Retina Microstructure and Network Organization of the Microvasculature in the Human Macula Paula K. Yu,1,2 Chandrakumar Balaratnasingam,1,2 Stephen J. Cringle,1,2 Ian L. McAllister,1 Jan Provis,3 and Dao-Yi Yu1,2 PURPOSE. To characterize the topography and cellular structure gion, with high-resolution visual acuity and oxygen uptake in of the macular microvasculature using a recently developed the macula even higher than in the remaining retina.5 To technique of arterial cannulation, perfusion, fixation, and stain- maintain energy-dependent processes and to clear away meta- ing of human donor eyes. bolic byproducts produced by neuronal activity, a well-regu- METHODS. Sixteen human donor eyes were used. The central lated blood flow within the brain and retina is vital. The vascular and nervous systems communicate closely; when dys- retinal artery was cannulated and perfused with Ringer’s, then 6 fixative, membrane permeabilizing, and selected labeling solu- regulated, this may contribute to many important diseases. It has been demonstrated that even relatively small reductions in tions. The eyes were immersion fixed, and the retina was flat 7 mounted for confocal microscopy. The macular area, including blood flow can have deleterious effects. The human retina is the foveola, fovea, and parafovea, was sampled. The intracel- vulnerable to a wide range of retinal diseases with a vascular lular cytoskeleton of vascular endothelial and smooth muscle component and angiogenic ocular conditions, representing the cells was studied in different orders of arterioles and venules leading cause of irreversible vision loss in developed coun- tries.8 Such diseases include diabetic retinopathy, vascular oc- and in the capillaries. To evaluate the degree of asymmetry 9–13 within vascular networks, the distribution of generation num- clusion, and age-related macular degeneration. bers and the Horton-Strahler approach to vessel naming were The vulnerability of the retina presumably stems in part compared. from the need to limit the extent of retinal vasculature to allow a clear light path to the photoreceptors. The outer retinal RESULTS. The distribution of the microvascular network in the layers are completely avascular and are dependent on meta- macular region was complex but followed a general theme. bolic support by diffusion from the retinal and choroidal vas- The parafoveal region was supplied by dense vasculature with cular beds.5 approximately nine closely arranged pairs of arterioles and The macula lutea contains specialized regions, the bound- venules. Each arteriole had abundant branches and a high aries of which are not well defined. Perhaps the clearest divi- degree of asymmetry (ϳ10 generations and 3.5 orders within 14 ϳ sion of regions was developed by Hogan, who subdivided the 1.2-mm length). Only a few arterioles (average 2.9) supplied macular region into the foveola, fovea, parafovea, and perifo- the terminal capillary ring. Very long spindle endothelial cells veal area.14 The foveola corresponds approximately to the were seen in the superficial and deep capillaries. Significant foveal avascular zone (FAZ) and includes the base of the foveal heterogeneity of distribution and shape of the endothelial and pit, where there is a peak spatial density of cone photorecep- smooth muscle cells was evident in different orders of the tors, for high-acuity functions. It is surrounded by terminal macular vasculature. capillaries on the slope of the pit. In the parafoveal region, the CONCLUSIONS. The authors have demonstrated for the first time retina has a maximum thickness because of the high densities the cellular structure and topographic features of the macular of neural elements in all the retinal layers. Development of the microvasculature in human donor eyes. (Invest Ophthalmol retinal vasculature, particularly in the macular area, has been Vis Sci. 2010;51:6735–6743) DOI:10.1167/iovs.10-5415 studied in the primates and human.15–17 Much of the literature has attempted to understand the role of retinal vasculature in he energy demand of the retina on a per gram basis has macular function and diseases.18 However, substantial varia- Tbeen described as higher than that of the brain.1–4 The tion of topologic, morphologic, hemodynamic, and functional macula is recognized as the exquisitely specialized retinal re- parameters in the retinal vascular network makes it difficult to understand and describe the properties and behavior of the vasculature, particularly in the macular area. There is still a lack From the 1Centre for Ophthalmology and Visual Science, and the of information about the structure and function of the vascular 2ARC Centre of Excellence in Vision Science, The University of West- endothelium and smooth muscle cells in the macular micro- 3 ern Australia, Perth, Australia; and the ANU Medical School and ARC vasculature and no consensus on vascular network topogra- Centre of Excellence in Vision Science, The Australian National Uni- phy. Fortunately, our recently developed technique19 allows us versity, Canberra, Australia. Supported by the National Health and Medical Research Council to detail the spatial distribution of retinal microvasculature and its relationship to neurons and glial cells at the cellular level in of Australia and the Australian Research Council Centre of Excellence 19 in Vision Science. human donor eyes. In the present study, we focus on the Submitted for publication February 22, 2010; revised April 30 and microstructure and network distribution of microvasculature June 22, 2010; accepted July 1, 2010. in the macular area. Disclosure: P.K. Yu, None; C. Balaratnasingam, None; S.J. Cringle, None; I.L. McAllister, None; J. Provis, None; D.-Y. Yu, None MATERIALS AND METHODS Corresponding author: Dao-Yi Yu, Centre for Ophthalmology and Visual Science and the ARC Centre of Excellence in Vision Science, The This study was approved by the human research ethics committee at University of Western Australia, Nedlands, Western Australia 6009; the University of Western Australia. All human tissue was handled [email protected]. according to the tenets of the Declaration of Helsinki. Investigative Ophthalmology & Visual Science, December 2010, Vol. 51, No. 12 Copyright © Association for Research in Vision and Ophthalmology 6735 Downloaded from tvst.arvojournals.org on 09/26/2021 6736 Yu et al. IOVS, December 2010, Vol. 51, No. 12 Human Donor Eyes the different wavelengths, with emission signals separated into differ- ent channels. Imaging began at low magnification. Specific regions A total of 16 human eyes from 13 postmortem donors were used. All were examined in using higher power objective lenses (ϫ40, ϫ60 plan eyes were obtained from the Lions Eye Bank of Western Australia or apochromatic oil lenses) for detailed imaging. Z-series were taken Donate West, the West Australian agency for organ donation. Nine eyes through a depth of 120 ␮m using a ϫ4 objective lens and up to 90 ␮m were received after the removal of corneal buttons for transplantation. using a ϫ10 objective lens to obtain three-dimensional information on None of the donors had a known history of eye disease. Demographic retinal microvascular architecture and to study labeled structures in the data, cause of death, and postmortem time to eye perfusion for each vascular endothelium and smooth muscle cells. donor are presented in Table 1. In this study we selected eyes that did not have a foveal capillary crossing the foveola to ensure that we were Topography Study characterizing the more common situation in which a distinct foveal avascular zone is present.19 A low-magnification (ϫ2) epifluorescent image was taken of the mac- ular region. Then using a ϫ4 objective lens (Plan Apo; Carl Zeiss, Preparations Oberkochen, Germany), an optical stack was collected in an area measuring 3180 ϫ 3180 ␮m square. The macular microvasculature was Details of the method of perfusion staining of retinal microvasculature studied in different regions, as defined by Hogan.14 The foveola was have been published previously.19 Briefly, the central retinal artery was designated to extend out to a radius of 175 ␮m from its center. The cannulated, and residual blood was washed out with oxygenated Ringer’s foveal zone extended out a further 750 ␮m, with the outer boundary solution with 1% bovine serum albumin. After the 20-minute Ringer’s delineated by a circle of 925 ␮m radius. The parafoveal area was wash, 4% paraformaldehyde in 0.1 M phosphate buffer was perfused designated to extend a further 500 ␮m, to lie within a circle of 1425 for at least 1 hour for fixation. An aldehyde-based detergent, 0.1% ␮m radius. Vessel orders were defined according to the convergence Triton-X-100 in 0.1 M phosphate-buffered solution, was then perfused of smaller branches. Levels of branching were defined as generations in for 5 to 7 minutes to aid in the permeation of endothelial cell mem- the vascular tree. Topologic description of trees by the Horton-Strahler branes. The detergent was washed out by a further 30 minutes of 0.1 and generation nomenclatures was performed in this study.20 The M phosphate buffer perfusion. Then the microfilament and cell nuclei Horton-Strahler scheme starts at the capillary level and proceeds cen- were labeled over the course of 2 hours using a mixture of phalloidin tripetally. The order is increased if two segments of equal order join at conjugated to Alexa Fluor 546 (30 U; A22283; Invitrogen, Carlsbad, a bifurcation. The aim is to group vessels with similar characteristics CA) and cell culture reagent (bisbenzimide H 33258; 1.2 ␮g/mL; into one order. The generation (centrifugal) scheme starts from the Sigma-Aldrich, St. Louis, MO). Residual label was cleared from the most central vessel considered and proceeds to the capillary level, vasculature by further perfusion of 0.1 M phosphate buffer, and the increasing the generation by one at every branch point.