iris arterioles originate. *Polysciences Ltd, 24 Low Farm Place, Moulton Park, Northampton NN3 IHY. .... 16 Tano Y, Chandler DB, Machemer R. Vascular casts.
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Recently, latex studies have been superceded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
ANTERIOR SEGMENT VASCULAR CASTING
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
ANTERIOR SEGMENT VASCULAR CASTING
vascular arterial anastomoses as seen on corrosion casts by scanning electron microscopy. Plas Recon
assistance. This work is supported by the Francis and Renee Hock Trust.
struc Surg1979, 64: 59-64.
References I da Vinci L: c1508. The brain injected to demonstrate
18
Nopanitaya W, Aghajanian JG, Gray LD: An improved plastic mixture for corrosion casting of the gastro-intestinal microvascular system. SEM 1979: Part Ill, SEM, Inc AMF O'Hare., 11 60666,
19
Simoen P. In Simoens P (thesis): Morphologic study
the shape of the cerebral ventricles. Royal Library, Windsor Castle. Cat19127. R. Anatomical draw ings from the Royal Collection. Royal Academy of
751-755.
Arts, London1977, p 48 and plate10.
of the vasculature in the orbit and eyeball of the 20
Br J Ophthalmol1952,36: 465-81. eye in man.
J Anat1954, 88: 94--101.
course.
and
intra-cranial
Part
II.
the
course.
Br J 21
Br J Ophthalmol1951, 35: 291303. Part II. Aqueous veins cont. Br J Ophthalmol 1952, 36: 265-fJ7. Part III. Arterial relations of Schlemm's Canal. Br J Ophthalmol1953, 37:57786. Aqueous veins.
Nowell JA, Pangborn J, Tyler WS: Scanning electron
surgery in a patient with chronic lymphocytic leu kaemia. 23
Reinecke RD: Anterior segment ischaemia fol
0, lIT Research Institute Chicago Illinois 1970,249-56.
lowing strabismus surgery.
the blood vessels.
J Paed Ophthalmol & Strab1984, 21: 179-84. (Discussion by France TD p185 ).
24
Arch Histol Jap1971,32: 445-
AAPO&S experience. J Paed Ophthalmol & Strab
1986, 23: 87-91.
Woodlief NF: Initial observations on the Ocular Microcirculation in Man.
Arch Ophthalmol1980,
25
98: 1268-72. casting
and
human
ocular
scanning
electron
vascular
microscopy of
abnormalities.
26
Arch
28
Freeman HM, Hawkins W R, Schepens CL: Anterior
J Paed Ophthalmol & Strab1978, 15: 197-204.
Shimizu K and Ujiie K: Structure of Ocular Vessels. Morrison JC and Van Buskirk M: Anterior collateral
segment necrosis. An experimental study. 29
Ophthalmology
Ophthalmology1987,28:1325-
the
rabbit:
A
fluorescein
Can J Ophthalmol1975, 10:
Wilcox LM, Keough EM, Connolly RJ: Regional following selective tenotomy in primates.
31
Lab Invest1983, 48:
Virdi PS and Hayreh SS: Anterior segment ischaemia after recession of various recti. An experimental study.
Ohkuma H and Ryan SJ: Vascular casts of experi
Exp Eye
Res1981,33: 353-fJ0.
169-80. 32
Ophthalmology1987,94: 10 1258-7l.
Hayreh SS and Scott WE: Fluorescein Iris Angiogra
mental subretinal neovascularisation in monkeys.
phy. II. Disturbance in iris circulation following
Invest Ophthalmol1983, 24: 481-90.
strabismus operations on various recti.
Am J
Ophthalmol1981; 92: 110--20. van Gelder P A and Klopper PJ: Healing of micro-
Arch
Ophthalmol1978, 96: 1390-400.
Tano Y, Chandler DB, Machemer R. Vascular casts of experimental retinal neovascularisation.
17
in
ischaemia and compensatory vascular dynamics
Burger PC, Chandler DB, Klintworth GK: Corneal neovascularisation as studied by scanning electron
16
ischaemia
255-62. 30
40.
15
Kottow M, Wassilewa P, Hendrickson P: Provoked iris
Morrison JC, DeFrank MP, Van Buskirk EM: Com parative Microvascular Anatomy of Mammalian
Arch
Ophthalmol1966, 75: 644--50.
angiographic study.
microscopy of vascular casts.
Can J
iridis.
Hiatt RL: Production of anterior scgment ischaemia.
1983,90: 707-15.
14
necrosis and rubeosis
27
Arch Ophthalmol 1987, 195:
681-7.
Ciliary Processes.
Anderson DM and Morin JD: Experimental anterior
OphthalmoI1971, 6: 196--201.
Yoncya S, Tso M OM: Angioarchitecture of the
circulation in the primate eye.
Am J
Ophthalmol1987, 103: 833-4. segment
Ophthalmol1985, 103: 118-20.
Igaku-Shoin Medical Publishers, New York,1978.
!3
Elsas FJ and Witherspoon CD: Anterior segment ischaemia after strabismus surgery in a child.
Fryczkowski AW, Grimson BS, Peiffer RL: Vascular
human choroid.
12
France TD and Simon JW: Anterior segment isch aemia syndrome following muscle surgery: The
54.
II
Ophthalmic Surg1976, 7: 42-8.
Simon JW, Price EC, Krohel GB, Poulin RW,
microscopy of the avian lung in Scanning Electron
microscope to the study of the fine distribution of
!O
Am J Ophthalmol1982, 93: 34-48.
Jacobs DS, Vastine DW, Urist MJ: Anterior segment ischaemia and sector iris atrophy. After strabismus
Murakami T: Application of the scanning electron
9
International
Saunders RA and Sandall GS: Antcrior segment isch position.
22
Microscopy. Ed. Johari
8
the
aemia syndrome following rectus muscle trans
aqueous veins by means of neoprene casts. Part I.
7
of
1978, 375-80.
Ashton N: Anatomical study of Schlemm's canal and
6
Meeting
Kyoto, Japan. New York, Grune & Stratton, Inc,
Ophthalmol1962, 46: 212-47. 5
Third
Strabismological Association, May 10--12, 1978,
and intra-canalicular
Intra-orbital
In
Reinecke RD (Ed): Strabismus. Proceedigns of
Hayreh SS and Dass R: The ophthalmic artery. Part I. Origin
Fells P and Marsh RJ: Anterior segment ischaemia following surgery on two rectus muscles.
Wybar KC: A study of the choroidal circulation of the
3
4
pig. 1985. State University of Ghent, pp 217.
Ashton N: Observations on the choroidal circulation.
2
33
Meyer PAR and Watson P: Low dose fluorescein angiography of the conjunctiva and episclera.
Ophthalmol1987, 71: 2-10.
Br J