Neural-Vascular Relationships in Central Retina of Macaque Monkeys (Macaca fascicularis)

Transcription

1 The Journal of Neuroscience, April 1992, 12(4): 1169-l 193 Neural-Vascular Relaionships in Cenral Reina of Macaque Monkeys (Macaca fascicularis) D. Max Snodderly,1,2,3 Richard S. Weinhaus,1*2*a and John C. ChoPb Neuroscience Uni, Eye Research nsiue, Boson, Massachuses 2114 and *Deparmen of Ophhalmology and 3Program in Neuroscience, Harvard Medical School, Boson, Massachuses 2115 The relaionship of he vasculaure o he neuronal layers was sudied in whole-mouns and in secions of macaque reinas. Like oher cenral nervous srucures, primae reinas have local variaions in vasculariy ha reflec local variaions in meabolism, raher han simply issue hickness or volume. A special feaure of he reina is a dense vascular plexus in he nerve fiber layer, which is unmyelinaed and hence mus generae a subsanial meabolic demand for ion pumping. Much of he reinal vasculaure is laminaed and locaed a specific layer boundaries. Throughou he cenral reina, wo planes of capillaries bracke he inner nuclear layer o form a sclerad capillary nework. n some regions, especially near he fovea, a second, more viread nework brackes he ganglion cell layer wih anoher pair of capillary planes. Wherever he nerve fiber layer is hick, he viread nework becomes less planar and is mulilayered. When surrounded by nerve fibers, capillaries end o orien parallel o he fibers; when adjacen o ganglion cell bodies, he capillaries are less sysemaically oriened. A he border beween he nerve fiber layer and he ganglion cell layer, rows of ganglion ceils ofen inerdigiae wih nerve fiber bundles, resuling in local perurbaions of capillary orienaion. The volume of he sclerad capillary nework is relaively consan a differen locaions, bu he volume of he viread nework increases dramaically where he nerve fiber layer is hick. As a resul, he vasculariy of he reina is greaes in he peripapillary region near he opic disk, even hough he oal hickness of he peripapillary reina is comparable o he reinal hickness near he fovea1 cres. As many as 6-7% of he phoons passing hrough he reina in he peripapillary region will encouner one or more capillaries before reaching a phoorecepor. Median capillary diameer increases wih reinal deph from Received June 7, 1991; revised Oc. 28, 1991; acceped Nov. 5, This work was suppored by NH Grans EY6591 and T32 EY774. Prining coss for he color plae were parially defrayed by a donaion from Allergan Pharmaceuicals. Mar&a Mullan-Sandsrom and Richard. Land, Jr. provided exper echnical assisance. James D. Broderick consruced he drawings of peripapillary regions P-P4. Dr. Margare S. Livingsone generously donaed reinal issue. John C. Choi submied porions ofhis work as an honors hesis a Harvard Medical School. Correspondence should be addressed o D. Max Snodderly, Neuroscience Uni, Eve Research nsiue. 2 Saniford Sree. Boson. MA = Presen addresses: Eye Research nsiue, Visual Services, Harvard Communiy Healh Plan, and General Eye Service of Massachuses Eye and Ear nfirmary. b Presen address: Deparmen of Ophhalmology, Universiy of Souhern California, Los Angeles, CA 933. Copyrigh 1992 Sociey for Neuroscience /92/ $5./O pm in he nerve fiber layer o 5. pm a he sclerad border of he inner nuclear layer. Capillary diameer in he nerve fiber layer also increases near he opic disk. Wihin he cenral reina of diurnal primaes, he opographic variaions in he neuronal layers creae large differences in he proporions of he cellular elemens a differen reinal loci. n he cener ofhe fovea, he reina is hin and only phoorecepors are presen. A he fovea1 cres, he ganglion cell layer reaches is greaes hickness, bu he opic nerve fiber layer is very hin. n he viciniy of he opic disk, he ganglion cell layer is hin, bu he nerve fiber layer is hick. The oal hickness of he reina is a combined funcion of hese widely varying componen layers. has long been appreciaed ha he hickness of he reina is one deerminan of is vasculariy (Michaelson, 1954; Chase, 1982; Buery e al., 199 1). f he reina is hin (as in he fovea1 cener), he oxygen demand can be me by diffusion from he choroidal circulaion, bu in more eccenric loci where he reina is hicker, an inrareinal blood supply wih muliple layers becomes prominen (Michaelson, 1954; Shimizu and Ujiie, 1978; Snodderly and Weinhaus, 199). The spaial disribuion of he vascular nework of primae reinas, including humans, has been he subjec of surprizingly lile quaniaive sudy. This is parly due o conflicing conclusions from earlier work (Michaelson, 1954; Toussain e al., 196 1; Wise e al., 197 1; Shimizu and Ujiie, 1978) abou he relaionships beween he reinal capillary neworks and he neuronal layers. Wihou a clear undersanding of he neuralvascular relaionships, i is difficul o accoun for he large regional variaions in he vasculaure. More recen work has shown an inricae and precise relaionship beween he reinal capillary neworks and he neuronal layers in he fovea1 region. n human reinas he capillaries of he ganglion cell layer are locaed a a border or in he middle of he layer, depending on he reinal eccenriciy and he hickness of he layer (wasaki and nomaa, 1986). Wihin he fovea1 depression of squirrel monkeys, we have idenified four disinc capillary planes ha are locaed near he borders of he inner nuclear layer and he ganglion cell layer (Snodderly and Weinhaus, 199). Wih increasing eccenriciy, he mos superficial (i.e., mos viread) capillary plane moves ino he nerve fiber layer. As a resul of his arrangemen, he capillary planes are appropriaely locaed near regions of high meabolic demand as indicaed by he cyochrome oxidase disribuion (Kageyama and Wong-Riley, 1984). n he presen aricle, we exend our analysis of neural-vas-

2 117 Snodderly e al. l Neural-Vascular Relaionships in Macaque Reina cular relaionships o addiional loci wihin he cenral reina, including he peripapillary region around he opic disk. We have chosen macaque reinas for his sudy because of he similariy of heir vasculaure o ha of human reinas (Henkind, 1967; Anderson, 1971; D. M. Snodderly and R. S. Weinhaus, unpublished observaions). Our resuls show ha he nerve fiber layer conribues more o reinal vasculariy han would be prediced from is conribuion o oal reinal hickness. This finding suggess ha reinal vasculariy, like he vasculariy of oher cenral nervous srucures, is more srongly relaed o local meabolic aciviy han o issue volume (Borowsky and Collins, 1989; Zheng e al., 1991). Maerials and Mehods Cynomolgous monkeys (Mucucufasciculuris), impored primarily from he Phillipines, were used. Reinas were coded by species (F forfusciculuris), a wo-digi idenificaion number, and righ(r) or lef(l) eye. Four reinas from hree monkeys were sudied inensively in whole-mouns, and measuremens of he vascular nework and he hickness of he neuronal layers were made. The sex and weigh a he ime of deah of hese hree animals were as follows: F22: female, 2.9 kg; F46: male, 2.8 kg: F54: male, 2.9 kg. n addiion, reinal whole-mouns from hree oher animals (F4, 648, and F53) and serial secions from reinas of five animals (FO 1, F4, Fl, F22, and F48) were sudied o confirm he generaliy of he paerns observed. Preparaion of rpina1 issue The mehods for preparaion of reinal issue have been published (Snodderly and Weinhaus, 199) and will only be described briefly here along wih subsequen modificaions. Monkeys were killed by injecion of an overdose of sodium penobarbial. The inensively sudied reinas were from animals whose reinas were hen immediaely fixed by ranscardial perfusion of he animal wih.9% sodium chloride conaining.5o/a sodium nirie followed by half-srengh Kamovsky s fixaive (2% paraformaldehyde and 2.5% gluaraldehyde in.1 M phosphae buffer, ph ). Less inensively sudied reinas were fixed wih sligh variaions in he saline prewash and he fixaive composiion. The eyes were enucleaed, opened a he pars plana, and posfixed in half-srengh Kamovsky s fixaive for an addiional 2-24 hr. Afer removal of he vireous, an 8 x 1 mm poserior segmen was cu from he globe ha exended superiorly, inferiorly, and emporally for a leas 3.5 mm from he fovea1 cener and for a leas.5 mm beyond he nasal margin of he disk. This cenral segmen of he globe was placed in.1 M phosphae buffer. To separae he reina from he choroid and sclera, he opic nerve fibers were genly severed a he level of he lamina choroidalis wih a microsurgical spaula. The reina was hen freezeproeced by ransfer o increasing concenraions of glycerol (12.5%, 25%, and 5%) in buffer. A his sage, he pigmen epihelium was sill adheren o he reina. Toal reinal hickness of wo reinas (F46R and F54R) was measured as described below, before dissecing off he pigmen epihelium. All oher reinas were immediaely disseced furher o opimize visualizaion of inrareinal feaures. The pigmen epihelium was removed by freezing he reina and scraping off he melanin-conaining issue (Snodderly and Weinhaus, 199). For microscopic sudy, he issue was placed viread side up in a shallow well urn deeo and COverSliDDed (Snodderlv and Weinhaus, 199). The well was filled wih 5% glycerol in buffer, wih sodium azide (O.O4O%r or.8%) added as a preservaive. Even wih he added azide, small paricles, presumably microorganisms, could be observed moving in he liquid phase afer several monhs. When his occurred, he issue was dismouned, soaked in fixaive for several hours o disinfec i, rinsed in buffer, and remouned. Whole-mouns were sored a -8 C o -9 C excep when being sudied. Despie repeaed cycles of freezing and hawing, he capillary nework and he neuronal layers were clearly visible for periods greaer han 18 monhs. Slow evaporaion of he fluid in he well caused gradual deerioraion in he visibiliy of reinal feaures. When he reina was remouned in a fresh soluion, however, he iniial clariy was resored. To confirm ha he microscopic images of he reinal whole-moun permied accurae localizaion of he reinal capillary planes in relaion o he neuronal layers, porions of seleced whole-mouned reinas were dismouned and serial secions 4 pm hick were prepared (Snodderly and Weinhaus, 199). Drawings The vasculaure in whole-mouns was drawn by using drawing ubes mouned on an Olympus BHS microscope wih a viewing magnificaion of 33 x or a Zeiss UEM microscope a 256 x magnificaion wih sandard brigh-field opics. Four cenimeers in he drawing plane corresponded o 1 pm on he reina. A coninuiy diagram of he vessels was raced by changing he focus, and he vessel segmens were assigned o heir respecive deph planes. Ambiguous relaionships were clarified a higher magnificaion wih Nomarski differenial inerference conras (DC) opics using he Zeiss sysem. The DC opics provided increased conras and decreased deph of field. Anaomical feaures visible in reinal secions were drawn wih DC opics a a magnificaion of 2 x. n hese relaively hick secions, only a single focal plane close o he coverslip was drawn so ha he layer borders could be represened as single lines in a hin opical plane. When he borders were irregular, a visual average was skeched as a smooh line. Quaniaive measures of he capillary nework Quaniaive measures of he capillary nework were made in he reinal whole-mouns in 1 x 1 pm sampling areas. The 87 sampling regions were approximaely midway beween areries and veins, a leas 6 pm from major vessels, o minimize local perurbaions of he capillary nework. Selecion and repeaed evaluaion of he sampling regions were faciliaed by fiing he Zeiss UEM microscope wih a sage wih moor conrol of he x- and y-axes (New England Affiliaed Technologies, Lawrence, MA), and a linear encoder (Dynamics Research, Wilmingon, MA) on he z-axis, so ha a coninuous digial display of he x, y, and z coordinaes was available. The digial oupu of he display was recorded by a microcompuer for furher analysis. Cupillmy screening ureas. The percenage of he reinal area covered by capillaries was esimaed afer enlarging he drawing of each 1 x 1 pm sampling area o a square 1 x 1 cm. The area devoid of vessel profiles was measured wih a Zeiss Videoplan digiizing sysem and subraced from he oal area o derive he reinal area opically screened by one or more capillaries. Capillary lenghs, diameers, and volumes. Lenghs of capillary segmens in he x-y plane were measured from drawings using he Videoplan digiizing sysem. Diameers ofcapillary lumens were direcly measured a he microscope wih DC imaging a 8 x magnificaion by superimposing he image of he capillary in he whole-moun on a scale imaged hrough he drawing ube. For each segmen, he mean of diameers measured a hree locaions disribued along he segmen was recorded. Volume esimaes were calculaed as he produc of he capillary segmen lengh and is cross-secional area, assuming a cylindrical shape wih a circular cross secion of uniform diameer. To verify ha reamen as a uniform cylinder was realisic, he diameers of more han 7 capillary segmens of F46R and F54R were measured a locaions all he way from he arerial o he venous end of he segmen. No consisen rends in diameer along he segmens were found. ndividual segmen volumes were summed o obain esimaes of oal capillary volume in each capillary grouping. Our complee sample included 1628 capillary segmens. Because our measuremens of capillary segmen lenghs were made from wo-dimensional maps in he x-y plane, and do no incorporae any verical componen, he derived volumes are underesimaes. To assess he error involved, we measured a he microscope he verical (z) componen of lengh for each capillary segmen in a subse of he sampling areas. The hypoenuse of he lengh in he x-y plane and he orhogonal componen in he z-axis were hen calculaed o obain an improved esimae of capillary segmen lengh and volume. The resuls showed ha we underesimaed capillary volume by an average of 7% in he peripapillay region and 3% in oher sampling areas. No correcions have been made for hese small errors. Measuremen hicknesses of reinal hickness and neuronal layer For wo reinas (F46R and F54R), oal reinal hickness and he hickness of he ouer reina were measured in whole-mouns before removal

3 The Journal of Neuroscience, April 1992, 72(4) 1171 of he pigmen epihelium. Thickness measuremens were made by focusing hrough he issue wih a 4 x waer-immersion objecive. The oal hickness was esimaed as he disance beween he planes of bes focus of he inner limiing membrane and of he viread border of he reinal pigmen epihelium. Here he epihelial cell mosaic was visible and melanin granules firs came ino sharp focus. For macaque reinas, he uppermos pigmen granules are abou 8 pm from Bruch s membrane, he boundary a which he reina usually separaes from he choroid in our dissecions. Conrol measuremens were done on one reina whose hickness was firs measured in whole-moun and hen remeasured in a side view of a slice cu from he reina wih a razor blade. Thickness values obained from he whole-moun were abou 12% less han hose measured from he side view. This discrepancy is oo small o affec our conclusions, and no correcion has been applied o he daa. The hickness of he ouer reina was measured as he disance from he capillary plane a he deep (sclerad) border of he inner nuclear layer o he viread border of he pigmen epihelium. For he oher wo whole-mouns from which quaniaive daa were obained (F22R and F22L), mos of he pigmen epihelium was disseced off immediaely so ha only small exrafoveal paches remained. n hese reinas, when a sampling area was near a pach of pigmen epihelium, a measuremen of ouer reinal hickness from he region of he pach was used. For sampling areas in he fovea1 region, where all pigmen epihelium was removed, an esimae of ouer reinal hickness was used by aking an average of measuremens a comparable loci from F46R and F54R. Values for oal reinal hickness of F22R and F22L were derived by adding he hickness of he ouer reina esimaed in his way o he measured hickness of he inner reina. Wih he pigmen epihelium removed, he hickness of each layer of he inner reina was measured for each 1 x 1 pm sampling area for all reinas. n sampling areas where he ganglion cell/nerve fiber boundary was irregular (a common occurrence), an average of he values a several locaions was used. Resuls Laminar disribuion of he vasculaure For each reinal whole-moun, a phoographic monage or a drawing was prepared o consruc a map of he large vessels, he fovea1 avascular zone, and he opic disk for use as landmarks (Fig. 1). Subsequen figures refer o hese maps o illusrae opographic relaionships. n his aricle, we have concenraed on he cenral reina, which we define as he approximaely circular region cenered on he fovea and exending along he verical meridian o include he superior and inferior emporal reinal areries (Fine and Yanoff, 1979). On he horizonal meridian, we have sudied he reina as far nasally from he fovea as he opic disk and for an equal disance emporally. The organizaion of he vascular nework of he macaque fovea is similar o ha already described for he squirrel monkey (Snodderly and Weinhaus, 199). n he whole-mouns, he vessel lumens appear as inerconnecing ubes differing in conras from he surrounding reinal issue (Snodderly and Weinhaus, 199). Radially oriened areries and veins in he ganglion cell layer are conneced by capillaries ha are locaed a preferred issue planes. Four planes of capillaries can be recognized a many locaions. Because of heir differen characerisics, we will divide hese four planes ino wo groups, a sclerad nework ha brackes he inner nuclear layer, and a viread nework ha brackes he ganglion cell layer or lies wihin he ganglion cell and nerve fiber layers (Fig. 2). n our illusraions and our erminology, we have adoped he convenion ha he reina is oriened vireous surface up, phoorecepors down. Thus, he erms shallow, superficial, and viread mean a common direcion in space, opposie o deep and sclerad. This convenion faciliaes in vivolin viro comparisons, since he reina is usually viewed in vivo wih he vireous surface oward he observer, so ha objecs deeper in he reina are more sclerad. Sclerad nework Throughou he cenral reina, capillaries a boh borders of he inner nuclear layer are visible as planar arrays in whole-moun images (Fig. 2) and in reinal secions (see below). These capillary planes, called he deep inner nuclear plane and he shallow inner nuclear plane (Snodderly and Weinhaus, 199), form he sclerad nework. Few capillaries are seen in he inerior of he inner nuclear layer; in regions of he whole-mouns where cloudiness made visualizaion of cell bodies difficul, he boundaries of he inner nuclear layer could readily be deermined by focusing on is bordering capillary planes. The only excepion o his rule occurs in an annulus approximaely 5-75 pm wide surrounding he fovea1 avascular zone; here he inner nuclear layer is relaively hin and capillaries someimes lie wihin i. Viread nework The viread nework is also planar in some local regions (Figs. 2,3). n he fovea1 region especially, he wo planes of capillaries forming he viread nework ighly bracke he ganglion cell layer. Here, he reina has four sric planes of capillaries, one pair brackeing he ganglion cell layer and he oher pair brackeing he inner nuclear layer [Fig. 4, 4 Planes (second phoo in row l)]. However, a greaer eccenriciies, he viread nework becomes less planar and i is srongly affeced by reinal opography. This is illusraed in rows 2 and 3 of Figure 4, which depic a secion ha exends from he cener of he fovea o 4 mm eccenriciy. The key facors influencing he viread nework appear o be variaions in he hickness of he ganglion cell and nerve fiber layers and he orienaion of he nerve fiber bundles. The border beween he ganglion cell layer and he nerve fiber layer. The border beween he ganglion cell layer and he nerve fiber layer is consisenly nonplanar in many reinal regions where boh layers are well developed. A he inerface beween hese wo layers, he ganglion cells are ofen arranged in irregular rows one o hree cells wide ha inerdigiae wih nerve fiber bundles (Fig. 3, upper righ). This gives he border an undulaing characer, wih small hills of ganglion cells creaing valleys in which he nerve fibers run. We are no aware of a previous descripion of his sriking reinal feaure. Vascularizaion of he ganglion cell layer. Wihin he fovea1 depression, a capillary plane lies a or near he deep border of he ganglion cell layer, along wih he major vessels, as previously described for he squirrel monkey (Snodderly and Weinhaus, 199). The close associaion of his plane of capillaries wih he large vessels promped us o use he locaions of he large vessels as an aid o idenify he ganglion cell capillary plane in he exrafoveal reina. was helpful o recognize ha major vessel runks in he viread nework lie mainly in he ganglion cell layer. To esablish his fac, we sudied whole-mouns of four reinas (F22L, F46R, F53R, and F54R) and confirmed he laminar relaionships in secions of a fifh reina (F48R). Medium-sized vessel runks could be readily assigned o he appropriae neural layer. The more difficul cases were vessels sufficienly large o prorude ino more han one layer and hose wih an asymmeric cross secion. The large veins in paricular ofen have obviously noncircular or asymmeric cross secions. n he whole-mouns, asymmery was eviden when he focal planes for he boom and he op surfaces of he vessels were

4 1172 Snodderly e al.. Neural-Vascular Relaionships in Macaque Reina Figure 1. Reinal regions where he vasculaure was illusraed or quaniaively measured. Seleced large vessels, he fovea1 avascular zone surrounded by he erminal capillaries, he oulines of he opic disks, and he animals idenificaion numbers are shown. The reinas are oriened according o sandard fundus phoographic convenion, wih he opic disk on he nasal side. The complee vascular neworks wihin he doed regions of F22R and F22L are displayed in Figure 5. Sampling areas. Small squares indicae he 1 x 1 pm sampling areas wihin which

5 The Journal of Neuroscience, April 1992, 12(4) 1173 no equidisan from he focal plane where he runk was wides. For he example shown in Figure 3 (lef column), he wides par of he runk of he large vein was abou hree imes as far from he boom surface of he vein as i was from he op surface. Deparures from roundness of he large vessels are also implied by he oblong profiles visible in secions orhogonal o he vessel axes (Fig. 4). Oher examples can be seen in Wise e al. (197 ), heir Figures 2-6 and 3-2, and Shimizu and Ujiie (1978), heir Figures 17, 36, 98, and 13. To be consisen, we assigned all major vessels o he lamina where heir runk was wides. The paern derived wih his crierion is relaively simple: when he opic nerve fiber layer was hick, near he opic disk, he major vessels were in he nerve fiber layer [see Fig. 4, Thick NF (row 4)]. As he major vessels moved away from he edge of he disk, hey descended ino he ganglion cell layer wihin.5 mm, wih one excepion, an arery ha raveled for.8 mm before descending o he ganglion cell layer. The only oher cases where major vessels were displaced from he ganglion cell layer occurred where veins and areries crossed each oher. For example, such displacemens occurred rouinely where venous ribuaries crossed he superior and inferior emporal reinal areries. Even here, he vessel runks reurned o he ganglion cell layer wihin.2 mm of he crossings. Where he large vessels are in he ganglion cell layer, he mos prominen capillary plane in he ganglion cell layer is a abou he same reinal deph as he large vessels. This capillary plane is disinc from he nerve fiber capillary grouping above i in hree ways: (1) i is presen hroughou he enire cenral reina including he fovea, excep for he fovea1 avascular zone and a surrounding annulus 5-7 wrn wide; (2) is capillaries are ofen no parallel o he nerve fiber bundles; and (3) i is presen in exrafoveal regions where he nerve fiber layer is hin or indisinguishable [Fig. 4, Temporal secion, TS (row 4)]. Wih increasing reinal eccenriciy ouside he fovea, he ganglion cell capillary plane gradually migraes viread relaive o he ganglion cell layer boundaries. This change of posiion occurs as he cell layer becomes hinner and he overlying nerve fiber layer hickens. Ou o an eccenriciy of abou 1.3 mm, he capillaries lie near he deep border of he ganglion cell layer. Along he fovea1 plaeau from abou 1.3 o 1.7 mm eccenriciy, hey are ofen in he inerior of he cell layer. A greaer eccenriciies, from abou 1.7 o 2.5 mm, hey are more frequenly locaed near he shallow (viread) border of he cell layer. A eccenriciies greaer han abou 2.5 mm, he ganglion cell layer hins o wo cells or less in hickness, and he capillaries can inermienly ouch eiher he deep or shallow border of he cell layer. They end o lie closer o he shallow border wherever he overlying nerve fiber layer is hicker han abou 5 pm. All capillaries having he characerisics described above were assigned o he ganglion cell plane (Fig. 2) and were coded in he same color in our coninuiy maps of he vasculaure (Fig. 5). Where his principal plane was superficial (viread) o he deep border of he ganglion cell layer, here were someimes a few addiional capillaries a he deep border of he cell layer. They were also assigned o he same ganglion cell nework. The capillary plexus of he nervejber layer. Capillaries near he border beween he ganglion cell and nerve fiber layers can be sandwiched beween cell bodies and nerve fiber bundles, surrounded by cell bodies, or surrounded by fiber bundles. f hey are surrounded by fiber bundles, he long capillary segmens orien mainly parallel o he bundles (Figs. 2, op; 3, lower righ). Slighly deeper capillary segmens sandwiched beween fiber bundles and cell bodies a he booms of he border valleys are ofen no parallel o he fibers. Shorer inerconnecing segmens beween neighboring capillaries or beween capillaries and major vessels can be oriened diagonal or orhogonal o he fiber bundles. n erms of heir physiological funcion, he capillaries in his border region mus supply nuriens o boh he ganglion cell and nerve fiber layers since he diffusion disances are shor. Furhermore, because nerve fibers iniially ravel hrough he ganglion cell layer before enering he nerve fiber layer proper, hey oo are served by capillaries in he superficial par of he ganglion cell layer. Wihou implying ha hey supply he nerve fibers exclusively, we have assigned capillaries o he nerve fiber capillary plexus on he basis of he following anaomical characerisics: (1) hey are locaed superficial o he principal capillary plane of he ganglion cell layer, eiher wihin he nerve fiber layer or near he ganglion cell/nerve fiber layer boundary; (2) long segmens are parallel o he nerve fibers excep when connecing o major vessels or when adjacen o ganglion cells; and (3) hese capillaries are infrequen or absen where he nerve fiber layer is hin, especially emporal o he fovea near he horizonal raphe (Fig. 4, Temporal secion, TS). One consequence of using hese crieria is ha occasional capillaries wihin he superficial porion of he ganglion cell layer are assigned o he nerve fiber plexusprimarily wihin an annulus from abou.7 mm o 1 mm reinal eccenriciy where he ganglion cell layer is very hick. This has lile effec on our measuremens of capillary volumes because he conribuion of hese few capillaries is quaniaively minor compared o he dense nework ha is unambiguously wihin he nerve fiber layer (see below). The capillaries in he nerve fiber layer are no organized in planes like hose of he sclerad nework brackeing he inner nuclear layer. As he nerve fiber layer builds up oward he disk, we have observed as many as eigh overlying layers of nerve fiber capillaries. The progressive addiion of capillaries as he nerve fiber layer hickens is illusraed in phoographs and drawings of reinal secions in Figure 4. The nerve fiber capillaries form a hree-dimensional plexus, wih long, horizonal capillary segmens conneced by shor verical or oblique segmens, as previously described by Shimizu and Ujiie (1978). As he hree-dimensional plexus becomes more and more elaborae, some of he complex feaures ha can be observed capillary parameers were quaniaively measured. Daa from adjacen sampling areas wih bars alongside hem were averaged ogeher. Locaions of he sampling regions are coded by he abbreviaions, inferior; N, nasal; S, superior; SN, superonasal; ST, superoemporal; r, emporal; and P, peripapillary. Phoographic locaions. For Figures 2 and 3, phoographs of F22R were aken a sampling locaions SN and SN.2; addiional phoos, bu no measuremens, were aken a locaions PH in reinas F22R and F46R. Secion locaions. The locaions and he orienaion of reinal secions illusraed in Figure 4 are indicaed by narrow verical recangles, a emporal one (TS) from he upper righ comer of F22L, and fovea1 (FS) and peripapillary ones (PS and PS2) from F48R. Arrowheads poin o major vessels ha are similarly idenified in he drawings in Figure 4. The open riangle jus above he label FS demarcaes where he drawing of he fovea1 secion in Figure 4 was divided o fi he page.

6 1174 Snodderly e al. l Neural-Vascular Relaionships in Macaque Reina in he horizonal (angenial) plane can also be found oriened in he orhogonal plane (verically). For example, compare he horizonally oriened loop in he whole-moun view in he op righ corner of he op panel of Figure 2 wih he verically oriened loop in he hird phoo of Figure 4. Vascular connecions and opography By successively focusing hrough he whole-mouns, one can visualize he relaionships among he planes of capillaries and he neuronal layers. Only a fragmenary picure of he relaionships can be obained phoographically, however, because he capillaries inerconnec by verical segmens and because he issue planes are parallel o he focal plane of he microscope only over shor disances. This can be appreciaed by comparing he phoographs in Figure 2 wih he coninuiy diagram of he same region (SN2) in he upper righ corner of Figure 5. Consequenly, he coninuiy diagrams form he basis for our analyses. The cenral porion of Figure 5 is a coninuiy diagram of he complee vascular nework of a fovea and is surround. illusraes he dramaic changes in he capillary nework ha occur as he inner reina hickens in he fovea1 region. A he corners of he figure are coninuiy diagrams of complee vascular neworks of hree exrafoveal regions wih differen hicknesses of he nerve fiber layer (P4 > SN2 > T). These diagrams give a visual impression of he influence of he nerve fiber layer on reinal vasculariy. A he boom of he fovea1 map near V4, and in he superonasal region SN2, wo layers of nerve fiber capillaries are presen. This ype of layering is illusraed in reinal secions in Figure 4 (upper lef corner, under he invered open riangle). For visual clariy, we coded hese wo vessel layers in differen colors in Figure 5. We coninued o use only wo colors o code he more elaborae capillary plexus in he nerve fiber layer of he peripapillary region (P4). The layer of nerve fiber capillaries (NF) closes o he ganglion cell capillaries is coded in yellow, and all more superficial capillaries are coded in red (NFs). This is inended o illusrae he grea proliferaion of he more superficial capillaries in reinal regions wih a hicker nerve fiber layer. Wih a magnifying glass, more deails of he inerconnecions of he vascular neworks can be seen. For readers wishing o sudy he geomery of he possible blood flow pahs, we can provide a larger color prin of his figure a a reasonable cos. The fovea1 region The fovea1 vascular nework of he macaque reina is similar in many respecs o ha of he squirrel monkey described previously (Snodderly and Weinhaus, 199). Figure 5 covers a greaer reinal expanse han our earlier drawing of a squirrel monkey fovea, so more of he fovea1 cres and he surrounding plaeau is included. Figure 2. Phoographs wih DC opics of he principal capillary planes in he whole-moun of reina F22R a locaion SN2 (2 mm eccenriciy) indicaed in Figure 1. Capillary neworks in he nerve fiber layer (N;?, in he ganglion cell layer (GC), a he shallow border of he inner nuclear layer (SN), and he deep border of he inner nuclear layer (DN) are shown. No well-focused plane exiss wihin he inner nuclear layer (N). A whie do near he op, lef, or boom border of four of he frames indicaes a feaure ha is idenified in Figure 5 (inse SN2) by a similar black do on a color-coded coninuiy diagram of his region.

7 The Journal of Neuroscience, April 1992, 12(4) pm Figure 3. Neural-vascular relaionships in he ganglion cell and nerve fiber layers phoographed in whole-moun (DC opics) a locaions indicaed in Figure 1. Lef column. A large vein in F46R abou 2.5 mm superonasal o he fovea1 cener a locaion PH in Figure 1. Four focal planes a successively deeper locaions show he following reinal feaures: () fiber bundles (F), a glial sepum (arrowhead), and he nerve fiber plane of capillaries jus above he ganglion cell layer; (2) he op (viread) surface of he vein, 11 pm lower, where some fiber bundles are sill visible, inerspersed wih rows of ganglion cells; (3) he wides par of he vein, anoher 12 pm lower, wih he vessel wall in bes focus surrounded by cell bodies; and (4) he boom (sclerad) surface of he vein, anoher 35 pm lower, receiving a ribuary vessel from he sclerad capillary nework (arrowhead). Righ column: Top, nerdigiaing rows of cell bodies and fiber bundles in F22R a he boundary beween he ganglion cell layer and he nerve fiber layer a inferoemporal locaion PH in Figure 1. Boom, Capillaries in he nerve fiber/ganglion cell boundary region of F22R a locaion SNl in Figure 1.

8 1 mm- 3mm- SUPEROR

9 The Journal of Neuroscience, April 1992, 72(4) 1177 An avascular zone a he cener of he fovea is surrounded by capillary neworks inerconnecing an approximaely radial array of alernaing areries and veins. We have only seen one example of a macaque reina lacking a clear fovea1 avascular zone; i will be described in a subsequen publicaion. As expeced, a zone of reduced capillary densiy is apparen around he areries. An experienced observer can deec his immediaely because i resuls in few capillaries crossing above he areries. Since he areries are in he ganglion cell layer, hey are far enough above he sclerad capillary nework o have less impac on i, and many capillaries can be seen o cross underneah he areries. The side branches of he areries ofen exi abruply, nearly orhogonal o he main runk (Wise e al., 197 1). The horizonal side branches send secondary verical branches up and down o feed he viread and sclerad capillary neworks as previously illusraed (Snodderly and Weinhaus, 199). However, among he erminal arerial branches, his paern is modified and he angles assumed by he branches become less abrup and more dichoomous. This is paricularly apparen in Figure 5 for A6 and for he long inferior branch of A2. The venous ribuaries have a quie differen disribuion and geomery. On he venous side of he circulaion, vessels in each of he capillary planes usually converge ono ribuaries ha hen drain ino he main venous runk. A paricularly large ribuary in he deep inner nuclear plane drains ino he main runk in he ganglion cell layer, ofen near he fovea1 cres, a around pm eccenriciy (Vl, V3, V4, V6). Near he horizonal meridian, his convergence poin can be closer o he cener of he fovea (V5) or farher away (V2). A he convergence beween he ribuary in he deep inner nuclear plane and he main runk in he ganglion cell plane, he shallow inner nuclear plane usually drains ino he main venous runk as well. This is a deparure from he paern in he squirrel monkey, where he shallow inner nuclear plane usually drains ino smaller vessels in he ganglion cell plane insead of direcly ino he main runk (Snodderly and Weinhaus, 199). The nerve fiber capillary plane can drain ino he main venous runk a abou he same poin as he oher capillary planes, creaing a quadriconvergence of all capillary planes (V 1, V6), or more commonly, i can drain ino he main runk more eccenrically. When his occurs, here can be a riconvergence of he deep inner nuclear, shallow inner nuclear, and ganglion cell planes ono he main runk a one eccenriciy followed by drainage of he nerve fiber capillary plane ino he main runk a a slighly greaer eccenriciy (V2, V3, V4). As previously noed (Shimizu and Ujiie, 1978) here are also many insances of individual capillaries draining direcly ino he main venous runk from he differen capillary planes. This conrass wih he siuaion on he arerial side ofhe circulaion, where a juncion beween he main vessel runk and an individual capillary is rare. Exrafoveal regions For he hree exrafoveal regions, he drawings in Figure 5 are oo small o include he major vessels so ha he connecions o hem are no shown. Region T is from he emporal reina near he raphe (Fig. 1) where he nerve fiber layer was only 9 pm hick; he nerve fiber capillaries were absen excep for one capillary segmen in he lower righ. Region SN2, from he superonasal reina where he nerve fiber layer was 57 pm hick, had wo layers of nerve fiber capillaries. Region P4, from he peripapillary reina where he nerve fiber layer was 11 yrn hick, had a dense nerve fiber capillary plexus ha was mulilayered. Noe ha in he drawings of all reinal regions he capillary segmens have a relaively uniform diameer. This conrass wih he siuaion in skeleal muscle, where he capillaries are apered; heir diameer increases as hey pass from he arerial o he venous side of he circulaion (Caro e al., 1978; Wiedeman, 1984). The aper may be relaed o he greaer lengh of capillary segmens in muscle (5-1 wrn) compared o hose in reina. Orienaion of nerve fiber capillaries n all regions of he reina, he long segmens of he nerve fiber capillary plexus were predominanly oriened parallel o he nerve fibers coursing oward he opic disk. This is eviden in all he drawings of Figure 5. Because of our crieria for assignmen o his capillary grouping, however, here are excepions o his rule when he capillaries are no immediaely surrounded by nerve fiber bundles. To illusrae he nerve fiber capillary grouping of he fovea1 region more clearly, i has been absraced in Figure 6. The rajecories of he nerve fiber bundles were drawn separaely and hen superimposed on he vascular nework as a series of Figure 4. Neural-vascular relaionships seen in verically oriened secions perpendicular o he reinal surface. See Figure 1 for secion locaions in F48R and F22L. Superior is o he righ for all secions. Phoographs oflocal regions (DC opics), The op row shows phoos of seleced regions of he secions drawn in he lower rows, in addiion o one secion no drawn. The deph of field of he phoographs is greaer and he focal planes are slighly differen from hose of he drawings, so some deails in he phoos are no presen in he drawings. Drawings, The ouermos boundaries drawn for he secions are he vireous surface and he inner segmen/ouer segmen juncion of he phoorecepors. The inner reinal layers are indicaed as follows: NF, nerve fiber layer; GC, ganglion cell layer; ZP, inner plexiform layer; N, inner nuclear layer. The fovea1 secion (FS) from F48R requires wo rows because of is lengh. Row 2 is is cenral segmen and row 3 is is more eccenric coninuaion. Reinal eccenriciy is marked above he drawing a 1 mm inervals. Jus beyond 2 mm eccenriciy in row 2, he drawing is inerruped a he invered open riangle; afer a small region of overlap indicaed by he repea of he riangle, he secion coninues in row 3. Arrowheads poin o major vessel inersecions similarly indicaed in Figure 1. Row 4 illusraes a emporal secion (ES ) from F22L (boom lef) and a peripapillary secion (PS) from F48R (boom righ). Relaionships beween phoos and drawings, The lefmos wo phoographic frames in row 1 are from he fovea1 secion (FS) in rows 2 and 3. To illusrae he laminar relaionships clearly, he phoographs are a wice he magnificaion of he oher panels of he figure. The firs frame (NFs), aken a he locaion indicaed by he large invered open riangle jus beyond 2 mm eccenriciy, illusraes he region where he nerve fiber capillary nework increases o more han a single layer (see Fig. 5 for illusraions of his from he whole-moun view). The second frame (4 PLANES) illusraes he region jus before 1 mm eccenriciy where he capillary bracke he nuclear layers. Small open riangles on he phoo poin o capillaries cu in cross secion. Solid riangles in he drawing immediaely beneah he phoo in row 2 poin o he lefmos and righmos capillary in each plane. The hird and fourh phoos are from peripapillary regions PS2 and PS in F48R (Fig. 1). The hird phoo (PSZ) illusraes a verical loop (large whie arrow) in he nerve fiber layer (region no drawn). The fourh frame (THCK NF) illusraes a mulilayered capillary nework wihin he nerve fiber layer. is drawn in he lower righ corner of he page (F48R PERPAPLLARY SECTON, PS). The arrowhead a he upper righ corner of he phoo corresponds o he righmos arrowhead above he drawing.

10 1178 Snodderly e al * Neural-Vascular Relaonshps n Macaque Rema SN2 SN R VJ T - 2pm Figure 5. Deph-coded coninuiy diagrams of he enire reinal vasculaure of four reinal regions: he fovea (cener), a emporal region (r), and a superonasal region (SN2) of F22R, and a peripapillary region (P4) of F22L, all drawn o he same scale and correcly oriened. A black do marks he cener of he fovea, and areries A l-a6 and veins V-k 6 are individually idenified for reference. The reinal areas of which he drawings were done are enclosed in doed lines in Figure 1. Phoos a differen deph planes from region SN2 are shown in Figure 2. Each whie do on he phoos of Figure 2 idenifies a disincive feaure of each capillary plane ha is marked by a corresponding black do in he drawing of SN2. The wo squares formed by he dashed lines in SN2 (visible wih a magnifier) are he sampling areas used for quaniaive measures of he capillary nework. Abbreviaions for he capillary groupings in he color key are he same as in Figure 2 excep for he addiion of NFs, he more superficial capillaries in he nerve fiber layer. Where an overlying vessel occludes pars of he nework, he pahs of he vessels crossing underneah are oulined by broken dark lines. Verical inerconnecions are indicaed by open circles drawn inside he ubular vessels. The original drawing of he fovea1 nework is 1 m x m and is a composie derived from more han 8 microscope fields.

11 The Journal of Neuroscience, April 1992, 72(4) Figure 6. Capillaries in he nerve fiber layer (NF and NFs) and major areries and veins of F22R absraced from he complee coninuiy diagram of Figure 5. Doed lines show he orienaion of he nerve fiber bundles coursing oward he disk. doed lines. This comparison confirms ha in mos ofhe fovea1 region he long segmens of capillaries assigned o he nerve fiber grouping are parallel o he pah of he nerve fibers. A region exhibiing excepions o his rule is eviden beween V 1 and A2, however. Less exensive excepions can also be seen beween V6 and A 1 and V5 and A5. The capillaries no parallel o he nerve fibers are generally eiher in he ganglion cell layer above (superficial o) he main ganglion cell plane, or a he border beween he ganglion cell layer and he nerve fiber layer, as illusraed in Figure 3. Quaniaive measures of he capillary nework Alhough i is generally expeced ha he capillary volume of he reina should increase wih reinal hickness (Michaelson, 1954; Chase, 1982) he diagrams of Figure 5 imply ha variaions in he hickness of he nerve fiber layer could play an unexpecedly large role. To invesigae his, we calculaed for each sampling area he capillary volume in each of he capillary groupings, as well as he oal capillary volume of he reina. The resuls for four reinal secors are ploed in Figures 7-1. Alhough he sampling locaions depar from sric linear geomery, for simpliciy of presenaion we have ploed he daa along a single eccenriciy axis as a schemaic profile. Daa from he fovea, a peripapillary region, and a emporal region near he horizonal raphe have been juxaposed o faciliae comparisons among reinal loci wih widely differing characerisics. The peripapillary regions were chosen o mach heir oal reinal hickness as closely as possible o he oal reinal hickness near he fovea1 cres. n his way we compared vasculariy of reinal regions wih he same oal hickness bu very differen hicknesses of individual layers. Toal capillary volume of he reina Figure 7 summarizes he effecs of reinal locaion on oal capillary volume of he reina. For F22, R and L (op row), we compare samples from he superonasal secor of he reina (righ), where he hickness of he nerve fiber layer increases seeply wih eccenriciy, wih samples from he superoemporal secor (lef) where he nerve fiber layer is less prominen. For F46R and F54R (boom row), he majoriy of he samples were disribued verically. n all cases, he capillary volume increases seadily wih eccenriciy from he erminal capillary ring surrounding he fovea1 avascular zone o he fovea1 cres, near 1 mm eccenriciy. n reinal secors where he nerve fiber layer does no reach is maximum hickness (superoemporal, superior, and inferior), he capillary volume changes relaively lile from abou 1 o 3 mm eccenriciy (Fig. 7, upper lef, lower lef, lower righ). Where he nerve fiber layer is hickes, in he superonasal secor (upper righ) and in he peripapillary regions (all panels), he capillary volume is greaes. This is rue in spie of he fac ha he oal reinal hickness of he peripapillary regions is comparable o he oal hickness of he reina a he fovea1 cres. The capillary volume is leas in he emporal loci where boh he nerve fiber layer and he oal reina are relaively hin. These daa show ha in he exrafoveal reina, he volume of capillaries is no a simple funcion of eccenriciy. The samples in he superior, peripapillary, and emporal regions of F46R and hose from he inferior, peripapillary, and emporal regions of F54R were all wihin 1% of he same eccenriciy. The key facors appear o be he hickness and composiion of individual layers of he inner reina.

12 ST3 8 F22 SuPERoTBNPORAL 8 SN3 F22 # SUPERONASU SN2 d SNl R L TmPORAL TEMPORAL ST1 ST2 ST? P2.L bc SUPEROR SNl SN2 SN3 '4 P4 RT NF NF GC P N RT = NF GC 5 - N GC p - N RT = NF GC E - N - y- OF , F54R NFEROR l -r SUPEROR! ;.. l NFEROR 8 P l El J _ P RT T <T = NF :C :P - :N zr :N RT - 3C - P - N 3R ECCENTRCTY (mm) ECCENTRCTY (mm)

13 The Journal of Neuroscience, April 1992, 72(4) 1181 Volumes of he separae capillary groupings The volumes of he four capillary groupings associaed wih individual reinal layers are illusraed in Figure 8. These daa are drawn from he same sampling regions as Figure 7 and are ploed in he same forma o faciliae comparisons wih neuronal layer hicknesses. The volume of he nerve fiber capillary plexus closely follows variaions in hickness of he nerve fiber layer. is low in he emporal reina where he nerve fiber layer is hin, and i increases in he nasal reina, being greaes in he peripapillary region where he nerve fiber layer is hickes. Wihin he fovea, he nerve fiber grouping is less voluminous han he oher capillary planes and i increases more slowly in volume wih reinal eccenriciy. does no become more voluminous han he neighboring ganglion cell plane unil well ouside he fovea1 depression. Sampling variabiliy precludes any precise comparisons of he wo componens of he viread nework in he parafovea1 regions. n he sclerad capillary nework, he deep inner nuclear plane is usually more voluminous han he neighboring shallow inner nuclear plane. The volume of he deep inner nuclear capillary plane increases rapidly wih eccenriciy in he fovea and hen remains a a relaively consan level hroughou he cenral reina. is ineresing o noe ha he cone pedicles, which are jus sclerad o hese capillaries, reach heir maximum densiy a abou he same eccenriciy where he volume of his capillary plane has climbed o is plaeau (Schein, 1988). The shallow inner nuclear plane shows individual variaion from one animal o anoher. is less voluminous han he deep inner nuclear plane in all reinal secors of F22, including he nasal and inferior secors no illusraed. For F46, many sampling regions have comparable values for he wo planes. F54 shows a mixed picure, wih he shallow inner nuclear plane less voluminous in mos samples bu abou equal o he deep plane in ohers. Volumes of he viread and sclerad capillary neworks To reduce he variabiliy shown by he individual capillary groupings, we pooled he daa as a sclerad nework brackeing he inner nuclear layer and a viread nework associaed wih he ganglion cell and nerve fiber layers (Fig. 9). We fel ha merging he ganglion cell and nerve fiber capillaries was funcionally jusified because of he frequen inermingling of ganglion cells and nerve fibers, as discussed earlier. Furhermore, combining he wo planes brackeing he inner nuclear layer did no obscure any of he poins we wished o emphasize. n fac, he sampling variabiliy diminished, he differences among animals were minor, and we were able o analyze he effecs of he hickness of reinal subdivisions. The paerns ha emerged are as follows. Wihin he fovea, he sclerad nework is more voluminous; he volumes of boh neworks increase rapidly wih eccenriciy. Ouside he fovea, he volume of he sclerad nework is similar from one animal o anoher and varies lile wih reinal locaion. n conras, he viread nework varies drasically wih reinal locaion, increasing where he reina and he nerve fiber layer are hick, and decreasing where hey are hin. As a resul, he sclerad nework is he more voluminous one in he emporal reina and he viread nework dominaes in he peripapillary region and he superonasal region. The wo neworks have similar volumes near he verical meridian where nerve fiber layer and oal reinal hickness are inermediae (represened by he samples from F46 and F54). Percenage of he issue volume occupied by capillary lumens The quesion ha arose from his analysis was wha caused he opographic variaions in he volume of he viread nework. We considered wo main possibiliies. Firs, differences in he hickness of he ouer reina creae variaions in he pah lengh for diffusion of oxygen (and perhaps oher nuriens) from he choroid. However, he paern of opographic variaions is no consisen wih his view (see below). The more likely possibiliy is ha he opographic variaions in capillary volume are linked o local variaions in demand for oxygen and oher meabolies. We paricularly waned o know wheher here migh be a higher densiy of capillaries in he viread nework, which would cause variaions in nerve fiber and ganglion cell layer hickness o conribue disproporionaely o he opographic variaions in he vasculaure. To evaluae his possibiliy, we calculaed for each sampling area he percenage of he issue volume occupied by capillary lumens (called percen capillary volume) for wo reinal subdivisions and for he oal reina. The percen capillary volume of he issue supplied by he viread nework was calculaed using he volume of he reina beween he vireous surface and he deep border of he ganglion cell layer. The percen capillary volume of he inner reina was calculaed using he volume of he reina beween he vireous surface and he deep border of he inner nuclear layer. For comparison, he percen capillary volume of he oal reina was calculaed using he volume of he reina beween he vireous surface and he viread border of he reinal pigmen epihelium. The resuls are summarized in Figure 1, using he same forma as Figures 7 and 9. The percen capillary volume of he oal reina is lowes in he fovea and highes in he peripapillary region. The values for region P2 of F22L are no as high as he oher peripapillary Figure 7. Toal reinal hickness (RT), neuronal layer boundaries, and oal capillary volume as a funcion of reinal locaion. n each panel, schemaic drawings (no o scale) absraced from Figure 1 idenify he sampling areas from which he daa were derived. The cener of he fovea is indicaed by an aserisk in a circle ha represens he fovea1 avascular zone. For each panel, he lef verical uxis gives he scale for he hickness of he reina and he disance of he layer boundaries from he reinal pigmen epihelium, indicaed by curves formed by solid lines. The ouer reina (OR), comprised of he phoorecepors and he ouer plexiform layer, and he individual layers of he inner reina are labeled (see Fig. 4 for abbrevaions). The righ verical axis is he scale for oal capillary volume of he reina, indicaed by he solid circles. The horizonal axis indicaes eccenriciy from he fovea1 cener; he axis is segmened by double verical lines afer 3.2 mm so ha peripapillary and emporal loci can be juxaposed o he oher regions. For F22 he reinal hickness of a p&papillary region was mached wihin 5% wih he reinal hickness a he fovea1 cres, by pairing peripapillary regions from he lef eye wih fovea1 regions from he righ eye. For he oher wo animals, he wors mismach occurred for FMR, where he reinal hickness in he peripapillary region was abou 1% greaer han a he fovea1 cres. Because of lack of space in he represenaions of he peripapillary regions P2 and P4 in he upper panels, labels are omied for RT and OR in he upper lef panel, and for OR in he upper righ panel. The nerve fiber layer was no visible in he emporal region represened in he lower righ panel.