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MICROSCOPY RESEARCH AND TECHNIQUE 77:138142 (2014)

Improved Method of Producing Satisfactory Sections


of Whole Eyeball by Routine Histology
BENJAMIN ARKO-BOHAM,1,2 JOHN AHENKORAH,1 BISMARCK AFEDO HOTTOR,1 ESTHER DENNIS,1
1
AND FREDERICK KWAKU ADDAI *
1
2

Department of Anatomy, University of Ghana Medical School, Accra, Ghana


Department of Medical Laboratories, School of Allied Health Sciences, University of Ghana, Korle-Bu Campus, Accra, Ghana

KEY WORDS

antero-posterior; ocular globe; paraffin wax; phenol; light microscopy

ABSTRACT
To overcome the loss of structural integrity when eyeball sections are prepared
by wax embedding, we experimentally modified the routine histological procedure and report
satisfactorily well-preserved antero-posterior sections of whole eyeballs for teaching/learning
purposes. Presently histological sections of whole eyeballs are not readily available because substantial structural distortions attributable to variable consistency of tissue components (and
their undesired differential shrinkage) result from routine processing. Notably, at the dehydration stage of processing, the soft, gel-like vitreous humor considerably shrinks relative to the
tough fibrous sclera causing collapse of the ocular globe. Additionally, the combined effects of fixation, dehydration, and embedding at 60 C renders the eye lens too hard for microtome slicing at
thicknesses suitable for light microscopy. We satisfactorily preserved intact antero-posterior sections of eyeballs via routine paraffin wax processing procedure entailing two main modifications;
(i) careful needle aspiration of vitreous humor and replacement with molten wax prior to wax
infiltration; (ii) softening of lens in trimmed wax block by placing a drop of concentrated liquid
phenol on it for 3 h during microtomy. These variations of the routine histological method produced intact whole eyeball sections with retinal detachment as the only structural distortion.
Intact sections of the eyeball obtained compares well with the laborious, expensive, and 8-week
long celloidin method. Our method has wider potential usability than costly freeze drying
method which requires special skills and equipment (cryotome) and does not produce whole
eyeball sections. Microsc. Res. Tech. 77:138142, 2014. V 2013 Wiley Periodicals, Inc.
C

INTRODUCTION
The standard histological processing method comprising fixation, dehydration, embedding, sectioning,
staining, and mounting produces excellent histological
slides for most tissues, even when partly mechanised.
However, preparation of histological sections of the
eyeball by routine processing procedure fails to produce satisfactory results. Since a pioneering effort 127
years ago (Barrett, 1886) to produce histological sections of the eyeball by infiltrating and embedding in
paraffin, literature is apparently devoid of any later
attempt. Rather, a celloidin method also described in
the Barrett (1886) publication has become the preferred procedure for preparing sections of parts of the
eye for histology. Consequently whole sections of the
eyeball are not readily available for teaching and
learning. The variable consistency of the tissue components of the eyeball and their differential shrinkage
during processing (Slomianka, 2006) has been proposed as accounting for its apparent unsuitability for
processing by the routine method.
An intact eyeball is roughly spherical in shape
(Nowak and KeR pi
nska, 2006) and has a wall of three
distinct tissue tunics. From without inwards these are;
the fibrous (corneo-scleral) layer, the vascular (uveal)
layer, and the neural (retinal) layer (Moore and Dalley,
C
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2013 WILEY PERIODICALS, INC.

2006; Slomianka, 2006). The interior of the eye has


two fluid-containing compartments separated by the
lens and its suspensory ligaments. The anterior compartment extends from the inside of the cornea to the
anterior surface of the lens and is occupied by fluid
(the aqueous humor). The posterior compartment
extends from the posterior surface of the lens to the
retina and contains the vitreous humor forming
approximately 80% of the eyeball (Montgomery, 2006).
The pressure generated by these fluids fills out the
eyeball and helps to maintain its spherical shape
(Garrity, 2006).
The difficulty in preparing whole sections of the eyeball by routine paraffin embedding method arises
chiefly because of differential shrinkage between the
vitreous humor and the sclera (Slomianka, 2006). The
soft vitreous humor, consisting of more than 99% water
*Correspondence to: Frederick K. Addai, Department of Anatomy, University
of Ghana Medical School, P.O. Box GP 4236, Accra, Ghana. E-mail:
faddai@chs.edu.gh
Received 19 July 2013; accepted in revised form 7 November 2013
REVIEW EDITOR: Prof. George Ruben
Contract grant sponsor: Carnegie funded University of Ghana Next Generation of Academics in Africa Project.
DOI 10.1002/jemt.22320
Published online 18 November 2013 in Wiley Online Library (wileyonlinelibrary.
com).

EYEBALL SECTIONS FROM WAX EMBEDDED BLOCKS

but having a gelatinous viscosity two to four times that


of water (Henrikson et al., 1997) undergoes considerable shrinkage during the dehydration stage of routine
histological tissue processing. The sclera, however, is
tough and undergoes less shrinkage relative to the vitreous humor. This differential shrinkage leads to the
collapse of the globe making it difficult to maintain the
spherical shape of the eyeball for embedding and sectioning. As a result many artifacts occur during processing, including loss of globular structure, spatial
dislocation of the iris, lens, ciliary body, and detachment of the retina from the pigment epithelium. Thus,
histological preparation of only parts of the eyeball has
become a way to overcome at least some of the problems (Slomianka, 2006).
Successful methods for producing histological sections of the eyeball exist. However, these are far more
expensive than the routine method, and require
reagents and equipment that are not easily obtainable
by resource-challenged laboratories. For instance the
celloidin method which produces good whole eyeball
sections involves the use of expensive (Landes et al.,
2005; Ritman, 2000) and potentially explosive chemicals. The celloidin method is also laborious because
sections are produced in nonribbon fashion (Drury and
Willington, 1980); and time-consuming because it
takes 8 weeks to complete (Landes et al., 2005).
There is also a freeze drying method for producing sections of the eye. Freeze drying requires expensive liquid nitrogen/cryotome, highly skilled (cryosectioning)
expertise, and is not suitable for producing whole
sections.
We report manual modifications of the routine tissue
processing procedure that produced substantially wellpreserved whole sections of eyeballs with minimal
distortions.
MATERIALS AND METHODS
This work was approved by the Ethical and Protocol
Review Committee of the University of Ghana Medical
School. Eyeballs were obtained from guinea pigs
(Cavia sp.). Animals were tagged young if they were
58 weeks old; and old if they were above 10 weeks
in age. Procedures involving the care and use of guinea
pigs conformed to the institutional guidelines in compliance with national and international laws and
guidelines for the use of animals in biomedical
research. The animals were anesthetized with chloroform prior to perfusion fixation via intracardiac canulation with 10% neutral buffered formalin (BDH
Chemicals Poole, England). Eyeballs were dissected
out from perfused animals and postfixed for 35 days
in either 10% neutral buffered formalin or Bouins
solution. Following fixation, eyeballs were dehydrated
through graded ethanols (BDH Chemicals, Poole,
England).
Prior to paraffin wax infiltration, the vitreous
humor in the eyeball was gently aspirated using a
syringe fitted with a hypodermic needle (JMK, 0.5 x
16 mm). The needle was inserted through the optic
nerve in order to enter the vitreous space by the optic
disc to avoid puncturing the fine and delicate retina. A
similar hypodermic syringe and needle was used via
the same optic nerve route to inject molten wax into
the evacuated vitreous chamber to replace the aspiMicroscopy Research and Technique

139

rated fluid. This procedure needed to be carried out


extremely swiftly before the molten wax solidified to
clog the tiny needle bore. The eyeball was prewarmed
by placement in molten wax in an oven at 60 C for one
minute before the molten wax was quickly injected to
replace aspirated vitreous humor via the optic disc.
The volume of wax injected into the vitreous space
was approximately the same as that of its aspirated
vitreous humor.
This study had a pilot phase in which several materials and variations of protocol were experimented
with. For instance, replacement of vitreous humor
with wax was done in some eyeballs before dehydration and in others after dehydration to determine the
better effect. The rationale for the vitreous substitution was to fill the eyeball with a substance that would
not shrink as much as the gel-like humor during dehydration, and so prevent significant structural distortion of the globe. It was also reckoned that the
substance substituted for vitreous humor must withstand the high temperature or wax embedding with
change (such as solidification) that filled the eyeball
and prevented its collapse. Hence materials used
besides molten paraffin wax in pilot experiments were
fresh (uncooked) cassava (Manihot spp.) starch, cooked
cassava starch, raw egg yolk, and raw egg albumin.
Checker board aqueous concentrations of these materials were used to find the ideal consistency for replacement of the vitreous body via hypodermic syringe and
needle. Apart from molten wax, all other substances
injected to replace the vitreous required hourly top-up
during subsequent processing to ensure that the eye
globe retained its shape. This was done to replace loss
of injected substance that may have arisen from leakage, or altered volume. For instance uncooked cassava
starch lost volume during dehydration and the hourly
top-up involved injecting just enough to restore the eye
globe to its original shape.
Since a certain degree of shrinkage during histological processing is attributable to fixation and varies
with the fixative used (Kiernan, 2000), we also experimented with two fixatives. Ten percent buffered formalin and Bouins solution were used to assess the degree
of tissue shrinkage, which was measured at both macroscopic and microscopic levels. At the macroscopic
level, whole eyeballs were measured before and after
tissue processing using a digital vernier calliper
(MAPLIN Electronics; Product No. N49FB). In the latter case, wax blocks were carefully broken to free
embedded eyeballs whose mid-coronal diameters were
then measured. At the microscopic level, diameters of
red blood cells were measured in fresh thin blood films
and in histological sections using a pre-calibrated
microscope stage micrometer (Graticules Tonebridge,
Kent. England).
After vitreous substitution, the eyeball was placed
in three changes of molten paraffin wax (2.5 h each)
for infiltration at 60 C in an oven. Finally each eyeball
was manually embedded in metallic moulds measuring
7 mm x 7mm. Wax embedded eyeballs were trimmed
using a rotary microtome (LEICA RM 2125RT; Leica
Microsystems; Ernst-Leitz-Strasse, Wetzlar, Germany)
along their antero-posterior axes until the lenses were
exposed. Use of razor blade to trim the wax block prior
to microtomy produced undesired results. Mainly,

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B. ARKO-BOHAM ET AL.

Fig. 1. Photograph of a wax block showing an embedded eyeball


trimmed to expose the lens. Inserts: Outer broken ring demarcates
extent of the whole lens; inner continuous ring demarcates the
nucleus of lens. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.].

razor trimming rendered the cutting face of the wax


block uneven, and this impeded efficient application of
a drop of phenol on the eye lens subsequently. Unevenness of razor trimmed block face arose in part from the
difficulty of generating adequate pressure with handheld razor to cut the hardened eye lens without worrisome distortion of the wax block. To soften the lens a
drop of concentrated (99%) liquid phenol (SIGMA,
Catalogue No. P-3653) was carefully placed at the
nucleus of the exposed lens (Fig. 1) and left for 3 h. The
duration of exposure was obtained from several trials
until the ideal was determined.
Microtomy of the wax block was resumed after phenol treatment of the lens, and full sections in parasagittal and mid-sagittal planes were cut at a
thickness of 10 lm. Sections thinner than 10 mm (particularly standard 57 mm) disintegrated when being
mounted of a glass slide. Ribbons of 10 mm sections
were picked and placed on warm (25 C) water in a
bath and subsequently transferred onto precleaned
glass slides (All Pro, Middlesex, England). The slides
with the eyeball sections were placed in a preheated
(30 C) oven overnight to facilitate good adhesion of
the sections to the glass to prevent them from falling
off during staining. Sections were manually stained
with haematoxylin and Eosin using standard procedures, and mounted with DPX (BDH, Poole, England). Slides of whole-eyeball sections were examined
and photographed using a digital microscope eyepiece
(Catalogue # MA 88; C & A Scientific, Manassas, Virginia) attached to a dissecting microscope (Nikon
84534).
RESULTS
Fixation and Dehydration
Fixation with Bouins solution produced 0.25 mm
(SE 60.07) reduction in mean mid-coronal diameter of
15 eyeballs while 10% neutral buffered formalin
shrunk the average diameter of 6 eyeballs by 0.42 mm
(SE 6 0.25). Compared with the diameter of unfixed
red blood cells of 8.5 mm (SE 6 0.8) there was a 20%
reduction in formalin fixed eyeballs (7.19 mm, SE 6 0.4)
and 17% (7.75 mm, SE 6 0.9) in Bouinssolution-fixed
eyeballs.

Substitution of Vitreous Humor


The volume of vitreous fluid in eyeballs of studied
guinea pigs had a mean of 0.6 ml (SE 6 0.1). An
approximately equal volume of studied materials
injected into aspirated vitreous humor just before
dehydration retained the shape of the eyeball somewhat. Hourly top-up of substituted egg yolk, egg albumin, and uncooked cassava starch during passage
through graded alcohols minimized gross structural
distortions compared with eyeballs without serial additions. Eyeballs in which vitreous humor was replaced
with molten wax showed the least structural distortions after dehydration and did not require serial topups. Except for paraffin wax-filled eyeballs, eyeballs
filled with all other study materials failed to produce
ribbons or intact slices useful for mounting on glass
slides.
Many problems attended wax substitution of vitreous humor before dehydration, with the key one being
distortion of the eyeball surface during passage
through graded alcohols and the clearing agent. Wax
substitution of vitreous body in eyeballs after dehydration and clearing eliminated the problems including
significant reduction in the structural displacement
and distortions of component parts (Figs. 2a and 2b).
Softening of Eye Lens With Phenol
Sectioning of eyeballs in which vitreous body was
replaced with molten wax produced incomplete sections because of hardness of the lens that either flaked
or fell out leaving a hollow at the centre of the capsule
(Fig. 2a). Treatment with a drop of concentrated phenol softened the lens to permit full intact sectioning of
the eyeball with a nicely sliced lens (Fig. 2b). This
result was consistent with sections prepared from 15
eyeballs, but detachment of retina persisted as the
only key structural distortion. The phenol treatment
produced better results on the lens of young compared with old animals.
DISCUSSION
The relatively milder shrinkage of eyeballs produced
by Bouins solution as a fixative may be explained by
the rapid cytoplasmic coagulation of picric acid
that augmented the protein cross-linking of formalin
(Labart-Moleur et al., 1998). Rapid coagulation of cytoplasm has two potential advantages. First, it could
restrict osmotic movement of fluid from the cells interior to the exterior; and second it could produce cellular rigidity which could limit pulling by contracting
cross-linked extracellular proteins (fibers). Our
reported improved technique for producing whole histological sections of eyeballs works best with Bouins
fixative.
Other study materials (egg albumin, egg yolk, and
cassava starch) failed to produce histological sections,
although they served the purpose of preventing eye
globe collapse during dehydration. Apparently, residual water in the materials after dehydration impeded
adequate wax infiltration which in turn prevented continuous ribbons from being obtained during sectioning.
Surface shrinkage of the eyeball in which the vitreous body was replaced with molten wax before dehydration may have arisen from reduction in the volume
Microscopy Research and Technique

EYEBALL SECTIONS FROM WAX EMBEDDED BLOCKS

141

mostly water (Henrikson et al., 1997) hence immiscible


with wax. It is plausible that a substantial amount of
the fluid still remained even after routine dehydration
and clearing during histological processing. There is
need therefore for the vitreous humor to be manually
removed and replaced with wax. The injection of paraffin wax into the vitreous space immediately prior to
the embedding stage of tissue processing protocol produced very satisfactory results.
On account of differential shrinkage leading to eventual collapse of ocular globe, the retina is highly prone
to detachment and disintegration during histological
processing. It is postulated that injecting paraffin wax
into the vitreous space as per our improved protocol
helped to hold the fragile retina in place and close to
the uvea in its intact and continuous state. Although
during the aspiration of the vitreous fluid, some noticeable collapse was evident, the prevailing conditions
were not too harsh on the eyeball to cause destruction
of the retina. Retinal detachment was however, not
completely eradicated but the extent was minimal
(Figs. 2a and 2b). The preservation of structural integrity of the retina with respect to its position in the
globe achieved by our innovative modifications (Fig.
2b) is far superior compared to what is typically
obtained via routine histological processing (Fig. 2c).
Although effort is being made to completely eliminate
the retinal detachment in our preparations, it is proposed that the present sections may serve as useful
teaching material for detached retina that occurs in
some people (Delyfer et al., 2011; Ivanisevic et al.,
2000; Li, 2003). It may also prove useful in experimental study of diseases of the retina and choroid such as
performed by Dundar et al., 2012.

Fig. 2. (ac) Photomicrographs of H&E stained antero-posterior


sections of eyeball. Eyeball in 2a and 2b were prepared according to
our method of vitreous homour aspiration and replacement with molten wax just before paraffin embedding. In 2a the lens was not
treated with concentrated liquid phenol. During sectioning, the overhardened lens got plucked out leaving its peripheral remnant (black
arrows) and a resultant void (demarcated by broken white ring). In
2b the lens was treated with phenol as per our protocol and shows an
intact lens in situ. In 2c eyeball was prepared as per standard histological processing protocol without any modifications. It shows a collapsed globe with displaced and disintegrated retina, and lens with a
void (*) at the center. Abbreviations: a, anterior chamber; v, posterior
chamber; c, cornea; i, iris; p, pupil; r, retina; su, sclera and uvea.
[Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.].

of paraffin. This could result from cooling in the graded


alcohols and/or possible leakage of wax through the
needle hole in the optic disc through which it was
injected into the vitreous space. Neither cooling nor
leakage of wax was expected to happen in eyeballs in
which vitreous substitution with wax was done after
dehydration. This is because the procedure was immediately followed by (pre-embedding) incubation in molten wax at 60 C.
In untreated eyeballs, it is almost impossible for
wax to get into the vitreous space. The vitreous fluid is
Microscopy Research and Technique

Phenol Application Made Lens Sectionable


The temperature range (5560 C) for wax infiltration in routine histological protocol has an undesired
effect on the lens of the eye (Barrett, 1886). The constituent crystalline proteins become transformed and
harden, especially at the sclerotic nucleus. The overhardened lens becomes unsuitable for sectioning with
a standard microtome blade or knife. The worst case is
that, the microtome blade is unable to slice through
the hardened lens. Impact of the lens with the microtome blade plucked off the lens nucleus from its position in the tissue/wax block, and sometimes broke the
block apart. Another observation is illustrated in the
controls in this study (Fig. 2a) where the lens was sectioned alright, but the central nucleus turned powdery
and flaked off leaving a void in the lens.
To overcome the problem of over-hardened lens, Barrett (1886) used paraffin that melted at 35 C but failed
to obtain satisfactory results. In the end, he concluded
that sections of parts of the eye without the lens of
young or embryonic eyes may be readily obtained by
infiltrating and embedding in paraffin using chloroform as clearing agent. Bancroft et al. (1996) proposed
addition of phenol to the alcohol used in dehydration,
particularly the lower grades (50 and 70%) to soften
the lens. Their suggestion was on the premise that, in
Molecular Biology, proteins and restriction enzymes
are removed by phenol and/or chloroform in disrupting
secondary structure formation of proteins causing
them to denature and precipitate from solution. If the

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B. ARKO-BOHAM ET AL.

protein precipitation and their secondary structure


formation are disrupted, in this case in the lens, it is
presumed that the lens will be made relatively softer
than it otherwise would, if the protein secondary structures had formed. The effect of phenol on the lens in
the present work, according to Bancroft et al. protocol
failed to produce satisfactory eyeball sections.
Application of concentrated phenol to the lens in the
present report yielded better results in eyeballs
obtained from relatively younger animals compared
with eyeballs belonging to older animals. The influence of age on the eye lens response to phenol is explicable by its unique structure and developmental
history. When the eye is developing in embryos, primary lens fibers first form when epithelial cells on the
anterior surface migrate to the equatorial region
where they transform into lenticular cells. In later
development throughout life, maturing primary lens
fibers are pushed more and more to the center (or
nucleus) of the lens which becomes surrounded by the
newly formed secondary fibers at the periphery (Kral,
1999; Standring et al., 2005). Thus, the older the animal, the more tightly packed the nuclear lens fibers,
leading to senile sclerosis in older eyeballs. We suggest that this phenomenon may explain why eyeballs
obtained from older animals failed to produce satisfactory effect of phenol on the lens for the 3 h exposure
period. Lens of eyeballs obtained from younger guinea
pigs, on the other hand, showed very satisfactory
response to the softening effect of phenol. When the
phenol was placed on exposed lenses of blocked eyeballs the result was desirable. The single phenol drop
placed at the center of the lens softened the nucleus,
relative to the cortex making sectioning possible and
uniform.
CONCLUSIONS
This work reports manual modifications of the routine paraffin wax method for tissue processing, which
produced whole histological sections of the eyeball far
superior to what otherwise could be obtained by the
procedure. The variations of routine (3-day long) histological method produced intact whole eyeball sections
with retinal detachment as the only residual structural distortion. Our technique produced intact sections of the eyeball that compares favorably with the
laborious, expensive, and 8-week duration celloidin
method. Being a modification of routine histological
technique, our method is potentially more widely usable than the highly skilled, special equipment- (cryotome)-dependent, and costly freeze drying method.
ACKNOWLEDGMENTS
The authors are grateful to the staff of the Department
of Anatomy, University of Ghana especially Messrs
Samuel Mensah, Archibald Danquah Amoah and Paul
Atiah for their technical support. The authors are also

grateful to Central Administration of the College of


Health Sciences, the University of Ghana Medical
School, and the School of Allied Health Sciences; all of
University of Ghana, for financial and administrative
support.
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Microscopy Research and Technique