were cut on a vibratome and examined by fluorescence microscopy. Results: New blood vessels penetrating Bruch’s membrane in the posterior half of an artificial deposit were independent of injection–related trauma. Many such new blood vessels were found in the samples examined, indicating that ECM deposits were the original cause. Conclusions: These findings clearly demonstrate that ECM deposits are able to induce CNV to penetrate Bruch’s membrane and thus provide evidence for a direct causal relationship between ECM deposits and CNV development.

Keywords: age–related macular degeneration • choroid: neovascularization • Bruch's membrane