Anterior Segment Vascular Casting - Nature

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 microscopy

and depth of focus, with which to examine the

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 tieth century latex was used to fill vessels which

The

normal

ocular

microcirculation

in

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

been only a few applications of vascular casting

Wybar3 and Hayreh4

examined the microcirculation of the posterior

techniques to examine pathological ocular or

segment of the eye and of the orbit. Ashton5

post-surgical

also studied the relationship of Schlemn's

include

canal to the anterior segment vasculature.

vascularisation,!4

Recently, latex studies have been superceded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

repair

vascular

circulations.

casts

of

These

corneal

experimental

neo

subretinal

Using this technique we have studied the

microscopy (SEM). SEM improves on light

normal

microscopy by allowing greater magnification

anterior segment in sheep to determine if this

microvascular

architecture

of the

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.

303

ANTERIOR SEGMENT VASCULAR CASTING

model can be used to help identify the mor phological changes following squint surgery.

roscopically in Figure 1 but full morphological 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 1b where

carotid arteries with phosphate buffered saline

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 Ilg into 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

(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

(peroxide) and accelerator

(de Trey and Alldrich). The proportions used

oval impressions, (see Figure 2) whilst veins

for each aliquot prepared are: base methacry

and

late 25 ml, catalyst 7.5 ml, promoter 0.7 ml,

impressions, (see Figure 3). The low viscosity

mono

methyl

methacrylate

12.5-15

venules

have

typical

more

rounded

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

The material was left one hour in water at

4).

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

ined using scanning electron microscopy.

anterior ciliary arteries.

arterial

circles

are

joined

by

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 defined in 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

structure.

anterior segment in mammals consists of a vir

deeper

collateral

circulation

of

the

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 flow through the ciliary

off the retractor bulbi muscles and from the

arteries

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. In contrast, mans and subhuman primatesI3

in

previously

tenotomised

rectus

anterior ciliary circulation might occur follow ing strabismus surgery and France in the dis

have anterior ciliary arteries predominantly of

cussion

muscular artery origin which connect directly

anecdotal evidence that the anterior ciliary

following

Simon's

paper

gave

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


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 this lies well within the iris. Spiral iris arterioles ( ) and posterior recurrent arteries to the anterior choroid (empty ) are seen arising from the incomplete arterial 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 offine capillaries traversing iris arteriole. (sheep x320)

305

Fig. 4. Scanning electron micrograph showing anterior aspect of ciliary processes (arrows) with fine iris capillaries and iris arteriole in the lower field. (sheep x 100)

Fig. 5. Scanning electron micrograph showing fine capillaries branching from arteriole. (sheep x 844)

306

J. M. OLVER AND A. C. E. MCCARTNEY

Fig. 6. Scanning electron micrograph of posterior recurrent artery to the anterior choroid showing charac teristic nuclear impressions. (sheep x844)

Fig. 7.

Scanning electron micrograph of ciliary pro cess vein with overlying venule showing characteristic 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 filling delay but

gained

that secondary vertical rectus muscle surgery

demonstrating the normal presence or absence

from

fluorescein

angiography

by

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 scientific and 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. Sheep

ocular

vasculature

shows

many

We suggest that casting is a suitable tech nique with which to study the changes to the

similarities with human ocular vasculature and

anterior

may be used to study basic vascular architec

strabismus surgery, particularly the circulation

ture.

around the altered muscle insertion, the

Sheep,

anterior

however,

ciliary

arteries,

lack

penetrating

suggesting

non

segment

vasculature

following

"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

307


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