Modified technique of whole retinal capillary network mounting for cells and capillaries counting

Abstract

Background Previous retinal vascular mounting only obtained part of retinal vessels for the study of retinal diseases,and thus it is difficult to comprehensively assess these diseases.So optimizing the trypsin digestion method to show the complete retinal capillary network is very important for the study of retinal diseases.Objective This study was to modify the preparing way of trypsin digested whole retinal capillary network and offer a basis for a quantitative analysis of cells and capillaries.Methods Thirty Wistar rats were divided into model group (20 rats) and control group (10 rats).Streptozotocinum(STZ) of 1.25％ dissolved in 0.05 mmol/L sodium citratehydrochloric acid buffer was intraperitoneally injected to establish diabetes models in the model group,and the equal volume of solvent was used in the same way in the control group.Eight months after injection,100 ml PBS was injected via ventriculus sinister and released via cut right atrium,and then 100 ml 4％ paraformaldehyde was injected into the ventriculus sinister.The rat retinas containing part of the optic nerve were entirely isolated,and digested by trypsin,and vitreous,inner limiting membrane and neural retinal tissue were removed.The whole retinal capillaries network was mounted on the slide.The ghost pericytes and acellular capillaries in the middle retinal area were identified and counted under the optical microscope.Results The retinal mount exhibited that the retinal vessels were stained by hematoxylin and periodic acid schiff.The vessels network presented with the entire type in shape with the radical central retinal arteries and veins and their branches.The capillary showed the shallow-and deep-layer networks between the small arteries and veins.Pericytes distributed and protruded vessel wall and formed the ghost cells without nuclei.The diameter of acellular capillaries was 20％ or more than that of near capillaries,and no cellular nuclei or ghost cell was found through the vessel.Conclusions The experimental technique for setting-up of cleaned vasculature and mounting vessels on glass microscopic slide provides intact vessels,which is helpful for the evaluation of retinal vascular morphology and quantitative analysis. Key words: Retina; Trypsin digestion method; Rat/Wistar; Retinal vessels/cytology