Eye (1989) 3, 302-307

Anterior Segment Vascular Casting

1. M. OLVER and A. C. E. McCARTNEY London

Summary Vascular corrosion casting provides a permanent three-dimensional record of the deeper vasculature of the anterior segment whereas fluorescein angiography allows clinical examination of superficial vessels. Morphological findings on scanning electron microscopy of vascular casts of the anterior segment in sheep are presented and compared with that of man by casting techniques. Sheep illustrate the basic mammalian anterior segment vascular archi­ tecture but lack anterior ciliary arteries which connect with the deeper collateral arterial circles. The casting technique is described and its use to answer comparative anatomical, pathological and clinical questions explained. These techniques are being applied to examine changes to the anterior ciliary arteries and the deeper vessels following strabismus surgery in an animal model. Scanning electron microscopy of vascular corrosion casts has given a new dimen­ sion to micro-vascular anatomy.

With the advancement of scanning electron and depth of focus, with which to examine the microscopy anatomical casting techniques microvascular cast three dimensionally. The have entered a new dimension. The original material now used, methyl methacrylate, has a anatomical castings were done by Leonardo da low viscosity which fills capillaries and with­ Vinci! in the early sixteenth century using wax stands electron bombardment during SEM, to fill the cerebral ventricles. In the mid twen­ The normal ocular microcirculation in tieth century latex was used to fillvessels which manH•9•1O and primates!!·!2.13 (whose anterior were viewed macroscopically following mac­ segment vasculature appears similar to man) eration of the surrounding tissue. Using this have been studied using SEM. There have technique, Ashton,2 Wybar3 and Hayreh4 been only a few applications of vascular casting examined the microcirculation of the posterior techniques to examine pathological ocular or segment of the eye and of the orbit. Ashton5 post-surgical repair circulations. These also studied the relationship of Schlemn's include vascular casts of corneal neo­ canal to the anterior segment vasculature. vascularisation,!4 experimental subretinal Recently, latex studies have been superceded neovascularisation!5.!6 and non-ocular arterial by low viscosity methyl methacrylate resins6•7 anastomoses.!7 used in conjunction' with scanning electron Using this technique we have studied the microscopy (SEM). SEM improves on light normal microvascular architecture of the microscopy by allowing greater magnification anterior segment in sheep to determine if this

This paper was presented as a poster at the annual meeting of the Ophthalmology Society of the United Kingdom in April 1988,

Correspondence to: Jane M, Olver FRCS, Institute of Ophthalmology. 17-25 Cayton Street, London EC1 V 9AT. ANTERIOR SEGMENT VASCULAR CASTING 303 model can be used to help identify the mor­ roscopically in Figure 1 but full morphological phological changes following squint surgery. interpretation is limited by the low resolution. The long posterior ciliary arteries are evident Casting methods in Figure la, coursing towards the iris where Adult CIun sheep were anaesthetised and they divided to form a virtually complete coro­ heparinised. The ocular vessels were flushed nally placed arterial circle. Part of the com­ free of blood by primary perfusion through the plete arterial circle is shown in Figure 1 b where carotid arteries with phosphate bufferedsaline it can be seen that no large vessels pass over it, at physiological temperature. Freshly pre­ and from which a second inner incomplete pared resin (secondary perfusate) was injected arterial circle is formed, off which the iris at a pressure of 90-120 mm Ilginto each carotid arterioles arise which are just visible (small artery until it appeared through the opened arrow). Detail of the posterior aspect of the jugular veins. The resin was prepared using a anterior segment showing the vessels of the modification of Nopanitaya's method.18 Com­ ciliary processes are shown in Figure 1c. mercially available Batson's No. 17, (Poly­ The depth of field and three dimensional sciences Ltd)* was used. This contains base viewing possible with SEM allowed full mor­ resin pre-polymerised methacrylate, separate phological interpretation. Arteries were dis­ catalyst (peroxide) and accelerator (n-N­ tinguishable from veins by the characteristic dimethylalinine). The resin was thinned by the appearance of the endothelial nuclear impres­ addition of monomeric methyl methacrylate sions; arteries and arterioles have elongated (de Trey and Alldrich). The proportions used oval impressions, (see Figure 2) whilst veins for each aliquot prepared are: base methacry­ and venules have typical more rounded late 25 ml, catalyst 7.5 ml, promoter 0.7 ml, impressions, (see Figure 3). The low viscosity mono methyl methacrylate 12.5-15 ml. of the resin allowed capillaries to be filled and Although coloured pigments may be added to identified by their size and lack of nuclear the clear resin, these were not used. "impressions" (see Figure 4). The material was left one hour in water at From the anterior view of the anterior seg­ 50°C for the resin to polymerise. The orbits ment vasculature in Figure 5, part of the com­ were excised and the tissue surrounding the plete arterial circle is seen (solid arrow), off cast was macerated in 6 M potassium hydrox­ which arises an incomplete arterial circle (hol­ ide. The resultant casts were washed thor­ low arrow). The incomplete arterial circle in oughly in tap water followed by distilled water turn gives rise to spiral iris arterioles (solid star) then air dried. The injection replicas of the vas­ and posterior recurrent arteries to the anterior cular lumen (corrosion casts) were sputter choroid (hollow star). Neither the complete or coated with a 50 nm coating of gold and exam­ incomplete arterial circles are joined by ined using scanning electron microscopy. anterior ciliary arteries. The vascular casts of the eye, especially the The anterior aspect of the ciliary processes anterior segment were extremely fragile, but (small white arrows) are shown in Figure 6 with could be examined at magnifications of up to the iris vessels in the foreground. The iris x 6,000 at an accelerating voltage of 20 kV for arterioles, barely visible in Figure 1b, are short periods of time. Working magnifications clearly definedin Figure 7 by their characteris­ were usually of the order of X 100 to x 3,000 at tic spiral shape. an accelerating voltage of 20 kY. Vessels were traced three-dimensionally and the cast micro­ Discussion dissected to reveal the underlying vascular The deeper collateral circulation of the structure. anterior segment in mammals consists of a vir­ tually complete arterial circle supplied by the Findings long posterior ciliary arteries, which gives rise Orientation of the overall vascular structure of to an incomplete arterial circle from which the the anterior segment is shown viewed mac- iris arterioles originate.

*Polysciences Ltd, 24 Low Farm Place, Moulton Park, Northampton NN3 IHY. 304 J. M. OLVER AND A. C. E. MCCARTNEY

Fig. 1. Macroscopic casts (la) Low magnification photomicrograph of long posterior ciliary arteries and complete arterial circle of the iris. (sheep) (lb) High magnification photomicrograph of part of complete arterial circle of the iris. Note absence of connecting anterior ciliary arteries. (sheep) (lc) High magnifica­ tion photomicrograph of posterior aspect of ciliary pro­ cesses. (sheep)

This study shows that in sheep the complete two or more rectus muscles in adults20-24 and and incomplete arterial circles lie on the iris. In has only rarely been described in children24.25 in mans and subhuman primates'3 the complete which there were other significant ocular arterial circle lies within the ciliary body (intra­ abnormalities. It is accepted surgical practice muscular arterial circle) and the incomplete to limit such operations to movement of two arterial circle (major arterial circle of the iris) muscles at any one time and postpone further lies at the iris root where it is sometimes visible surgery for 2-3 months. Although experi­ in the peripheral iris. In sheep there are only a mental studies can reproduce anterior segment few anterior ciliary arteries which remain ischaemia26-3'the changes in vascular architec­ superficially located and do not connect with ture which result in restored blood flow to the the deep arterial circles. In pigs'9 the anterior iris have not been determined. Saunders and ciliary arteries have a diverse origin from the SandeIF' suggested, from anecdotal evidence, superior and inferior rectus muscular arteries, that there was no blood flowthrough the ciliary off the retractor bulbi muscles and from the arteries in previously tenotomised rectus long and short posterior ciliary arteries. These muscles. In an opposing view, Simon et al.23 penetrate the sclera but do not connect directly suggested that re-establishment of the original with the arterial circles above. anterior ciliary circulation might occur follow­ In contrast, mans and subhuman primatesI3 ing strabismus surgery and France in the dis­ have anterior ciliary arteries predominantly of cussion following Simon's paper gave muscular artery origin which connect directly anecdotal evidence that the anterior ciliary with the intramuscular arterial circle either arteries recanalised. through direct perforators or through perfor­ Alterations in vascular dynamics have been ating branches from the anterior episcleral demonstrated following strabismus surgery by arterial circle. anterior segment fluorescein angiography.3' Anterior segment ischaemia is a rare but Characteristic iris segmental filling defects serious complication of strabismus surgery on have been demonstrated in adults following ANTERIOR SEGMENT VASCULAR CASTING 305

Fig. 2. Scanning electron micrograph oj part oJ com­ plete arterial circle of the iris (equivalent to the intra­ muscular arterial circle in man) (white arrow). In sheep Fig. 4. Scanning electron micrograph showing this lies well within the iris. Spiral iris arterioles ( * ) and anterior aspect of ciliary processes (arrows) with fine posterior recurrent arteries to the anterior choroid iris capillaries and iris arteriole in the lower field. (sheep 100) (empty * ) are seen arising fr om the incomplete arterial x circle (equivalent to major arterial circle of the iris in man) (empty arrow), anterior to the complete arterial circle. (sheep x 100).

Fig. 3. Scanning electron micrograph showing mass Fig. 5. Scanning electron micrograph showing fine offine capillaries traversing iris arteriole. (sheep x320) capillaries branching fr om arteriole. (sheep x844) 306 J. M. OLVER AND A. C. E. MCCARTNEY

Fig. 6. Scanning electron micrograph of posterior Fig. 7. Scanning electron micrograph of ciliary pro­ recurrent artery to the anterior choroid showing charac­ cess vein with overlying venule showing characteristic teristic nuclear impressions. (sheep x844) endothelial nuclear impressions. (sheep x2,080) vertical rectus muscle surgery.32 Recent work In summary, vascular casting extends our (Olver) has shown that primary vertical rectus knowledge of anterior segment circulation muscle surgery results in sector fillingdelay but gained from fluorescein angiography by that secondary vertical rectus muscle surgery demonstrating the normal presence or absence does not. Low dose fluorescein episcleral of anterior ciliary arteries connecting with angiography which allows a clear interpreta­ deeper arterial circles, clarifying the existence tion of the episcleral vessels33 has shown that of two deeper arterial circles, one complete there is no re-establishment of blood flow and one incomplete and the origin of the iris through the tenotomised rectus muscle (Olver, arterioles. Casting may both identify the unpublished data). normal ocular microcirculation and be applied These angiographic findings strongly sug­ to examine pathological ocular microcircula­ gest that recanalisation of the anterior ciliary tion, in particular the nature and morphology arteries does not occur. of new vessels thus helping to answer both Vascular casting can be used to observe the basic scientificand clinical questions. The limi­ effects of surgery on the anterior segment cir­ tation of this technique is that functional culation in an animal model and provide evi­ characteristics of vascular flow can only be dence of whether or not a new circulation is inferred from the structural findings. established through the muscle insertion. We suggest that casting is a suitable tech­ Sheep ocular vasculature shows many nique with which to study the changes to the similarities with human ocular vasculature and anterior segment vasculature following may be used to study basic vascular architec­ strabismus surgery, particularly the circulation ture. Sheep, however, lack penetrating around the altered muscle insertion, the anterior ciliary arteries, suggesting non­ "strabismus scar" . human primates are a better model to study changes in anterior segment vasculature fol­ lowing strabismus surgery. This future appli­ We are grateful to de Trey and Alldrich for their sup­ cation is underway and will be reported upon. port. Our thanks also to Robin Howes for technical ANTERIOR SEGMENT VASCULAR CASTING 307 assistance. This work is supported by the Francis and vascular arterial anastomoses as seen on corrosion Renee Hock Trust. casts by scanning electron microscopy. Plas Recon­ struc Surg 1979, 64: 59-64. 18 Nopanitaya W, Aghajanian JG, Gray LD: An References improved plastic mixture for corrosion casting of I da Vinci L: c1508. The brain injected to demonstrate the gastro-intestinal microvascular system. SEM the shape of the cerebral ventricles. Royal Library, 1979: Part Ill, SEM, Inc AMF O'Hare., 11 60666, Windsor Castle. Cat19127. R. Anatomical draw­ 751-755. ings from the Royal Collection. Royal Academy of 19 Simoen P. 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