Vet Pathol 27244-253 (1990)

The Effects of Phenylbutazone on the Morphology

and Prostaglandin Concentrations of the Pyloric
Mucosa of the Equine Stomach
C. L. MESCHTER, . GILBERT,. KROOK,
G. MAYLIN,AND R. CORRADINO
Department of Pathology and Equine Drug Testing and Toxicology,
College of Veterinary Medicine, Cornell University, Ithaca, W, and
American Health Foundation, Research Animal Facility,
One Dana Road, Valhalla, NY

Abstract. Phenylbutazone, a nonsteroidal anti-inflammatory drug known to produce gastric ulcers, was
administered intravenously (13.46 mdkg body weight) daily to 12 horses. Horses were euthanatized daily after
24,48,72, and 96 hours following the initial injection. Eight untreated horses served as controls. Small multifocal
pyloric erosions were seen after 24 hours and then progressed in severity over time. The erosions were characterized by sloughing of the surface epithelium, subepithelial bleb formation, necrosis of the lamina propria,
degeneration of the walls of subsurface capillaries, and microthrombosis of the capillaries of the pyloric mucosa.
Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Eroded
pyloric mucosa and adjacent macroscopically intact mucosa were examined ultrastructurally. In both the macroscopically eroded mucosa and multifocally in the adjacent macroscopicallyuneroded mucosa, there was cellular
swelling of the endothelium, pericytes, and smooth muscle cells of arterioles. In capillaries and post-capillary
venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and
edema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular
injury that is associated with the formation of pyloric erosions in horses.
The pyloric mucosa of six horses was assayed for prostacyclin and prostaglandin E, at 48 and 96 hours following
the initial injection. There was no statistically significant difference between prostaglandin concen-trations in the
mucosa of control and treated horses. It was concluded that there was little correlation between pyloric mucosal
prostaglandin concentrations and pyloric erosions after 48 hours.
Key words: Horse; phenylbutazone; prostaglandin; pyloric mucosa.

Phenylbutazone, a nonsteroidal, anti-inflammatory important roles in maintaining vascular and mucosal

drug commonly used in the horse as an analgesic, has perfusion and integrity22,32-38,4u8and exert cytoprotecbeen considered nontoxic. Phenylbutazone is toxic to tive
They also modulate motility, acid and
horses, however, when the maximum therapeutic dos- mucous secretion, electrolyte absorption and water abage of 8.8 mg/kg bodyweight daily has been exceedsorption, prevent ulcer formation, and accelerate ulcer
ed.4,21Hematologic ~ h a n g e s , ~oralJ~ .and~~ gastrointes- healing.32-35
tinal u l ~ e r a t i o n ,colonic~.~~ ~necrosis,~~~~ 17,21 renal crest Little work investigating prostaglandin
suppression
n e c r o ~ i s ,and~~ vasculitis2I~~.~~ have been described.
by phenylbutazone in the horse has been
performed.
Gastric ulceration is a common toxic effect of non- This study reports on the sequential histologic and
steroidal anti-inflammatory drugs and has been ultrastructural development of acute phenylbutazonedescribed in phenylbutazone-treated
induced pyloric lesions and the effects of phenylbutaThe mechanism by which phenylbutazone induces zone on pyloric mucosal prostaglandin levels in adult
ulceration is not well defined. Proposed mechan- horses.
isms include inhibition of cyclo-oxygenase leading to
Materials and Methods
loss of prostaglandin-mediated cytoprotective effects
For both parts of this study, adult horses (3 to 15 years
resulting in vasoconstriction with subsequent ischemia,4,33-35,46-48and microvascular damage.z1,26.28~29

old, Thoroughbreds and Standardbreds) were used in the

Prostaglandins are produced by the gastric m u ~ o s a , ~ ~ , ~ ~

endothelium,14 kidney,I3 and in sites of inflamma- ual box stalls and fed standard hay and grain rations with t i ~ n . *
. ~ JIn~ Jthel gastric mucosa, prostaglandins have free access to water.
treatment and control groups. They were housed in individ-

244

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Phenylbutazone-induced Gastric Ulcers in Horses

For the first part of the investigation, the morphologic study,

12 treated and eight control horses were used. Each horse in

the treated group was administered phenylbutazone (PhenButa-Vet, 13.46 mg/kg, Anthony Products Co., Ar-cadia, CA)
intravenously daily for up to 4 days. The control horses
received no phenylbutazone but received 30 ml nor-mal saline
intravenously daily in order to sham phenylbu-tazone
injections.
The purpose of the study was to examine the early lesions of
pyloric mucosal erosion formation. Horses were euthan-atized
as follows: 24 hours after a single injection (24 hours); after
two injections at 24 hour intervals (48 hours); after three
injections at 24 hour intervals (72 hours); and after four
injections at 24 hour intervals (96 hours). Two treated horses
and two control horses were euthanatized at 24 and 72 hours,
and four treated and two control horses were euthanatized at
48 and 96 hours for 4 consecutive days following the initial
administration of phenylbutazone.
The administration of the phenylbutazone was to mimic
daily clinical administration of the drug. All horses were
examined morphologically and biochemically while receiv-ing
phenylbutazone. A complete and systematic necropsy was
performed on each horse, and representative tissues were
retained for histopathologic and ultrastructural evaluation. The
stomachs of all horses were thoroughly and systemati-cally
examined macroscopically and microscopically. No le-sions
were seen other than in the pylorus. Based on the macroscopic lesions, sites were selected for ultrastructural
sectioning and biochemical assay. The macroscopic, microscopic, and biochemical changes found in the other organs will
be presented in subsequent manuscripts.
For the morphologic study, tissues for histopathologic and
ultrastructural evaluation included representative sections from
the pyloric mucosa. Tissues were fixed for 24 hours in chilled
(4 C) 2.5%Karnovskys solution, embedded in par-affin
blocks, sectioned at 4-6 pm, and stained with hema-toxylin
and eosin.
For the ultrastructural phase of the morphologic study, two
treated horses and one control horse from 24, 48, 72, and 96
hours were used for perfusion fixation. The remaining four
treated horses and four control horses from the study were
necropsied but not perfused. The tissues from these horses
were included in the histologic phase. For the purposes of
achieving the best fixation possible for ultrastructural study, in
vivo perfusion with 2.5% Karnovskys solution was performed. In vivo perfusion, while laborious and time consuming, has been shown to produce excellent and reliable
fixation for electron microscopic studies in both the gastrointestinal tract and nervous system and is performed regularly on
horses and other species (John Cummings, Department of
Anatomy, Cornell University, College of Veterinary Medicine, Ithaca, NY,personal communication). The quality of
the fixation was evaluated at the time of perfusion and necropsy. Good to excellent fixation was characterized during
the early phase of the perfusion by muscular fasciculation and
a profound hardening of the carcass beginning at the nose and
proceeding caudally. The properly fixed gut was pale and
hose-like or leathery.
For the perfusion, horses were administered sodium heparin (Lyphomed, 50,000 units, Melrose Park, IL) intrave-

245

nously, approximately 45 minutes prior to euthanasia in or-der

to minimize blood clotting and allow for more complete
exsanguination. After 30 minutes, they were then administered acepromazine (Promace, 40 mg, Fort Dodge Co., Fort
Dodge, IA) intravenously to facilitate sedation and vasodilation, followed 10 minutes later with ketamine hydrochloride (Ketaset, 800 mg, Bristol-Myers, Evansville, IN) intravenously. When the horses became recumbent, deep
anesthesia was achieved via slow intravenous injection with
sodium pentobarbital (Fort Dodge Co., Fort Dodge, IA).
After deep anesthesia was obtained, the right ventral chest
wall was opened, forming a window approximately 20 cm by
20 cm. The lungs were retracted and the pericardium incised.
The right auricle was cut off and the left ventricle cannulated.
Approximately 20 liters of physiologic saline prewarmed to
body temperature (approximately 37 C) were infused by
means of an infusion pump (Manostat Variostatic Pump,
Manostat, New York, NY) under medium to mod-erately high
pressure in order to exsanguinate the carcass. It was then
followed by 40 to 50 liters of 2.5% Karnovskys solution
infused under medium pressure to achieve whole body in vivo
fixation. A successful perfusion took approxi-mately 30
minutes. Identical fixation techniques were used in both the
treated and control horses.
Blocks of tissue were collected from macroscopic pyloric
erosions and from macroscopically uneroded mucosa adjacent to the erosions. The fundus and antrum were examined
histologically but not ultrastructurally. There were no macroscopic lesions seen in the fundus or antrum.
Tissue for electron microscopy (pyloric mucosa) was fixed
24 hours in 2.5%Karnovskys solution, changed into 0.1 M
cacodylate buffer, post-fixed in 1% osmium tetroxide in 0.1 M
phosphate buffer (7.2 pH), dehydrated in a graded series of
ethanol, and embedded in Spurrs resin. The mucosa was
oriented longitudinally. The semi-thin Epon sections (1 pm)
and ultrathin sections (30-50 nm) were cut. Ultrathin sec-tions
were stained with uranyl acetate and lead citrate. Approximately 1,500 blocks (approximately 25 blocks per horse)
for a total of 7,500 grids were examined.
In the second part of the investigation, the biochemical
study for the analysis of pyloric mucosa for 6-keto-prostaglandin F,,, the stable breakdown product of prostacyclin and
prostaglandin E,, four treated horses and two control horses
were used. As in the morphologic study, the treated horses
were administered 13.46 mg/kg phenylbutazone intravenously daily for 4 days. The control horses received no
phenylbutazone but received 30 ml normal saline intravenously in order to sham phenylbutazone injections prior to
euthanasia. Two treated horses and one control horse were
euthanatized at 48 hours and at 96 hours. Complete necropsies were performed on these horses. The light microscopic
findings were included in morphologic study. In the control
and treated horses, multiple blocks of tissue from the pyloric
mucosa of each horse were processed according to the method of Whittle et al.46in preparation for radioimmunoassay.
The sites selected for prostaglandin assay corresponded to the
sites selected for electron microscopy, i.e., macroscopi-cally
eroded and adjacent macroscopically uneroded pyloric mucosa
(Fig. 1).
In preparation for radioimmunoassay, solid-phase tissue

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Meschter et al.

246

Fig. 1. Schematic diagram indicating the location and

onentation of erosions around the pylorus. For both
electron microscopy (perfusion fixed tissue) and
prostaglandin assay (unfixed tissue), small blocks of
Fig. 2. Pyloric mucosa, 24 hours; horse No. H-24- 1.
mucosa were collected from macroscopically eroded (1)
There is lifting off and separation of the epithelium from
and adjacent macroscopically un-eroded (2) tissue.
the foveo-lar crests (arrows). Small thrombi (T) are present
subjacent to the epithelium. There is congestion and
extraction was accomplished by extracting tissue supernatant hemorrhaging of the capillaries (H).
solutions with octadecyl (C18) solid phase columns (Applied
Separations, Lehigh Valley, PA). For radioimmunoassay for
prostacyclin and prostaglandin E,, prostaglandin E, and
6-keto-prostaglandinF,,-iodine1-25
radioimmunoassay kits were used (New England Nuclear,
Boston, MA). The pooled mean of prostaglandin concentrations
from the control horses were compared to the pooled mean of the
prostaglandin con-centrations of the treated horses using the t-test.

Results
Gross pathologic findings

Thorough and systematic examination showed that

mucosal lesions were limited to the pyloric region; no
parasites were present. At 24 and 48 hours, multiple,
irregular, small, eroded, and hemorrhagic foci were
scattered over the pylorus of all horses. The erosions
were fairly superficial in the mucosa and ranged from
0.2 to 0.5 cm in diameter. At 72 hours, there were mild
patchy areas of congestion on the pylorus of both horses.
At 96 hours, there were multiple eroded areas ranging
from small pinpoint areas of congestion to deeper more
extensive areas of erosion in all horses. The erosions
formed a radiating or spoke-like pattern around the
pyloric opening. These erosions were deeper than those
seen at 24 and 48 hours. They were between 0.2 and 1.5
cm in width and 1 and 3 cm in length. They were
somewhat cigar shaped and ranged between 4 and 12 in
number radiating around the pyloric opening. There were
no lesions in any of the control horses.

Histolopathologic findings

There were no significant histologic lesions in the

control horses. At 24 hours, there was separation of the
foveolar crest epithelium and microthrombosis of the
superficial lamina propria (Fig. 2). More severe lesions
were characterized by shallow erosions of the superficial
mucosa, and the lamina propria contained numerous foci
of necrosis (Fig. 3). Capillaries were thrombosed and
capillary walls were dense, eosino-philic and disrupted.
The most superficial lesions con-sisted of very shallow
erosions involving only the fove-olar crests. There were
subepithelial clefts and surface breaks in the mucosal
epithelium with the epithelium of the neck of the gland
first lifting off and then peeling back exposing the
lamina propria. Neutrophils, eosin-ophils, fibrin and
debris lay on the mucosal surface of the gastric lumen.
Individual or large rafts of sloughed epithelial cells and
fibrinous and cellular debris were free in the gastric
lumen. The cytoplasm of the colum-nar epithelial cells
varied from darkly eosinophilic to pale and finely
vacuolated. In many areas, particularly in the deeper
portions of the glands, the mucosal ep-ithelial cells
contained large vesicular nuclei and nu-merous mitotic
figures. In other areas, usually more superficially,
epithelial cells were cuboidal to flattened, suggesting
either attempts to cover eroded areas or

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Phenylbutazone-induced Gastric Ulcers in Horses

Fig. 3. Pyloric mucosa, 24 hours; horse No. H-24- 1. There

is superficial sloughing of the mucosal epithelium and neutrophils are scattered throughout the mucosa. Capillary walls
are hyalinized (arrows) and there is perivascular leakage and
thrombosis (T).

epithelial atrophy. The mucosal lamina propria subjacent to the erosions was edematous with engorged
capillary loops that often contained small thrombi and
marginated neutrophils and eosinophils. Extravasated
erythrocytes, neutrophils, and engorged capillaries
were present in the deeper lamina propria.
Epon semi-thin sections revealed a microvascular
lesion in the superficial mucosa. The microvascular
lesion was usually subjacent to the subepithelial clefts.
The capillary walls were hyalinized and there was disruption of these vessel walls. Numerous, small, fibrin
thrombi were present. The microvascular lesion was
confined to the most superficial subepithelial region,
and often appeared where there was little epithelial or
interstitial injury.
After 48 hours, the mucosal lesions were similar to
these described at 24 hours except that the erosions
were often larger, deeper, and more severe. The microvascular lesion present at 24 hours was more prominent and severe at 48 hours with pronounced thickening, dilation, increased density, and often disruption
of the walls of small vessels and capillaries. The
vessels contained fibrin, cellular debris and small
thrombi (Fig. 4). In the muscularis mucosa and
submucosa under-lying erosions, many arterial walls
were disrupted. Large numbers of neutrophils and
mononuclear cells were present both within these
vessel walls and perivascu-larly. Tissue not associated
with eroded areas was his-tologically normal.

247

Fig. 4. Pyloric mucosa, 48 hours; horse No. H-48-2. There

is more extensive sloughing and erosion of the mucosa and
infiltration with neutrophils. Sloughed epithelium and debris
are present on the surfxe. There is more extensive damage to
capillary walls, thrombosis (T) and necrosis.

At 72 hours, the pyloric mucosal lesions were limited

to a patchy mucosal congestion. No erosions were seen.
After 96 hours, the histologic lesions were similar
but more severe and extensive than those in the 48
hour group. Deep mucosal erosions extended to but
did not penetrate the muscularis mucosa. There was
severe neutrophilic infiltration and necrosis of the
mu-cosa. The microvascular lesions were more severe
and extensive than those seen at 48 hours. There was
severe hemorrhage and thrombosis of the mucosa.
Ultrastructural findings

No ultrastructural lesions were seen in the control

horses. After 24 hours, in the macroscopically un-eroded
pylorus adjacent to eroded mucosa, there were wellpreserved glands consisting of columnar epithelial cells
containing mucus granules and occasional en-docrine
cells. The mucus secreting enterocytes con-tained a
moderate number of apically located secretory granules;
numerous, well-formed microvilli; numer-ous, welldefined mitochondria; and abundant, rough endoplasmic
reticulum. The interstitium contained fi-broblasts, loose
collagen, and a few inconspicuous, nor-mal capillaries.
Occasionally, small arteries with swol-len endothelium
and convoluted nuclei were seen.
In other macroscopically uneroded areas, however,
there were mucus secreting enterocytes with loose vacuolated cytoplasm, swollen mitochondria, dilated en-

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248

Mesch.ter et al.

Fig. 6. Electron micrograph, 24 hours; horse No. H-241. There is abundant necrotic debris (E). An erythocyte

(R) contacts the basement membrane (bm). Bar = 5 pm.

Fig. 5. Electron micrograph, 24 hours; horse No. H- 24-1.

The glandular epithelium (GE) is flattened and there are
swollen mitochondria (m) and a cytoplasmic bleb. The endothelium (EC) of a small vessel is swollen and cleared,
re-ducing the vessel lumen diameter. Erythrocytes (r),
eosino-phi1 (e), and a neutrophil (n) are free in the
interstitium. Note the glandular lumen (GL). Bar = 5 pm.

doplasmic reticulum, and Golgi complex. The adjoining interstitium was loose and edematous. In many
capillaries and small vessels, the endothelium was
swollen and vacuolated, reducing the diameters of the
capillary lumina. Vessels were engorged, there was
ex-travasation of erythrocytes, and platelet masses adhered to capillary walls.
In macroscopically eroded areas, capillary and small
vessel endothelium and pericyte and smooth muscle cell
cytoplasm were often severely swollen. The cyto-plasm
of the mucus secreting enterocyteswas clear and swollen
and contained only a very few organelles. Neu-trophils
and platelets adhered to the endothelial cell surface. The
lumina of many small arteries were ob-structed by
swollen endothelial cells (Fig. 5). In some vessels, the
endothelium was completely necrotic and lysed, and the
vessel lumina contained cellular debris and fibrin (Fig.
6). The basement membrane integrity was lost, and the
membrane was frequently split or

laminated. Within the interstitium were numerous

erythrocytes, eosinophils, neutrophils, and fibrin. The
cytoplasm of fibroblasts often was clear and slightly
swollen, and the interstitium was loose and
vacuolated. In nearby glands, changes in the
epithelium varied from columnar to flattened (Fig. 5),
and there was swelling and clearing of cytoplasm with
dilated en-doplasmic reticulum and swollen
mitochondria. Mi-crovilli were sparse and short, and
membrane-covered, cytoplasmic blebs often protruded
into the gland lu-men. Tight junctions were intact
between enterocytes. Many enterocytes, which were
depleted of mucus gran-ules, contained membranous
whorls or lamellar bodies within both the cytoplasm
and mitochondria.. Mito-chondria were often swollen
with disrupted cristae and occasionally contained dark
granular material inter-preted as precipitated calcium.
Numerous subepithe-lial clefts were present. Within
many columnar epi-thelial cells were numerous,
basally located, vacuolar spaces separatingthe
epithelial cells from the basement membrane.
After 48 hours, the ultrastructural lesions were similar to those seen at 24 hours, except that those at 48
hours were more severe and extensive than those at
24 hours. After 72 hours, the lesions were very minimal; cellular swelling of epithelial and endothelial
cells, mild edema of the lamina propria, and vascular
congestion were observed. Ultrastructural lesions observed at 96 hours were similar to those seen at 48
hours but were more severe. There was also severe
necrosis of the lamina propria.

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Phenylbutazone-inducedGastric Ulcers in Horses

249

Table 1. Pyloric mucosal concentration of prostaglandin (ng/mg tissue) and microscopic pylonc mucosal lesions in
horses treated at 24-hour intervals with phenylbutazone.*
Horse
Number?

Site One

C-48-1
C-96-2
H-48-1
H-48-2
H-96-1
H-96-2

18.7
13.83
53.2
27.9
16.8
47.7

Prostacyclin
site TWO
x

16.6
51.4
23.1
23.4
5.6
99.9

SEM~:

17.65 f 0.74
32.6 f 13.29
38.15 f 15.17
25.65 f 1.59
11.20 f 3.96
73.8 f 18.45

Prostaglandin E,
Site One Site Two
X = SEM*

1.59
1.55
1.84
3.02
0.86
2.69

1.60
4.30
3.47
3.90
0.59
3.30

1.60 f 0.003
2.93 f 0.97
2.55 f 0.66
3.46 f 2.45
0.73 k 0.31
2.55 f 0.22

Microscopic Pyloric
Mucosal Lesions

No significant lesions
No significant lesions
Mild multifocal erosions
Mild multifocal erosions
Large multifocal erosions
Mild multifocal erosions

* There was no significant difference (P< 0.1) between pyloric mucosal prostacyclin and prostaglandin E, concentrations in control and
treated horses.

t Horses were administered 13.46 mdkg of phenylbutazone intravenously. Following two injections (48 hours), three horses were

euthanatized (two treated and one control) and then following four injections (96 hours), three horses (two treated and one control) were euthanatized . Two blocks
of mucosa were collected at each site.

Standard error of the mean.

Prostaglandin assays

The effects of phenylbutazone on pyloric mucosal

prostaglandin concentrations are presented in Table 1.
In the pyloric mucosa, prostacyclin was the predominant prostaglandin. There was no statistically significant difference between the prostaglandin concentrations in the treated and control animals. There was
little correlation between prostacyclin and prostaglandin E, concentrations and the presence of pyloric erosions.
Discussion
Phenylbutazone is toxic to adult horses when administered intravenously at doses of 13.46 mg/kg daily. This dose is 50% larger than the currently recommended maximal dose of 8.8 mg/kg of body weight
daily.
Macroscopic lesions were present in all horses at
24, 48 and 96 hours. Mucosal congestion, but not
erosion, was seen at 72 hours. This is probably due to
the small group size, the low dose administered, and
individual variation. All horses were subjected to a
total necropsy, and the 72 hour horses as well as the
other horses displayed macroscopic lesions in the
duodenum, co-lon, and kidney. These findings will be
incorporated into subsequent manuscripts.
Pyloric erosions were present after 24 hours. The
purpose of this study was to evaluate the very earliest
mucosal lesions occurring in order to evaluate the
pathogenesis of phenylbutazone-induced erosion formation. It appears that erosions develop first in the
pylorus. In other s t u d i e ~ , ~deepJ~, ~and~ large,
even perforating, ulcers have been seen. In these studies,
much higher doses and longer administration of phenylbutazone were given. The lower daily dosage in this
study was used both to mimic clinical administration and
to cause less severe lesions that could be evaluated

in a more detailed way than large florid lesions. Ini-tially,

multifocal, pinpoint pyloric erosions occurred, which then
increased in severity over time. Histolog-ically,
progressive microvascular damage and mucosal epithelial
damage resulted in destruction and sloughing of the
superficial mucosa. The primary ultrastructural lesion was
microvascular damage that caused hemo-dynamic
derangements, microhemorrhage, micro-thrombosis, and
subsequent mucosal ischemia, which thereby led to
erosions. The microvascular lesion oc-curred either
independently of epithelial injury or pre-ceded epithelial
injury and was comparatively more severe than the
epithelial injury. The lesion was char-acterized by
endothelial swelling that progressed to necrosis and lysis
of the vessel walls of small arterioles, capillaries, and
small venules. These swollen vessels occluded blood
supply, which led to a low-flow state with retrograde
slowing and sludging of the capillary blood flow.
Mucosal ischemia and necrosis with sub-sequent erosion
followed. The necrotic and lysed en-dothelial cells
resulted in a loss of integrity of the mi-crovasculature,
microhemorrhage, thrombosis, and destruction of the
mucosa. Similar microvascular le-sions have been
described in the stomach of ethanol3s4I and aspirintreated rat^,^^,^^,^^ and indomethacin-treat-ed
pigs.28Microvascular lesions have been observed within
15 minutes of administration of certain non-steroidal,
anti-inflammatory drug^.^^,^^ These investi-gators
concluded that microvascular damage with focal ischemia
is the initial lesion in nonsteroidal, anti-in-flammatory
drug-induced gastric erosions. This is compatible with
what was observed in this study, and it is concluded that
phenylbutazone causes a direct toxic injury to gastric
mucosal epithelium. Microvas-cular lesions were
observed in eroded mucosa and were present multifocally
in adjacent uneroded mucosa. This has been observed but
not explained in other

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250

Meschter et al.

The lesions in uneroded areas probably represent prodromal changes.

Nonsteroidal anti-inflammatory drugs reduce the blood
flow in the stomach possibly by causing vaso-constriction
due to prostaglandin i n h i b i t i ~ n . ,Retro~,~~-grade
congestion causes rupture of capillaries and microhemorrhage of the mid - muc~sa .Although~ some
microhemorrhage of the mid-mucosa was observed in one
treated horse, the ultrastructural findings in this study
indicate that endothelial swelling and damage rather that
vasoconstriction of the microvasculature occurs. The
endothelial lesions do not resemble those seen in either
infarction or reperfusion injury of colonic mucosa (Diane
Craig, personal communication). The microhemorrhage is
associated with leakage from damaged vessels rather than
leakage due to back pres-sure from vasoconstriction.

Gastric lesions developed first in the pylorus, where

the lesions were arranged in spoke-like rays around
the circumference of the pylorus. Similar distributions
of pyloric erosion have been de~cribed.~'Muscular
con-traction around the pylorus contributes to the
distri-bution of these lesion^.^^^^^ Phenylbutazone
increases gastric contractility, and hemorrhage occurs
at sites of mucosal compression.2oIn the horse,
contractive peri-staltic actions of the pylorus could
produce compres-sion and a local reduction in
circulation. Ischemia of the mucosa coupled with
endothelial damage could lead to the distribution of
erosions radiating around the pyloric opening.
In this study, as well as in several others,
phenylbutazoneZ0or other nonsteroidal anti-inflammatory drug^^^,^^,^^ were given parenterally, and mucosal erosions developed, indicating that erosions do
not necessarily develop because of the direct contact of
phenylbutazone or other nonsteroidal anti-inflammatory drugs with the gastric mucosa. Because of its
low pKA, phenylbutazone accumulates in acidic environments such as the stomach and renal medulla., It
is likely that the microdomain of the gastric mucosal
capillary endothelium is the initial tissue in the stomach with which phenylbutazone interacts. Injury to the
capillary endothelium, then, would lead to the development of mucosal erosions. In the case of other orally
administered nonsteroidal anti-inflammatory drugs26
or ethan01,~'very early endothelial cell injury is also
observed, indicating that endothelial injury is an important component of ulcerogenesis whether these ulcerogenic agents are administered parenterally or orally.
The formation of subepithelial clefts was observed
concurrently with the microvascular changes. This lesion preceded mucosal erosion and sloughing. Clefts
were generally associated with subjacent damaged capillaries. Subepithelial clefts are considered character-

istic of and are initially indicative of ischemic small and

large intestinal damage.21s44,45Similar clefts in the
gastric mucosa would also be indicative of ischemic or
low-flow injury. It was theorized that clefts developed as
a result of the accumulation of edema fluid between the
epithelial cells and lamina p r ~ p r i aSubsequent.~~
work suggests that the cleft formation occurs as a result
of the formation of toxic free radicals generated during
low-flow or reperfusion ~ t a t e s . ~The,'~ ?
generation~~ of superoxide radicals may account for
cleft formation and epithelial sloughing by degrading
hyaluronic acid, the primary component of the epithelial
basement memb~ane . ~The.'~ superoxide radicals also
damage cellular components such as mitochondria1 and
cellular membranes by lipid peroxidation.
Generalized damage to the mucus secreting enterocytes, including discharge of the mucus granule, mitochondrial injury, formation of lamellar bodies, and
microvillus injury seen in this study and el~ewhere,,~
probably represents direct toxic injury to the columnar
epithelial cell by phenylbutazone. Phenylbutazone and
some salicylates have been shown to increase apical
shedding of epithelial cells without increasing the renewal rate,18 to decrease mucin production,18damage
parietal cells2' and to accelerate gastric apical granule
as was seen in this study. These factors
also contribute to mucosal erosion.
Although phenylbutazone is commonly considered
a prostaglandin or cyclo-oxygenase this study
indicates that phenylbutazone may damage cells
through non-prostaglandin mediated pathways. Phenylbutazone exerts numerous non-prostaglandin mediated effects, including a variety of toxic
effects'6,25,38,42 and enzyme inhibitory These effects
may result in damage to energy-producing pathways, oxidative phosphorylation, and biochemical pathways that
use the mitochondria, such as the Kreb's cycle. Depletion of adenosine triphosphate, leading to a reduction in energy potential and inhibition of biochemical
pathways, would lead to cellular swelling and account
for some of the ultrastructural lesions observed, particularly the cell damage and swelling. It has been demonstrated that the metabolism of phenylbutazone re-sults
in the production of toxic oxygen species.3LThe
generation of free radicals causes local tissue damage
and contributes to the toxic effects of the drug.31
The pyloric mucosa was assayed by radioimmunoassay for the two predominant gastric prostaglan-dins,
prostacyclin and prostaglandin E,. In the pyloric mucosa
of the horse, as in other specie^,^^,^^ prosta-cyclin was
found to be the predominant prostaglandin. As there was
no statistical differencein mucosal prosta-glandin
concentrations between control and treated horses, it is
concluded that phenylbutazone may not cause a
reduction in mucosal prostaglandin concen-

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Phenylbutazone-induced Gastric Ulcers in Horses

tration after 96 hours of treatment. A correlation be-

25 1

effect of phenylbutazone on the gastric mucosa. A sec-

tween the development of gastric erosion associated

ond explanation is that there may be an early phenylbuwith phenylbutazone administration and alterations in tazone-induced prostaglandin reduction, but mucosal
the concentration of prostaglandin in the pyloric muprostaglandin concentrations either then undergo a
cosa does not seem to exist based on the data present. compensatory increase46or are obscured by the intense
This is in conflict with some reports28,46,48but in agree- inflammatory cell infiltration. Finally, the group of
ment with other~.3.15.28,29.41,46 horses assayed was small ( n = 4)and may not be comThere was considerable variation in the mucosal,
pletely representative; however, in studies with cats ( n
site-to-site concentrations of prostaglandin within in= 6 )using the same techniques employed in this study,
dividual horses and between horses. This has also been profound decreases in prostaglandin concentrations
seen in the duodenum and colon of phenylbutazonewere seen in all aspirin-treated cats.48
treated horses (Carol L. Meschter, unpublished reIt is concluded, therefore, that phenylbutazone-insults). Within the cortex and pelvis of the kidney, how- duced gastric erosions in horses are primarily due to
ever, prostaglandin concentrations appear to be fairly a direct toxic effect of the drug on the endothelium and
uniform (Carol L. Meschter, unpublished results). Lit- mucosa and that the development of erosions is not tle
is known about the behavior of the gastric/pyloric mediated entirely by prostaglandin reduction. microdomain
of the horse, or other species for that
matter. The reason for the variation in mucosal prosAcknowledgements
taglandin concentrations is not known. It may be a
The author thanks Mr. J. Barbato and Mr. M. Blank for
reflection of an intrinsically uneven distribution of
their photographic expertise and Ms. J. Fox Davidson for
prostaglandin within the mucosa. Secondly, it may be her editorial assistance.
a reflection of a variation in the microperfusion of the
mucosa. It was noted in the morphologic study that
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Request reprints from Dr. C. Meschter, American Health Foundation, Research Animal Facility, One Dana Road, Valhalla,
NY 10595 (USA).
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