Eye (1989) 3, 121-127

The Circulation of the Human Limbus

PAUL A. R. MEYER Cambridge

Summary The superficial circulations of the anterior segment of the human have been studied by the following techniques: photographic and video low-dose fluorescein angiography, video-microscopy using red-free and stereoscopic colour pho­ tography. The results are combined to give a dynamic description of the Iimbal circulation, including its arterial supply and venous drainage.

Limbal vessels we know more about the control of In order to achieve optical clarity, blood growth here than at any other site in the body. vessels are excluded from the in most Vascular invasion of is inhibited by species of mammal: this is possible because its a factor extractable from bovine ,8 metabolic activity is matched by the delivery thought to be chondroitin sulphate.9 This of glucose and. oxygen from aqueous and forms about 30 per cent of corneal pro­ tears.! However, the requirements of the cor­ teoglycan,! and the penetration of clear cor­ neo-scleral junction are substantially greater. nea by limbal may be suppressed in is largely renewed from the same way, although other hypotheses limbal stem cells,2.3 and there is evidence that have been proposed.lO the drainage of aqueous may be metabolically When placed within stromal pockets, demanding.4 tumour angiogenic factor,II,12 fibroblast These demands are met by an elaborate growth factor, 13 epidermal growth factor,n limbal capillary bed. The corneal epithelium catecholamines14 and prostaglandins (particu­ is supplied by a monolayer of superficial l)15. 6 arcades, and beneath these a receding bank of larly PGE 1 all stimulate limbal neo­ capillaries reaches down to the Canal of vascularisation. Hypoxia also appears to Schlemm. cause limbal vessels to proliferate, possibly as Capillaries of the limbus have thicker endo­ a result of the accumulation of ADP or lactic thelium and fewer fenestrations than those of acid. 17 This may account for the corneal vas­ the .5 They are anchored within cularisation that accompanies epithelial kera­ the corneal collagen matrix by a highly ­ tinisation in vitamin A deficiency.IS ised adhesive of proteoglycan.6.7 Activated release angiogenic mediators,19 which may explain the neovascu­ Capillary growth and regression lar response to infection, corneal foreign This unusual circulation, much of which is bodies and grafts. This process is facilitated by imprisoned in collagen, might be considered granulocytes, 19.20 but not dependent upon unsuitable for studies of mediators affecting them. 21,22 vessel growth. However, the transparency of Therefore there is no single map of the lim­ the cornea has attracted many researchers and bal arcades. They form a cylindrical bank of

Correspondence to: Paul A. R. Meyer, MRCP, Addenbrookes Hospital, Cambridge. 122 P. A. R. MEYER capillaries which may wax and wane according in the vertical meridian and the long posterior to environmental influences. ciliary in the horizontal meridian.34 However, a recent study, in which emissary and anterior were dis­ Vascular relationships tinguished by anterior segment fluorescein According to Leber's classical account,23 the videoangiography, has shown that, both in the anterior segment of the human eye has deep horizontal and vertical meridians, most and superficial circulations which both arise arteries and all veins do carry blood away from the ophthalmic : from the limbus.35 The circulations of the The long posterior ciliary arteries penetrate anterior ocular surface are therefore predomi­ the behind the equator, run forward nantly supplied from within the globe. within , and cross the supra-choroidal The classical, static anatomical techniques space to join the major circle of the . This of indian ink injections33 and vascular cast­ intra-ocular arterial circle (which has recently ing36.37 do form the foundation for our know­ been shown to have an additional intra-mus­ ledge of the limbal vasculature.23.38.39 cular component24) supplies the iris and ciliary However, when circulatory dynamics are to apparatus. be described, there is no substitute for imag­ The anterior ciliary arteries-superficial, ing a living circulation using techniques such anterior continuations of the arteries to the as fluorescein angiography35.40 and video rectus muscles-were thought to supply the recording in red-free light. 41 limbus, anterior conjunctiva and episclera. In 1903 Leber was clear that the superficial Low dose anterior segment fluorescein and deep circulations were joined anteriorly angiography by substantial arteries that perforated the fluorescein angiography has been sclera and crossed the supra-choroidal space. practised since 1960,42 but extravasation of His findings have since been confirmed by 4 fluorescein has limited the value of anterior vascular castings,2 .25 but the direction of flow segment studies. However, by restricting the in these vessels has excited controversy. dose of fluorescein to prevent the saturation Whether studied by fluorescein angiogra­ of available ionic binding to circulating phy,26 or by the injection of radio-labelled albumin,43.44Ieakage from conjunctival capill­ microspheres,27 iris perfusion is found to aries may be reduced and resolution decline after section of the rectus muscles. enhanced.40 Low dose fluorescein angiograms This has been considered to indicate that the may be recorded photographically to give muscular/anterior ciliary arteries contribute high spatial resolution, or by video to achieve to the deep circulation.26.27 higher temporal resolution. Photographic fluorescein angiography of the anterior ciliary arteries themselves has yielded conflicting evidence. A number of Video recording in red-free light investigators have published angiographic The clinical use of red-free light to observe sequences in which anterior ciliary arteries blood flowin the anterior segment of the eye is appeared to fill away from the limbus.28.29.30 commonplace. However, this is difficult to Others have argued31.32 that these represent record by video since restriction of the band­ emissary veins, known to drain the deep cir­ width of illumination lowers light intensity, culations of the ciliary apparatus into the and most video cameras have enhanced sen­ superficial episcleral venous system.33 sitivity to longer wavelengths. The technique Squint surgery upon the vertical, but not used for this study represents a compromise, the horizontal, rectus muscles may give rise tv in which a broad waveband of illumination perfusion defects in iris fluorescein angio­ below 600 nm is recorded by a CCD camera grams.34 This has prompted the suggestion which achieves peak sensitivity at 520 nm. that the circle of arteries in the iris root and This account of the limbal circulation con­ (intra-ocular arterial circle) catenates the results of recent studies using may receive blood from the muscular arteries both techniques, paying particular regard to THE CIRCULAnON OF THE HUMAN LIMBUS 123 flow dynamics, arterial supply and venous normal subjects. Three hundred and sixty drainage. degree surveys were performed, with special reference to the changes in flow characteris­ Methods tics that accompany transit of blood through Photographic low dose fluorescein the limbal capillary bed and the of angiography venous drainage. The descriptions below are based upon find­ Illumination was by a 2S-watt incandescent ings in eight normal subjects, which have been lamp, filtered by a SOO-S80nm band-pass reported previously.40 Each volunteer interference filter. received one angiogram and colour photo­ Recordings were made on low-band graphs of the angiographic field. U-matic videotape using a COHU 4712 CCD Sodium fluorescein (120 mg) was injected camera. briskly into an ante-cubital fossa . The eye was illuminated at 1.S second intervals by 720 Results and Discussion Wsec flashesfrom an off-axis xenon flashunit, Delivery of Blood to the Anterior Segment mounted on a Zeiss photo-. The (low-dose fluorescein videoangiography) exciting radiation was filtered by a 420- Using low-dose fluorescein videoangiogra­ 490 nm interference band pass filter; a long phy, arteries and veins may be distinguished pass filter (TSO = S1Snm) was placed in the by their flow characteristics and direction of imaging pathway. were taken flow can be recorded even when velocity is using the x 16 setting on the objective turret high. The velocity of an advancing fluorescein and a x2 magnifier (a magnification factor of front may be estimated, but this is not 3.S at the camera back). 400 ASA mono­ reliable. chrome film (Kodak Tri-X) was used, but was A study of 40 radial arteries from IS sub­ developed to 1200 ASA using Acuspeed jects demonstrated that more than 60% of developer (Patterson). anterior ciliary arteries filledfrom scleral per­ forations close to the limbus and flowed back Low dose fluorescein videoangiography towards the rectus muscles. They gave rise to These results are taken from studies of IS circumferential branches (the episcleral normal volunteers.35 The subjects received arterial circle) and radial vascular trunks, angiograms at low (13 mm/vertical screen) �ome of which met muscular arteries flowing and high (8 mm/vertical screen) magnifica­ in the opposite direction. This pattern of per­ tion, in addition to 360 degree stereo colour fusion was found in both the horizontal and photographic surveys. vertical meridians. Sodium fluorescein (1.S mg/kg) was If arteries communicate directly (end to injected as a brisk bolus. The eye was illumi­ side or end to end), they may be presumed to nated by a 2S watt incandescent bulb. A 4SO- support the free shunting of blood. Such 490 nm band pass filterwas used for excitation arterial communications can be detected by and a long pass filter (TSO = SIS nm) for imag­ videoangiography when each vessel gives rise ing. The angiograms were recorded on low­ to a separate run-off: the intervening column band U-matic videotape, using an image­ of blood remains static, non-fluoresceinated intensified nuvicon (Visual Contacts) and gently pulsating. mounted on the video of a Zeiss clinical This sign was observed close to the inser­ slit lamp. The images were then transferred tions of the horizontal and vertical rectus onto one inch videotape and slowed to one­ muscles, and within perforating anterior cili­ fifth real time for analysis. ary arteries. This indicates that the saggittal arterial ring, composed of long posterior cili­ Red-free video-microscopy of limbal ary arteries (lpca) and perforating scleral capillaries arteries, muscular arteries and anterior ciliary The limbal circulation of one eye was arteries (aca), may indeed support the shunt­ observed at low (8 mm/vertical field)and high ing of arterial blood. Direct arterial communi­ (1.S mm/vertical field) magnification in four cation is also demonstrable in the episcleral 124 P. A. R. MEYER arterial circle (eac) , indicating that this, too, is ary arteries meet each other in the episcleral a true arterial shunt circulation. arterial circle, which lies 1 to 5 mm behind the Therefore the arterial circulation of the limbus. In man this may have superficial and anterior segment is composed of superficial deep components, giving the appearance of and deep coronal arterial circles (the eac and discontinuity. the major circle of the iris + intramuscular Photographic fluorescein angiography has circle, respectively). These are fed and joined shown that the eac distributes blood to the by a saggittal arterial ring Opca + perforating anterior surface of the globe. Posterior roots scleral arteries + aca/muscular arteries) supply the episcleral capillary net, but (Fig. 1). Blood is most commonly delivered to anterior roots run forwards to the reflectionof both coronal circles from within the globe, the conjunctiva. There they divide into fine presumably by the long posterior ciliary conjunctival that loop back radially, arteries. and more delicate vessels that feed the limbal capillaries. Limbal Vascular Anatomy (Figs. 1 and 2) (episcleral arterial circle [photographic! Limbal Capillaries video-fluorescein angiography] red-free (photographic fluorescein angiography; red­ video-microscopy) fr ee video-microscopy) The circumferential branches of anterior cili- Each limbal supplies between one and three superficial limbal capillaries. These repeatedly divide and coalesce, forming a net­ work of interconnected vessels one to four tiers deep. Whereas conjunctival vessels leak fluorescein from discrete fenestrations, the limbal arcades remain competent until late in an angiogram, when they become uniformly obscured by fluorescein,possibly as a result of diffusion from the subconjunctival space.

Limbal Veins (stereoscopic colour photography; red-free video-microscopy) Immediately behind the limbal arcades, and central to the episcleral arterial circle, lies a circle of fine veins which may be called the limbal venous circle. It can be composed of up to three communicating, parallel vessels. They collect blood from the anterior con­ junctival veins and limbal arcades, and drain into radial episcleral collecting veins which flow towards the rectus muscles. As they course across the anterior sclera, radial collecting veins are joined by anterior Fig. 1. Delivery of blood to the anterior segment. episcleral veins and veins that gradually oa . emerge from the sclera (presumably draining lpca long posterior ciliary artery. } (saggittal rna artery of rectus muscle. arterial the deeper limbal capillaries). They also meet aca anterior ciliary artery. ring) the larger emissary veins that emerge from ... episcleral arterial circle (superficial coronal scleral foramina. They leave the anterior sur­ arterial circle). of the globe over the rectus muscles. -0- major circle of iris+intra-muscular arterial circle (deep coronal arterial circle). Veins draining aqueous surface into both ec episcleral capillaries. the limbal venous circle and the radial collect­ mc capillaries of rectus muscle. ing veins. THE CIRCULATION OF THE HUMAN LIMBUS 125

ev Ipca

Fig. 2. The al circulation. lpca long posterior ciliary artery. aca anterior ciliary artery. -0- episcleral arterial circle. ... limb al venous circle. ev radial episcleral collecting vein.

Blood Flow Dynamics Limbal arcades Anterior ciliary arteries (red-free video-microscopy) (low-dose fluorescein videoangiography) Perfusion may follow one of two patterns: Anterior ciliary arteries usually fill early in a (1) Blood is delivered into the apices of the videoangiogram. They carry blood with arcades and then meanders towards the most pulsatile flow and velocities of up to 100 mml proximal limbal vessels, which are broadened sec. However, close to sites of direct arterial to form the limbal venous circle. communication arteries may fluoresce very (2) More rarely, arterial blood is delivered to late, often after the adjacent veins. In these both proximal and distal capillaries. The circumstances pulsation occurs, but blood venous circle then bisects the limbal arcades. flow is minimal (vs). Blood is injected into the limbal arcades 126 P. A. R. MEYER with pulsatile flow and at high velocity, but The limbal venous circle drains into radial flowis slowed and smoothed early in the capil­ episcleral collecting veins, as do the deep lim­ lary phase. The passage of cells through the bal circulation and the episcleral capillary net. arcades may be extremely slow, and the capill­ Collecting veins join with each other, and with aries are often perfused by serum alone. This emissary veins, before leaving the globe over indicates that they filter cells, which is con­ the rectus muscles. firmed when collecting converge on Aqueous veins surface into both the limbal the limbal venous circle or radial collecting venous circle and the radial collecting veins. veins: streaming may arise as a result of the These techniques are simple and non-inva­ differing haematocrits in the contributary sive. In this study they have been used to vessels. elucidate the anatomy and dynamics of the normal limbal circulation, but they are Limbal venous circle; radial episcleral equally applicable to the investigation of local collecting veins and systemic vascular pathology. (red-free video-microscopy; low-dose fluorescein videoangiography) I wish to thank all the subjects who participated in these studies. I am also grateful to Mr. A. Quested for These vessels very rarely demonstrate pulsa­ advice on the slowing of video images, and to Mr. A. tion, and support laminar flow. This becomes Lacey who prepared animations for the presentation apparent as a result of the separation of cells upon which this paper is based. from serum within converging columns of The videoangiographic studies were supported by the Iris Fund for the Prevention of Blindness. blood. However, it is most spectacular where Paul Meyer is in receipt of a Wellcome Research veins receive blood of differing haematocrit, Fellowship. or aqueous. Reversal of blood flow may arise within the References limbal venous circle and ra<;Jjal episcleral col­ I Friend 1: Physiology of the cornea: metabolism and lecting veins, and is commonly precipitated by biochemistry. In Smolin G, Thoft RA eds. The cornea: Boston, Toronto: Little, Brown & Co. . It demonstrates free shunting of 1983: 17-3 1. venous blood between adjacent radial collect­ 2 Shapiro MS, Friend 1. Thoft RA: Corneal re-epi­ ing veins. thelialisation from the conjunctiva. Invest Fluorescein videoangiography indicates Ophthalmol Vis Sci 1981,21: 135-42. 3 Kinoshita S, Friend 1, Thoft RA: Sex chromatin of that the arterio-venous transit time is not con­ donor corneal epithelium in rabbits. Invest sistent throughout an angiographic field. Ophthalmol Vis Sci 1981,21: 434-41. 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