Oval corneal opacities in beagles

III. Histochemical demonstration of stromal lipids

without hyperlipidemia
Alan M. Roth, Marilyn B. Ekins, George O. Waring III, Lata M. Gupta,
and Leon S. Rosenblatt

We found oval stromal avascular corneal opacities in 128 eyes of 75 beagles from 497 studied.
There were three morphologic types that progressed in severity with time: nebular, racetrack,
and white arc. Histochemical sti'^n r "the earliest morphologic type (nebular) revealed neutral
fats, cholesterol, phospholipids, and sometimes fatty acids both intracellularly and extracel-
lularly. We found no elevation of serum cholesterol or triglycerides except in dogs with the most
advanced inorphologic type (white arc) and no alteration in thyrometabolic function. We think
that oval stromal opacities in beagle corneas are a primary disorder ofcorneal lipid metabolism
closely resembling the central crystalline dystrophy of Schnyder and may be an animal model
for this human disease.

Key words: corneal metabolism, primary lipid dystrophy, Schnyder's crystalline

dystrophy, lipid histochemistry, animal model

c <orneal dystrophies and degenerations

have been described rarely in dogs.1 Al-
though sporadic cases have been reported,
none has resembled the oval stromal corneal
opacities we previously described in 128 eyes
of 75 purebred beagles from a colony of 497
dogs.2 These anterior stromal opacities were
oval, about 3 by 5 mm, located at the junction
of the middle and inferior thirds of the avas-
cular cornea, and were usually bilaterally
From the Departments of Ophthalmology (Drs.. Roth,
Ekins, Waring, and Ms. Gupta) and Pathology (Dr.
Roth), School of Medicine, and the Radiobiology Lab-
oratory, University of California, Davis, and Genti-
con, Walnut Creek, Calif. (Dr. Rosenblatt).
Supported by National Eye Institute Grant EY03302 and
grants from the National Society for the Prevention of
Blindness, the U.S. Department of Energy, and the
Medical Research Council of Canada.
Submitted for publication Nov. 2, 1979.
Reprint requests: George O. Waring, M.D., Emory
University Clinic, Department of Ophthalmology,
1365 Clifton Road N.E., Atlanta, Ga. 30322.
0146-0404/81/070095+12$01.20/0 1981 Assoc. for Res. in Vis. and Ophthal., Inc.
symmetrical. We observed three morphologic
types: nebular, with a homogenous gray ap-
pearance (Fig. 1); racetrack, with a gray oval
surrounding a slightly depressed, brownish
center (Fig. 2); and white arc, with subepi-
thelial, dense, white plaques of granular or
spicule-shaped material (Fig. 3). These three
patterns represent different stages in the
natural history of the disorder, progressing
from the nebular (least severe) to the white
arc (most advanced).3 Because the colony was
outbred,4 we could not establish a hereditary
pattern. We have also seen similar corneal
opacities in beagles that were not part of this
colony (Fig. 4).
When we initially described the clinical
appearance of oval stromal corneal opacities
in beagles,2 we did not know what material
composed these lesions. Negative findings
with standard histochemical stains and the
presence of vacuoles and crystals in trans-
mission electron micrographs led us to search
for lipids. Although we recognized that lipid
95

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 Invest. Ophthalmol. Vis. Sci,
96 Roth et at. July 1981

Fig. 1. Nebular oval corneal opacity has homogeneous gray appearance with indistinct margins.

Fig. 2. Racetrack oval opacity has slightly depressed, brownish center.

histochemistry is an imprecise art and that
only a few techniques define lipids with cer-
tainty, we decided to perform a large battery
of techniques to give the most comprehen-
sive data. These may then be interpreted in
light of the limitations of such testing and
correlated with more precise biochemical
analysis. We report here the histopathologic
findings and histochemical demonstration of
lipids in these opacities and show that serum
lipid levels and thyroid hormone assays are
similar in both affected beagles and age- and
sex-matched controls.
Materials and methods
All beagles came from a colony involved in
lifelong radionuclide toxicity studies at the Radio-
biology Laboratory, University of California,
Davis.1' 8 As each dog was scheduled for sacrifice,
we reviewed our previous records to determine

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 "'f--'"

Volume 21
Number 1, Part I Oval corneal opacities in beagles 97

Fig. 3. White arc oval opacity is densely white, granular subepithelial deposit.

Fig. 4. Oval opacity in beagle from outside the colony studied shows double row of gray
crystalloid deposits (left), which stand out prominently in retroillumination (right).

the type of corneal opacity present. After the dog
was anesthetized with intravenous sodium pen-
tobarbital, we verified the type of corneal opacity
by inspection with a penlight and enucleated the
eyes. We immediately removed a corneosclerat
shell by cutting into the epichoroidal space 4 mm
posterior to the limbus, excising the shell with
scissors, and disinserting the ciliary body from the
scleral spur. The cornea was placed epithelial side
clown on a silicone block and trisected through the
oval opacity; one third was fixed in cold 2% glu-
taraldehyde in Millonig's buffer for electron mi-
croscopy, one third was frozen in liquid nitrogen
for biochemical determinations, and one third was
fixed in Baker's fonnol-calcium solution7 for light
microscopy.
We recorded the nutritional and medical his-
tory of each dog. An experienced veterinary pa-
thologist performed a complete autopsy.
Histochetnistry. We studied 29 corneas from 16
affected beagles that had the nebular type opacity
and two corneas from two unaffected beagles.
Three specimens from two dogs were blocked in
paraffin only. Because routine histochemical stains

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 Invest. Ophthalmol. Vis. Sri.
98 Roth et al. July 1981

Table I. Histochemical stains done for lipid

Refer- Speci-
ence Type of reaction Lipid demonstrated ficity
Oil red 0 7 Lipid-soluble dye Neutral fat High
Suden black B 30 Lipid-soluble dye Neutral fat especially Moderate
phospholipid
Baker 28 Acid hematein with pyridine extraction Phospholipid Low
Pearse 31 Copper phthalocyanin Phospholipid Low
Menschik 32 Nile blue sulfate Phospholipid Low
Landing et al. 33 Phosphomolybdic acid Choline-containing Low
phospholipid
Fischler 27 Cupric soap-lithium hematoxylin Fatty acid Moderate
Holczinger 27 Cupric soap-rubeanic acid Fatty acid High
Schultz 27 Acetic-sulfuric acid Cholesterol Moderate
Adams 34 Perchloric acid-naphthoquinone Cholesterol High
Okamoto 27 Sulfuric-iodide Cholesterol Moderate
Bromine-Sudan black B 35 Bromination unmasking Unmasked cholesterol Low
Mukherji 27 Bromine-silver Unsaturated lipid Moderate
Norton 36 Bromine-silver Unsaturated lipid Moderate
Osmium tetroxide 28 Lipid-soluble reagent Unsaturated lipid Low
Seligman-Ashbel 7 Naphthoic hydrazide Active carbonyl lipids Low

of sections cut from these blocks revealed no ab-
normal substances, because the ultrastructural
studies on these specimens showed intracellular
vacuoles and crystals suggestive of lipids (Spang-
ler, Waring and Morrin, unpublished data), and
because the clinical appearance was compatible
with lipid deposits, we divided the formol-cal-
cium -fixed tissue from each of the remaining
26 corneas into two pieces. We embedded one of
these in paraffin and stained sections with hema-
toxylin and eosin, Masson's trichrome method, the
periodic acid-Schiff reaction, and Alcian blue at
pH 2.5. The other piece was frozen, and sections
were cut 10 to 15 /xm thick and stained by 16
histochemical methods for demonstrating lipids
(Table I). We stained sections of brain known to
contain the different lipids with each corneal spec-
imen as a control. Sections being analyzed for cho-
lesterol were examined within 15 min of staining
because of rapid oxidation of the reaction and be-
cause the sections were destroyed by the tech-
nique. Paraffin and frozen sections were also
examined under plane-polarized light for bire-
fringence.
Serum lipid measurements. We measured se-
rum cholesterol and triglycerides in affected
beagles and in age- and sex-matched control
beagles on two occasions, 1 year apart. In Decem-
ber 1977, we paired each of 48 affected beagles (28
males, 20 females) with an unaffected beagle of the
same age and sex (27 males, 21 females) in the
same radiation group (90Sr, 226Ra, or not radiated).
Some of the affected and control dogs were litter-
mates. In January 1979, we re-examined the 29
surviving affected beagles (18 male, 11 female) and
the 40 surviving controls (24 male, 16 female). The
mean ages of the control and affected dogs were
comparable for both sexes (male 12.5 years, female
12.6 years). After the clogs had fasted 6 hr, we
collected 10 ml of clotted blood with a 20-gauge
needle from the left jugular vein. The samples
were immediately centrifuged at 3000 rpm at 4 C;
the serum was removed and stored at 4 C. We
measured total cholesterol by the Technicon Au-
toAnalyzer II (Clinical Method No. 24/prelimi-
nary March 1972) for the first determination and
by the enzymatic method of Patsch8 for the
second. The high-density-lipoprotein cholesterol
(CHDL) <X cholesterol) was determined by NIH pro-
tocol,9 and triglycerides by Fletchers technique. l0
The combined low-density-, and very-low-den-
sity-lipoprotein cholesterol (C LD L and CVLDL> j3 and
pre-/3 cholesterol) was then calculated by subtract-
ing the CHDL from the total cholesterol. 9 All
solvents used were reagent grade. To confirm the
accuracy of our serum lipid measurements, we
employed standards of known lipid concentra-
tion and also sent random beagle serum samples to
an independent laboratory that used the same
methods.
We employed analysis of variance and regres-
sion analysis to compare serum lipid measure-
ments from the total number of affected and con-
trol dogs, to compare each of the three affected
subgroups (nebular, racetrack, and white arc) to
each other and to the controls, and to compare
sexes and ages. We used data only from beagles
whose serum lipids were measured twice. Each of

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 Volume 21
Number 1, Part 1
the affected dogs was classed according to the le-
sion of its more severely affected eye. Between the
first and second lipid determination, the opacities
in some of the affected dogs progressed; thus the
same dog may be in one affected subgroup in 1977
and in another in 1979.
Thyroid hormone assays. We tested thyromet-
abolic function in 24 affected beagles and in
39 age- and sex-matched control beagles. Blood
was collected from the jugular vein and the serum
was immediately separated as described above.
The samples were stored at 4 C for no longer than
48 hr before analysis. Thyroxine (T4) levels were
measured by the Quantimune T-4 Radioimmuno-
assay technique (Bio-Rad Laboratories Bulletin
4202, October 1978), and the binding capacity of
thyroid hormone carrier proteins was assayed by
the Quanta-Count T3 resin uptake method (Bio-
Rad Bulletin 4212, March 1977). The free thyroid
index (T7) was calculated by multiplying the T4
level and the T3 percent resin uptake (%RU).
We employed chi-square analysis to compare
the results between control and affected dogs, be-
tween control dogs and those in each affected sub-
group (nebular, racetrack and white arc), and be-
tween dogs in each subgroup. Statistical review
was performed by Dr. Rosenblatt.
Results
Complete autopsies on all dogs revealed no
changes suggestive of a systemic lipid meta-
bolic disorder.
Histology and general histochemistry. Par-
affin sections stained with hematoxylin and
eosin and with Masson's trichrome method
demonstrated that the epithelium and its
basal lamina generally were intact with occa-
sional focal acanthosis (facet formation). Al-
though Bowman's layer is not normally pres-
ent in beagle corneas (Morrin, Waring,
Spangler, unpublished data), the normal an-
terior stroma is hypocellular, with interlacing
collagen bundles. The major abnormality in
the affected corneas was in this area, with
disruption of collagen architecture, focal
areas of collagen fibril fragmentation, stromal
vacuole formation, and scattered enlarged
vacuolated keratocytes. No blood vessels
were seen.
Periodic acid-Schiff and Alcian blue reac-
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Oval cornea! opacities in beagles 99
tions revealed no gross abnormalities in glyco-
saminoglycans. Plane-polarized light showed
no birefringence in sections from paraffin
blocks. Frozen sections, however, contained
many doubly refractile crystals, primarily
within keratocytes, but occasionally in the ex-
tracellular stroma. The posterior stroma, Des-
cemet's membrane, and endothelium were
normal.
Lipid histochemistry. Table II shows the
results of the histochemical reactions for
lipids in 26 affected corneas. The right and
left corneas from each dog usually stained
similarly. All stains were positive on the
known control tissue and showed no reaction
in the two unaffected control corneas. The
majority of positive stains were intracellular
with many having extracellular staining as
well. No case showed only extracellular stain,
although a few had such dense overall reac-
tion that we could not ascertain its location.
Stains for neutral fats (oil red O and Sudan
black B) were positive in all corneas, primar-
ily in the anterior half. In general, the intra-
cellular stain was in large globules, whereas
the stromal stain was in finer granules (Fig.
5). At least one of the methods for demon-
strating cholesterol (Schultz, Okamoto, and
Adams) was positive in each cornea (Fig. 6).
The majority were positive by all three
techniques. All corneas demonstrated phos-
pholipids with at least one of four reactions
for phospholipid (Baker, Pearse, Landing,
and Menschik), and 12 of the 26 corneas
demonstrated fatty acids by at least one of the
tests (Fischler, Holczinger). The remainder
of the histochemical reactions were negative,
although sporadic intracellular staining was
sometimes present.
Serum lipid measurements. There was no
significant difference between affected and
control dogs in either 1977 or 1979 for total
cholesterol, CHDL, C L D L , and CVLDL, or tri-
glycerides (Table III). The influence of sex on
serum lipids was also negligible except that,
in 1979, affected females showed higher tri-
glycerides than affected males. The influence
of age on serum lipids was less clear-cut
(Table III). In 1977, there was a statistically
significant trend for serum cholesterol to rise
 Invest. Ophthalmol. Vis. Sci.
100 Roth et al. July 1981

Fig. 5. Neutral fat appears as large globules in keratocytes, whereas smaller extracellular
granules accumulate in anterior stroma. (Frozen section, oil red O; X200.)

Table II. Results of histochemical stains foi lipid

A EI C D I F

Dog and cornea No 1 2 3 4 5 6 7 8 9 10 11

Oil red O +
Suden black B ++
Bromine-Sudan black B ++
Schultz ++
Okamoto +
Adams +
Baker* ++
Pearse ++
Landing ++
Menschik
Fischler
Holczinger
OsO4
Norton
Mukherji
Seligman-Ashbel
- = Negative; + = mildly positive; ++ = moderately positive; + + + = strongly positive.
Dogs D and M supplied only one cornea each.
*With pyridine extraction control

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 Volume 21
Number 1, Part 1 Oval corneal opacities in beagles 101

Fig. 6. Cholesterol accumulates predominantly in keratocytes of anterior stroma. (Frozen

section, Okamoto's method; x200.)

G H I J K L M N
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

-f +

-f+ +

+ +

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 Invest. Ophthalmol. Vis. Sci.
102 Roth et al. July 1981

Table III. Serum lipid measurements (mg/dl) in beagles with oval opacities
and unaffected controls
Total cholesterol cHDL
First sample Second sample First sample Second sample

Control Opacity Control Opacity Control Opacity Control Opacity

Male:
Mean 202.6 222.2 221.3 188.4 163.1 165.8 165.9 142.1
S.D. 43.6 76.2 46.2 39.3 28.7 35.9 32.2 45.0
N 27 28 24 18 27 28 24 18
Female:
Mean 201.0 220.0 233.0 222.4 160.2 173.0 171.6 170.2
S.D. 56.9 61.1 54.8 79.8 40.6 35.4 31.7 50.2
N 21 20 16 11 21 20 16 11
Analysis of variance*:
By sex NS NS NS NS
By group NS NS NS NS
By sex and NS NS NS NS
group
Linear regression against age:
Male
r 0.40 0.53 0.32 0.27 0.25 0.40 -0.03 0.10
P(r) 0.04 0.004 NS NS NS 0.04 NS NS
Female
r 0.57 0.08 0.01 0.03 0.46 0.39 0.05 0.42
P(r) 0.01 NS NS NS 0.03 NS NS NS

First sample = 1977; second sample = 1979; NS = not significant = p >0.05.

* Normal vs. abnormal; male vs. female.

with age, but this was not confirmed in 1979. Comment

When we analyzed total serum cholesterol,
CHDL> C L D L , and CVLDL measurements in the
three different clinical patterns in 1977, dogs
with white arc opacities consistently had
higher absolute values, but we could not
confirm these differences in 1979 (Table IV).
Thyroid hormone assays. The 39 control
dogs had T 3 %RU of 35.2% 4.6, T4 level of
1.37/xg/100 ml 0.68, and T7 of 0.48. Eight
beagles with the nebular opacity had T 3 %RU
of 34.6% 5.4, T4 level of 1.45 jug/lOO
ml 5.4, and T7 of 0.51. Eight dogs with the
race track lesion showed T3 %RU of 36.6%
5.1, T4 levels of 1.46 ^g/100 ml 0.32 and
T7 of 0.55. Six animals with the white arc
corneal opacity had T3 %RU of 35.3% 4.2,
T4 level of 1.70 /Ag/100 ml 0.70 and T7 of
0.57. Chi-square analysis showed no sig-
nificant difference in T 3 %RU, T4, or T7 be-
tween control and affected dogs, between
control dogs and those in each subgroup, or
between beagles in each subgroup.
Relatively little is known about the com-
position, source, and metabolism of normal
corneal lipids. There is no evidence for lipid
storage in the normal cornea in vivo and
lipids are probably limited to corneal cell
membranes in the form of neutral fats (pre-
dominantly cholesterol and its esters) and
phospholipids (primarily sphingomyelin).11
These are relatively stable and there appears
to be a low lipid turnover rate. Normal cor-
neal stromal cells can synthesize lipids12' 13
and can incorporate labeled phosphorus into
phospholipids.14
Lipids appear abnormally in the human
cornea in a variety of patterns (Table V).
Three factors may contribute to these lipid
deposits: (1) stromal blood vessels such as
those in old scars or in limbal masses, (2) ab-
normal systemic lipid metabolism such as
type II hyperlipoproteinemia or Tangier dis-
ease, and (3) primary corneal abnormalities
such as central crystalline corneal dystrophy.

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 Volume 21
Number 1, Part 1 Oval corneal opacities in beagles 103

CL0Lana CvLDL Triglycerides

First sample Second sample First sample Second sample

Control Opacity Control Opacity Control Opacity Control Opacity

39.5 56.5 55.4 46.3 65.6 65.1 108.3 80.4

30.9 51.3 31.5 31.7 30.7 34.3 54.4 32.7
27 28 24 18 27 28 24 18

40.7 47.0 61.6 52.2 66.1 77.7 123.2 170.5

30.7 39.4 32.6 45.6 25.3 66.1 66.4 166.1
21 20 16 11 21 20 16 11

NS NS NS P = 0.02
NS NS NS NS
NS NS NS NS

0.33 0.51 0.50 0.19 0.16 0.06 0.15 0.39

0.01 0.006 0.01 NS NS NS NS NS

0.43 -0.22 0.13 -0.41 0.16 -0.10 0.46 0.15

0.05 NS NS NS NS NS NS NS

We searched for these three contributing fac-
tors in beagles with oval lipid corneal
opacities. The corneas were avascular both
clinically and histopathologically. In a few
cases, corneal ulceration had occurred in con-
junction with the oval opacities, and the cor-
neas vascularized secondarily.
Serum lipid measurements in affected dogs
were normal. The environment, nutrition,
and medical care of this colony were carefully
monitored.15' 16 A complete autopsy on each
dog revealed neither atherosclerosis nor ex-
cess lipid deposits in parenchymal organs.
Our initial analysis of serum cholesterol sug-
gested elevated CHDL i n dogs with the race-
track and white arc patterns.17 However,
further statistical analysis and a second sam-
pling of affected and control dogs failed to
reveal statistically significant differences (Ta-
ble IV). Moreover corneal opacities have not
been described in reports of hyperlipemic
beagles.18> 19 Therefore we concluded that a
systemic disorder of lipid metabolism did not
contribute to the oval lipid corneal opacities.
However, we did observe consistently
higher levels of serum cholesterol in dogs
with the white arc type opacity, although the
level was not always statistically significant
and the number of these dogs was small
(Table IV). It is possible that these sub-
epithelial white deposits, which we presume
are lipids but have not studied histochemi-
cally, are related to the elevated serum
lipids. On the other hand, although we found
clinical progression of these opacities from
nebular to racetrack to white arc types, we
have not been able to document either a con-
current rise in serum lipids or increased
serum lipids with age (Tables III and IV).
In hypothyroidism, serum cholesterol lev-
els may be elevated because of altered me-
tabolism; this metabolic effect has been re-
ported in beagles.20 Because our pilot study
suggested that affected beagles had elevated
serum cholesterol levels, we measured thy-
roxin and thyroglobin binding capacity, cal-

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 Invest. Ophthalmol. Vis. Sci.
104 Roth et al. Julij 1981

Table IV. Total serum cholesterol measurements in beagles with nebular, race track,
and white arc oval opacities
First sample Second sample

Control Nebular Racetrack White arc Control Nebular Racetrack White arc

Male:
Mean 202.6 210.1 148.0 290.2 221.3 186.0 184.2 204.3
S.D. 43.6 48.9 136.8 46.2 30.4 55.0 37.6
N 27 22 1 5 24 9 6 3
Female:
Mean 201.0 202.3 275.0 296.3 233.2 199.8 202.8 278.7
S.D. 56.9 53.9 30.0 54.8 38.8 58.7 133.8
N 21 16 1 3 16 4 4 3
Analysis of variance*
By sex NS NS
By type P = 0.001 NS
By sex and type NS NS

First sample = 1977; second sample = 1979; NS = not significant = p > 0.05.
* Normal vs. abnormal; male vs. female.

Table V. Corneal stromal lipid deposits in man

Type Reference Clinical appearance

Corneal arcus Gray paralimbal arc with lucid interval

Normal aging >40 years 37,38
Hyperlipoproteinemia II and III, 39
<40 years
Secondary (pre-existing stromal vessels) 40
Vascularized scar Gray-white, random pattern, spiculated margin
Limbal mass (e.g., dermoid, nevus) Gray arc, lucid interval
Primary (no known pre-existing corneal
pathology)
Limbal 41 D-shaped, yellow, solid, deep stromal, initially
vascular
Central 42 Round, yellow, initially avascular
White ring (of Coats) 40 Small, white, round with gray dots
Central crystalline dystrophy (of 21-26 Central anterior ring of fine, white crystals, hazy gray
Sch'nyder) background, corneal arcus
Systemic disorders
Tangier disease (decreased CHDL in serum) 43,44 Diffuse fine gray spots
Lecithin cholesterol acyltranferase 45-47 Diffuse fine gray spots
deficiency
Juvenile xanthogranuloma 48 Flat, yellow, vascularized limbal mass

dilated T7) and found no significant differ-
ences between affected and control dogs or
dogs with different subgroups of opacities.
Thus thyroid dysfunction does not play a part
in the pathogenesis of the oval opacities.
We think that oval stromal corneal opac-
ities in beagles represent a primary disorder
of corneal lipid metabolism. Of the human
disorders in which lipid is deposited in the
cornea, these opacities most closely resemble
the central crystalline corneal dystrophy of
Schnyder.21' 22 Both occur in avascular, non-
inflamed corneas, are bilateral, and appear
relatively symmetrical. The clinical appear-
ance of both includes a ring-shaped configu-
ration in which a central gray stromal opacity
that is most dense superficially is studded
with subepithelial white crystals. In ad-

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 Volume 21
Number 1, Part 1
vanced cases of both disorders, the opacity
may occupy full-thickness stroma. Histochem-
ically, both contain cholesterol and neutral
fats,15' 18< 19 whereas the beagle corneas also
have histochemical evidence of phospho-
lipids and sometimes fatty acids. Both have
similar ultrastructure with elongated crystals,
extracellular vacuoles and debris, disruption
of collagen lamellae, hyperplasia of kerato-
cyte organelles, and stromal cellular degen-
eration. 26 Hyperlipoproteinemia has been
found in some patients with central crystal-
line dystrophy and in family members of af-
fected individuals.21' 22 Although we did not
find hyperlipidemia in affected beagles, the
dogs with white arc opacities did have serum
cholesterol levels higher than the control
population, which may contribute to their
clinically more advanced opacity. Bron and
colleagues22 speculated that the stromal
keratocytes in central crystalline corneal dys-
trophy are unable to properly metabolize lip-
ids, so that local lipid deposits occur. Ele-
vated serum lipids then further overload the
cells and increase the lipid deposition. In
man, the disorder is autosomal dominant; it is
unfortunate that we cannot determine the
hereditary influences in our beagle colony
because of the carefully planned outbreed-
ing.4 Oval corneal lipid opacities in beagles
may be an animal model of the human cor-
neal dystrophy of Schnyder.
The demonstration of lipids by histochemi-
cal techniques is a tedious process. The
methods are not well documented, many
reagents are difficult to obtain, and only after
diligent search by laboratory personnel can a
meaningful battery of lipid stains be per-
formed (Table I). Even when histochemical
methods demonstrate the presence and lo-
cation of lipids, distinguishing among the
classes of lipids is difficult.27 Many of the
reactions are nonspecific for the different
types of lipid and can give false-positive
and/or false-negative results. The presence of
other types of compounds in the tissue may
cause capricious staining. Diffusion from the
sections can occur and subcellular fractiona-
tion has been found.28 Biochemical analysis of
lipids28' 29 is necessary to accurately identify
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Oval corneal opacities in beagles 105
and quantitate the varieties in the oval
opacities; we are presently performing these
studies on affected beagle corneas.
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