Microstructure and Network Organization of the Microvasculature in the Human Macula

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.

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Human Donor Eyes the different wavelengths, with emission signals separated into different 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 macular 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. The generation posterior chamber was immersion fixed in 4% paraformaldehyde over- scheme can evaluate the degree of asymmetry within vascular net- night. works using distributions of generation numbers in comparison with Flat Mounting of Retina the number of different order vessels. The posterior globe was dissected at the equator to allow viewing of Study of Detailed Intracellular the posterior retina. The macular region and the fovea were identified Microfilament Distribution by the anatomic orientation of the globe, yellow pigmentation around the fovea, and increased choroidal pigmentation in this region. The Optical sections of selected vascular segments were obtained at high retina was carefully dissected out without inclusion of the optic disc magnification to study the intracellular microfilament distribution in region. A few cuts were made to the peripheral retina to enable the detail. Projections of selected optical sections were made to demon- retina to lie flat. strate endothelial or smooth muscle cell microfilaments. Sketches of endothelium and smooth muscle cells were made to outline the cell Confocal Imaging shape and nucleus position. A camera equipped with three lasers (405, 488, and 532 nm; C1; Nikon, Tokyo, Japan) was used in conjunction with a microscope Statistical Analysis (E800; Nikon). Imaging was performed with the EZ-C1 software All statistical testing was performed using commercial software (Sig- (v.3.20; Nikon). Confocal imaging was performed simultaneously for maStat, v.3.1; SPSS, Chicago, IL). For the vascular topography param-

TABLE 1. Donor Eye Details and Time to Cannulation

Donor Age Time to Eye (y) Sex Eye Cause of Death Cannulation (h)

A 51 M L Myocardial infarction 9.5 B 61 M R Myocardial infarction 10 C 61 M L Myocardial infarction 10 D 53 M L Myocardial infarction 19 E 53 M R Myocardial infarction 19 F 66 M L Cardiac? 14 G 52 M L Myocardial infarction 17 H 78 M R Sepsis 20 I 27 M L Motor vehicle accident 13.5 J 28 M L Cardiac? 22 K 43 M R Collapse, unknown cause 18 L 43 M L Collapse, unknown cause 18 M 27 M L Motor vehicle accident 8 N 23 M L Suicide 22 O 51 M L Coronary 19 P 53 M L Myocardial infarction(?): Motor vehicle accident 14

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eters, Student t-testing was performed on all data that were normally network. The microvasculature network distribution appeared distributed. The Mann-Whitney rank sum test was performed if the random; however, some common properties in topographic normality test failed for the raw data pairs. The Spearman correlation distribution can be found. test was used to determine whether each of the measured parameters An arteriole was easily differentiated from a venule by its of endothelial cell and nuclei shape was correlated with vascular order thicker wall, which takes up stronger stain with a circular in the arterial or venous network. P Ͻ 0.05 was taken as significant. All pattern of smooth muscle cells. Arterioles also have slightly results are expressed as mean Ϯ SE unless otherwise stated. smaller diameters than the corresponding venules. It was also notable that the capillary free zone along the arteriole was less RESULTS apparent around the macular region than in the peripheral retina, as we previously reported.19 There was a one-to-one General relationship between a relatively large arteriole and a venule; the two were connected by a capillary plexus without the The results from this study demonstrated that when the central intervention of the shunt pathways present in other microcir- retinal artery was cannulated and perfused, the retinal micro- culatory systems.14 We carefully traced the vascular tree in all vasculature was well stained and the endothelial and smooth specimens but did not find any arteriolar-venular shunts in the muscle cells and nuclei of the retinal vasculature were clearly macular area. No evidence was found of a capillary arcade labeled. They allowed us to reliably obtain detailed information system, as seen in peripheral retinal vessels.21 Neither were of the microstructure and topography of the macular micro- 22 vasculature, superimposing anatomic terminology on fundus there connections between macular venules. Our results topography. suggest that there is no bypass between the macular arterioles and venules or connections between pairs of arterioles or Topography of Macular Microvasculature venules. Therefore, the hemodynamics of the macular vasculature appears to be mainly determined by the branching struc- Figure 1 shows a low-magnification projected confocal image tures. In general, the arterioles were more superficial than the from the macula region of the left eye of a 66-year-old donor. venules, as evidenced by the pseudocolored code properties The retinal microvasculature had been perfusion labeled for ϫ ␮ and the overlapped relationship. Numerous pairs of arterioles microfilament. The area imaged was 3180 3180 m square and venules were found in the macular area. The average and covered the foveola, fovea, and parafovea regions, indi- number of pairs of arterioles and venules was 8.9 Ϯ 0.23 (n ϭ cated by three concentric circles superimposed on the vascular 10) arranged in a radial pattern surrounding the foveola. Only a few arterioles (on average, 2.9 Ϯ 0.25; n ϭ 10) entered the foveal region, where they directly supplied the terminal capillary ring. The avascular region was surrounded by terminal capillaries, forming a terminal capillary ring that often had an irregular oval shape with a mean diameter of 362.3 Ϯ 49.7 ␮m (n ϭ 7) vertically and 410.8 Ϯ 80.7 ␮m(n ϭ 7) horizontally. However, there were no significant differences between the horizontal and vertical diameters (P ϭ 0.590). It was also evident that there were many bifurcations from each arteriole or venule, indicating that many generations oc- curred in a short segment length. Some arterioles and venules only reached the parafoveal or perifoveal region. Figure 2A shows a schematic of the nomenclature of vessel order and vessel generation. Figure 2B shows the nasal half of the macular region from the same eye shown in the area indicated by the white dashed line in Figure 1. The order was increased if two segments of equal order joined at a bifurcation (i.e., where two capillaries [c] joined together to form a first-order arteriole [a-1] and two a-1 arterioles joined together to form a second- order arteriole [a-2]). However, if we use the superior arteriole from 61-year-old donor B as an example, the generation (centrifugal) scheme started from the larger retinal arteriole (Fig. 2C) and proceeded to the capillary ring, increasing the generation by one at every branch point. Five generations in the parafoveal region and eight generations in the foveal region can be counted. FIGURE 1. Low-magnification confocal image from the macula region Higher order arterioles give off twigs of smaller branches of of the left eye of a 66-year-old donor. The retinal microvasculature had been perfusion labeled for microfilament. Optical sections were col- capillaries and arterioles that further bifurcate to form the lected through 120 ␮m of the retina at 4-␮m intervals. The area imaged capillary networks before converging on the venules. The measured 3180 by 3180 ␮m. The image stack was then split in half for average number of vessel generations in the macular area projection of two extended focus images that were pseudocolored imaged was 11.63 Ϯ 0.40 (n ϭ 32), and there were 3.56 Ϯ 0.14 (red for the superficial half, and green for the deeper half). The two (n ϭ 29) different orders of vessel. A significant asymmetry pseudocolored images were merged to give an overall impression of existed between the order and generation counts of vessels in the complete microvasculature at different depths. The retinal arte- the macular region (P Ͻ 0.001). The number of generations rioles (A) appeared to be more superficial than the venules (v), with was 0.85 Ϯ 0.04 per 100 ␮m(n ϭ 33) in the parafoveal region, few exceptions. Asterisk: center of the foveola. Dashed concentric 14 which was significantly less than that in the foveal region circles: foveola, fovea, and parafovea as defined by Hogan. The Ϯ ␮ ϭ Ͻ diameters of the three circles are 350, 1850, and 2850 ␮m, respec- (1.24 0.05 per 100 m; n 33; P 0.001). However, it was tively. The white and red dashed regions are shown at higher magni- also notable that the smaller arterioles were often seen as fication in subsequent figures. Scale bar, 300 ␮m. branches in the parafoveal region.

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FIGURE 2. Schematic description of vascular tree and a high-magnification confocal image of the nasal half of the macular microvasculature. (A) Sche- matic drawing showing the topological description of vascular trees using the Horton-Strahler and generation nomenclatures. The Horton-Strahler approach starts at the capillary (gray) level and proceeds centripe- tally. The order is increased if two segments of equal order join at a bifurcation. The generation (centrifugal) scheme starts from the most central vessel considered and proceeds to the capillary level, increasing the generation by one at every branch point. Drawing modified from Tuma RF, Duran WN, Ley K, eds. Hand- book of Physiology: Microcircula- tion. 2nd ed. Academic Press; 2008. (B) High-magnification confocal image of the nasal half of the macular region from Figure 1 showing arterioles (A), venules (v), and the subsequent branching of the retinal arteriole into smaller arterioles (a-2 and a-1) and capillaries (c). Two capillaries (c) join together to form a first-order arteriole (a-1), and two a-1 arterioles join together to form a second-order arteriole (a-2). The capillaries branching off the retinal arterioles are predominantly in the superficial half of the image stack (red) before connecting to the capillaries draining toward the retinal venules lying in the deeper half of the stack (green). Scale bar, 150 ␮m. (C) A projected image of a superior retinal arteriole traversing the parafoveal and foveal regions of the macula of a 61-year-old donor. Yellow lines: outer boundaries of the three zones as defined by Hogan.14 The retinal arteriole traversed the parafoveal region before entering the foveal region (top to the bottom) and stopped short of the foveola. Asterisk: point of branching used for counting the number of generations from this arteriole. Scale bar, 150 ␮m.

The capillaries that arose from the arterioles in the inner- cells. Although only one layer of smooth muscle cells may be most retina often were at right angles to the vessel of origin, seen encircling the a-1 arteriole, there is a denser presence of and the capillary meshes of the capillary plexus were rela- smooth muscle cell nuclei and processes seen in the a-3 arte- tively uniform with intercapillary anastomoses. The capillar- riole. In the larger retinal arteriole (a-4), smooth muscle cell ies branching off the retinal arteriole side were predominantly processes may be seen overlapping other smooth muscle cell in the upper half of the image stack (red pseudocolor) and then processes, suggesting more than one layer of smooth muscle connected to the capillaries draining toward the retinal venules cells in these larger retinal arterioles. In all orders of retinal lying in the deeper half of the stack (green pseudocolor; Figs. arterioles, the smooth muscle cells had a circumferential ar- 1, 2B). However, the capillaries were especially numerous in rangement that was perpendicular to the endothelial cell align- the perifoveal and parafoveal regions, diminished rapidly to- ment. ward the central macular region, and were absent in the fove- Figures 4A and 4B are the projected images from the optical ola. The spatial pattern and density of the macular microvas- stack of the inferior area of the macula, including part of the cular network therefore varied greatly in different regions of foveola, fovea, and parafoveal region. The FAZ is uppermost. the macula. Figure 4A shows the vascular pattern and distribution of the superficial capillary network in the inner half (vitreal side) of Microstructure of Macular Microvasculature the optical stack. The arteriole and both the side branches and Microstructural heterogeneity of the macular microvasculature the dichotomous branches came off the lateral surfaces of the was studied. Figures 3, 4, and 5 show the cell shape and arteriole and coursed horizontally in the superficial retina. intracellular structure of vascular endothelium and smooth Some branches then dipped into the deeper retinal tissue and muscle cells in arterioles, capillaries, and venules, respectively. anastomosed with the capillary plexus in the deeper network. Structural information of microvessels is essential for reliable The superficial capillary layer had a more three-dimensional determination of the orders and generations of different com- structure in comparison with the single-layer appearance of the ponents of the microvascular tree. deep capillaries. Individual capillaries in both networks were Figure 3 shows high-magnification confocal images of dif- relatively homogeneous and interconnected by anastomotic ferent orders of retinal arterioles (a-1 to a-4) labeled for micro- capillary bridges. There were, however, distinct differences in filament and counterstained using Hoechst to label the nuclei. vascular pattern and mesh distribution between the two net- Insets highlight the peripheral border microfilament staining of works. The deep capillary network appeared to be sparser than individual endothelial cells of the retinal arteriole. With increas- the superficial capillary network. Examination along the z-axis ing order of retinal arteriole (i.e., upstream) the shape of the of the confocal stack also highlighted the planar configuration endothelium and its nuclei became more slender. There was of the deep capillary network that was similar in morphology also an increasing presence of intracellular microfilament to a two-dimensional net (Fig. 4B). Figures 4C and 4D show (stress fiber) appearing as short and thick bundles that were high-magnification confocal images of individual capillaries more prominent with increasing order of arterioles and most sampled from the superficial and deep capillary network, re- prominent in a-4 vessels (blue arrows). Smooth muscle cells spectively, and insets highlighting the peripheral border stain- surrounding the endothelium were also present. With increasing of the endothelium. The capillary endothelium in both ing order of the retinal arteriole, there was a thickening and superficial and deep capillary networks have long peripheral widening of the microfilament bundle of the smooth muscle border microfilament distributions running in a longitudinal

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DISCUSSION The number of microvessels in the human body is extremely large, including approximately 2 ϫ 109 capillaries. The microvasculature topography and complexity varies in different or- gans, presumably reflecting the different structural and functional features of specific tissues. The macula is a unique functional and structural region of the retina. It is specialized for diurnal high-acuity vision that requires a high density of cone photoreceptors and a large number of inner retinal cells to participate in nonconverging circuits.16 The absence of large-diameter vessels from the macula underlines its important acuity function.23 The very center of the macula, the foveola, is presumably avascular to avoid compromising the light path to the high-density cone photoreceptor array. The present findings also indicate that specific conditions, or factors, gov- ern vessel diameter and branching patterns in the fovea and parafovea because we observed significantly different generations of vessels in these two regions. Indeed, antiangiogenic pigment epithelium-derived factor has been shown to be highly expressed in ganglion cells at the developing fovea and most likely plays a significant role in defining the foveal avascular zone and in regulating vessel growth around the fovea.24 The foveola also has a thinner inner retina, with many of the cells displaced into the foveal area. The high metabolic de- FIGURE 3. Confocal images of different orders of macular arterioles labeled for microfilament. Confocal images of different orders of retinal arterioles (a-1, a-2, a-3, and a-4) were labeled for AlexaFluor546 phalloidins (red) and counterstained using Hoechst to label the nuclei (blue) and were taken with a ϫ60 objective lens. Insets: highlighted peripheral border microfilament staining (outlined in yellow) of individual endothelium and smooth muscle cells (red arrowhead). With the increasing order of retinal arterioles (i.e., upstream), the shape of the endothelium and their nuclei became more slender. There was also an increasing presence of intracellular microfilaments, stress fibers, appearing as short and thick bundles that were more prominent with the increasing order of the arteriole and that were most prominent in a-4 vessels. Red arrowheads: microfilament of smooth muscle cells surrounding the endothelia. With the increasing order of the retinal arteriole, there was a thickening of the microfilament bundle of the smooth muscle cells. Although only one layer of smooth muscle cells may be seen encircling the a-1 arteriole, there is a denser presence of smooth muscle cell nuclei and processes seen in the a-3 arteriole. In the larger retinal arteriole (a-4), smooth muscle cell processes may be seen overlapping other smooth muscle cell processes, suggesting more than one layer of smooth muscle cells. In all orders of retinal arterioles, however, the smooth muscle cells had a circumferential arrangement perpendicular to the endothelial alignment. Scale bar, 50 ␮m.

fashion, indicating that capillaries have thin spindle-shaped capillary endothelial cells. Figure 5 shows high-magnification confocal images from different orders of retinal venules (v-1 to v-4), with further magnified insets highlighting the shape of the endothelium as indicated by peripheral border microfilament distribution. Ve- FIGURE 4. Confocal images of macular capillaries labeled for micro- nous endothelia displayed a slender morphology but were filament. (A, B) Projected images taken from the optical stack of the much shorter than arteriole endothelia (see Fig. 3), assuming a inferior region of the macula corresponding to the red marked region in Figure 1. The original image stack measured 62 ␮m in thickness and more rectangular shape in third-order vessels (v-3). 1270 ϫ 1270 ␮m in area. The superficial (A) and deeper (B) halves of Endothelial cell and nuclei lengths, widths, and aspect ra- the stack are shown separately. The superficial network contained tios are shown in Table 2 as a function of vessel order in the arterioles (a), venules (v), and capillaries, and the deep network con- arterial and venous system in the human macula. The Spear- tained only capillaries. There was a distinctive difference in the vascu- man correlation test revealed that endothelial cell length and lar pattern, mesh density, and presence or absence of larger vessels in nuclei length were correlated with vessel order in the arterial the two layers. The deep network had a mostly two-dimensional system in the macula (P Ͻ 0.05). In the venous system, the distribution and lower density of capillaries compared with the super- endothelial cell width and nuclei width were correlated with ficial network in which all the larger vessels were located, and the vessel order (P Ͻ 0.05). Conversely, endothelial cell width capillary distribution was more complex and had a higher density. ϭ ϭ (C, D) High-magnification confocal images of individual capillaries. (P 0.91) and nuclei width (P 0.61) were not correlated Insets: peripheral border staining of the endothelium. The capillary with vessel order in the arterial system, and endothelial cell endothelium in both the superficial (C) and the deep (D) capillaries length (P ϭ 0.35) and nuclei length (P ϭ 0.39) were not have a long peripheral border microfilament distribution (insets, correlated in the venous system. Endothelial and nuclei aspect dashed yellow outline) running in a longitudinal fashion. Scale bars: ratios displayed equivalent correlation properties. 200 ␮m(A, B); 50 ␮m(C, D).

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crovascular networks, particularly in the macular region, in both physiological and pathologic conditions. To gain such information, there are two fundamental factors, network topology and geometric irregularities, that affect network hemodynamics. Network topology investigates how individual vessel segments are connected to one another, whereas the geometry is determined by the lengths and diameters of the vessels. Based on these two fundamental factors, it becomes feasible to interpret many complex clinical phenomena. For example, modeling of the hemodynamic characteristics of the human macula may help us estimate the hemodynamic changes in the various orders in the macular microvasculature, such as perfusion pressure, blood flow, velocity, and hematocrit. Ultimately, we hope to be able to determine the influence of vascular changes on the perfusion of the macular area. We believe that knowledge of the vascular network topography and the geometry around the FAZ is a necessary first step to understanding macular perfusion. Although extensive studies have been performed in other vascular beds, the macular vasculature seems much less uniform. As a starting point, we have focused on the distribution and branch properties that can be reliably determined from such anatomic studies. Although considerable literature exists on this important and interesting subject, there is still no consensus about the FIGURE 5. Confocal images of different orders of macular venules basic relationships between the neural structure of the mac- labeled for microfilament. Confocal images from different orders of ula and its vascular supply.22,30 Studies in human eyes pro- macular venules (v-1, v-2, v-3, and v-4) with further magnified insets vide perhaps the most directly relevant information that is highlighting the shape of the endothelium, as indicated by peripheral valuable for understanding retinal pathology in human reti- border microfilament distribution. Microfilament has been labeled us- nal diseases. The purpose of the present study was to doc- ing AlexaFluor546 phalloidins (red) and cell nuclei counterstained using Hoechst (blue). The peripheral border microfilament staining ument the microstructure and network of the human mac- (yellow dashes) of individual endothelium and smooth muscle cells ular microvasculature. Our recently developed technique (red arrowheads). Unlike the macular arterioles, the venule endothe- allows us to study postmortem human eyes to investigate lium appeared to be shorter and more irregular with some spindle both spatial distribution and cellular structure in a great shapes (v-1, v-2, and v-4) or more rectangular (v-3), and there were no detail.19 An advantage of our preparation is that the entire intracellular stress fibers in macular venules. Few smooth muscle cells retinal microvasculature can be well fixed and stained. Here can be found in the macular venules. These cells were sparsely distrib- we focus on the macular microvasculature, which can be studied uted and circumferentially arranged with weak staining (v-1 and v-3). Scale bar, 50 ␮m. in detail in our flat mount preparation. We believe that this report provides some of the most detailed information regarding the macular microvasculature at the topographic and intracellular mand of the neural retina in the macula seems likely to demand level in human donor eyes. We have categorized the branching tight regulatory control to match local blood flow to tissue pattern of the macular microvasculature using the established requirements. It is generally believed that the retina has one of centripetal Horton-Strahler approach and compared this with highest metabolic demands per gram of tissue in the body.4,25 an analysis using the centrifugal generation scheme. This al- We and other groups5,26,27 have demonstrated that the inner lows an assessment of the degree of branching and the asym- segments of the photoreceptor and the inner and outer plexi- metry of the vascular network.20 The present study may be form layers have the highest metabolic demands within the seen as a first step in the analysis of capillary ensembles to retina. It is particularly interesting that cone photoreceptors obtain static and planar geometric properties of the macular have been suggested to have higher energy demands than rod microvasculature. photoreceptors.28,29 Our results clearly demonstrated that there are abundant It would be useful to know the detailed relationship be- blood vessels supplying the macular area, with approximately tween structural and hemodynamic heterogeneity of the mi- nine pairs of arterioles and venules distributed in the parafo-

TABLE 2. EC and ECN Dimensions According to Vessel Order

Aspect Ratio of Vessel EC Length EC Width EC Length and ECN Length ECN Width Order (␮m) (␮m) Width (␮m) (␮m)

A1 63.6 Ϯ 7.00 (4) 8.5 Ϯ 0.80 (4) 7.7 Ϯ 1.46 (4) 16.3 Ϯ 1.18 (4) 6.4 Ϯ 0.32 (4) A2 63.1 Ϯ 4.99 (12) 8.8 Ϯ 0.60 (12) 7.3 Ϯ 0.55 (12) 14.9 Ϯ 0.88 (12) 6.7 Ϯ 0.60 (12) A3 74.5 Ϯ 3.68 (21) 9.4 Ϯ 0.50 (21) 8.3 Ϯ 0.61 (21) 15.2 Ϯ 0.46 (20) 6.4 Ϯ 0.32 (20) A4 96.6 Ϯ 2.53 (14) 8.6 Ϯ 0.46 (14) 11.5 Ϯ 0.60 (14) 18.6 Ϯ 0.36 (14) 6.9 Ϯ 0.20 (14) V1 62.5 Ϯ 8.16 (6) 8.8 Ϯ 0.44 (6) 7.1 Ϯ 0.84 (6) 14.1 Ϯ 1.32 (6) 7.1 Ϯ 0.84 (6) V2 64.6 Ϯ 6.66 (7) 10.8 Ϯ 1.01 (7) 6.2 Ϯ 0.72 (7) 16.3 Ϯ 1.42 (7) 8.5 Ϯ 0.68 (7) V3 74.3 Ϯ 4.03 (26) 15.4 Ϯ 0.66 (26) 4.9 Ϯ 0.24 (26) 14.8 Ϯ 0.54 (26) 9.2 Ϯ 0.39 (26) V4 72.1 Ϯ 3.67 (24) 21.6 Ϯ 1.11 (24) 3.5 Ϯ 0.22 (24) 14.4 Ϯ 0.56 (24) 12.5 Ϯ 0.55 (24)

Values are mean Ϯ standard error (sample number). EC, endothelium; ECN, endothelium nuclei.

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veal regions. Rapid branching results in a relatively short dis- topological structures resulting from this approach are there- tance (few hundred microns) of capillary between the arteriole fore likely to be approximate but to contain some branch and venule. We have found that the arterioles in the macular properties. Generation numbers were assigned to the vessel area have a high number of generations (ϳ10) within a very segments on the basis of the number of bifurcations, reflecting short length (ϳ1.2 mm) but relatively few orders (3–4) when the topological structure of the network more accurately than compared with other vascular beds.31 There were typically order numbers. Most macular arterioles were found to have only two to four pairs of arterioles and venules found in the dichotomous branching structures that could be symmetric or foveola region supporting the terminal capillary ring. The venu- asymmetric. The present study sought to obtain information of lar and arteriolar portions of the macular vasculature were both order number and generation number for the macular closely associated. All these features create a high density of vasculature. capillaries in the parafoveal region and a lower density of The human donor eyes selected for this study were ob- capillaries in the fovea and foveal avascular zone. The capillary tained from donors with no previous history of ocular dis- network appears to be dense and three dimensional, particu- ease. The data obtained, therefore, are presumably repre- larly in the parafoveal area. sentative of the normal human eye. The retina lends itself The central role of the cardiovascular system is to maintain well to a study of vascular patterns. Perhaps no other tissue an adequate capillary flow.20 Perfusion and metabolic activity in the body can be removed with such a minimum of are heterogeneous and spatially correlated. It is difficult to dissection, providing a great opportunity for us to study examine the effects on flow distribution and oxygen level in macular microvasculature in intact macular tissue. The en- terminal vascular beds of inherent irregularity in structure, dothelial cells in the microvasculature of interest were ade- hemodynamic properties, and local variations in oxygen de- quately stained at all levels to reveal arterioles, venules, and mand.32 Mean macular capillary flow velocities have been capillaries at different depths through the macula. We were found to range from 1.37 to 3.3 mm/s.32–34 Apparently, mac- able to trace the blood vessels through the different orders ular capillary flow velocity is comparable to that in the brain, at various depths of the tissue from arteriole to venule. The which has been reported to range between 0.3 and 3.2 mm/s,35 quality of the images obtained is comparable with our pre- and higher than that in the mesentery, with a mean velocity of viously published data using fresh porcine and rat eyes.39–42 0.8 mm/s.20 Based on these flow velocity values and short The labeling of the intact microvasculature meant that en- lengths of capillaries in the macula, blood transit time in an dothelium and smooth muscle cells at different levels of the individual capillary would be well under 1 second. vasculature can be studied in the context of the surrounding To our knowledge, the intracellular cytoskeleton of human microenvironment. Intracellular structure such as endothe- macular microvasculature has not been reported before. lial f-actin and their arrangement at arteriole, venule, and Knowledge of the cellular structure of the macular vasculature capillary levels has been clearly demonstrated (Figs. 3–5). helps to reliably determine the topologic features by confirma- There are several limitations in the measuring of the shape tion of the vessel orders. It is also valuable for understanding of the endothelial cells, particularly in smaller vessels. Iden- macular physiology and pathology. tification of the endothelial cell border is dependent on Endothelial cells are subjected to an extremely dynamic peripheral border staining, which does not stain as strongly range of mechanical stresses, including fluid shear, hydrostatic as do other membrane stains. The endothelium is slender pressure, and cyclical stretch. Endothelial cells are sensitive to throughout the microvasculature and tends to curl around such stress and modify their cytoskeletal composition and the vascular lumen. Because the measurements were done architecture according to the different magnitude and type of on a two-dimensional projection of optical sections, it was stress experienced. The effect of shear stress on endothelial often difficult to find endothelium that has its whole cell cell morphology and function has been extensively studied in border within the plane of the image to allow accurate cultured cells under different shear stress conditions and in measurements. vivo at different regions where local blood flow patterns are Vascular smooth muscle cells have also been labeled with predictable.36–38 Those results indicate that under high fluid phalloidin perfusion because there is a large amount of f-actin shear stress, endothelial cells tend to elongate and orient them- present in these cells, enabling the study of smooth muscle cell selves along the flow direction. In addition, stress fibers of shape and arrangement at the different levels of the vascular actin filaments become more prominent and align along the tree. In addition to the roles of endothelial cytoskeleton we cell long axis. Endothelial cells undergo profound morphologic reported previously,39–42 there are a number of interesting changes in response to alterations in shear stresses that are features in the macular vasculature. First, a long spindle shape imposed on them by blood flow, and these responses have of the endothelial cells has been found in the both superficial important implications for physiologic and pathophysiologic and deep capillaries in the foveal and parafoveal regions. This function of blood vessels. may be related with their high exchange capability. Second, Our results suggest that there is reasonably high blood we were able to precisely define the relationship between the velocity and shear stress in the A-4 vessels, evidenced by the terminal arterioles, venules, and capillaries based on shape, long spindle shape of the endothelial cells and the presence of size, distribution, and structure of the endothelium and smooth intracellular stress fibers in the endothelium as previously re- muscle cells. Interestingly, the terminal arterioles are always ported in larger retinal arterioles.39–42 Our results indicate very close to each other and also close to the venules, thus there are some special features in the macular hemodynamics. minimizing the length of the capillaries. Perhaps this vascular Our growing knowledge of the roles and regulation of cytoskel- pattern is to provide sufficient capillary perfusion and high etal components in endothelium and further data from macular regulatory capability to adapt to functional demands of the hemodynamics may provide us with an improved understand- high density and functional requirements of the macular neu- ing of endothelial function in both health and disease. rons. Like most terminal vascular beds, macular vasculature is Our results demonstrated a high degree of structural expected to have a high degree of structural heterogeneity and heterogeneity and typically asymmetric and irregular distri- is typically asymmetric and irregular. The topological structure bution in the macular vasculature. Significant structural het- of the networks was analyzed using the Horton-Strahler tech- erogeneity is inevitable, particularly in the macular region, nique and a generation scheme. The Horton-Strahler nomen- because network structures must continually adapt to clature provides only a rather coarse classification, and the growth and changing functional demands. Local addition or

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