Three-dimensional quantification of the spatiotemporal co-evolution of vascular and neuronal networks within intact eyes

Retinopathy of prematurity (ROP) is a disease that affects blood vessel development and distribution in the eyes of infants born prematurely resulting in vision impairment and loss. While many techniques exist for investigating this disease, a common method used to investigate structure-function relationships in ROP is serial sectioning followed by two-dimensional image analysis. Often, serial sectioning is fraught with inconsistencies due to tissue tearing and folding which may introduce optical artifacts during imaging. Of note are the errors that occur when multiple physical sections are computationally reconstructed to quantify the spatial location of fluorescent labels within the original three-dimensional tissue. In this work we utilize passive CLARITY technique PACT, which renders tissue optically transparent through the establishment of a monomer hydrogel matrix and removal of light scattering lipids to generate optically transparent eyes. To measure fluorescent labels within these intact eyes, we have used a newly developed and unique digital scanned light sheet microscope (DSLM) specifically designed to quantify fluorescently labeled signaling molecules and structures within PACT treated samples. These two techniques combined provide a methodology to quantify the three-dimensional distribution of key signaling molecules and structures during development of the eye, bypassing issues inherent in serial sectioning, two-dimensional imaging and computational reconstruction. Here, we report quantification of the vascular and neuronal network structures in intact control and endotoxin diseased model rat eyes. We provide a comparative analysis evaluating both two-dimensional and three-dimensional imaging techniques and find that network features in developing eyes are more accurately quantified using our three-dimensional imaging approach.