176
PEYRON (C.) AND COLLABORATORS
Vacuolar hepatopathy in 43 French Scottish Terriers:
a morphological study.
C.PEYRON1, P.LECOINDRE1, M.CHEVALLIER2, S.GUERRET3, A.PAGNON3
Clinique Vétérinaire des Cerisioz, Route de Saint Symphorien d’Ozon, 69800 Saint Priest, FRANCE
Laboratoire Biomnis, Avenue Tony Garnier, 69007 Lyon, FRANCE
3
Laboratoire Novotec, Rue Jacques Monod, 69007 Lyon, FRANCE
1
2
* Corresponding author: [email protected]
SUMMARY
RESUME
Scottish Terriers (ST) are known to develop vacuolar hepatopathy (VH)
associated with elevated alkaline phosphatase (ALP) activity, which likely
reflects a genetic defect affecting steroidogenesis that might also explain
the appearance of hepatocellular carcinoma (HCCA) in these dogs. The
objective of the present study was to assess the hepatic lesions in a cohort
of French STs with VH using histological techniques and transmission
electron microscopy (TEM) in order to characterize cellular changes and
attempt to identify the pathogenesis of this disease. Forty-three ST dogs
were enrolled on the basis of their breed, elevated plasma ALP activity
and the presence of vacuolar hepatopathy in histological samples. Each
dog underwent complete serum biochemistry and hematology testing,
abdominal ultrasounds and liver biopsies. Overall, 54.6% of the dogs showed
moderate to severe vacuolation. Almost 90% of the dogs exhibited no or
minimal inflammation and extensive fibrosis was observed in 30% of the
dogs. Neither systematization nor zonal distribution was observed regarding
the location of the vacuolized hepatocytes. Vacuolated hepatocytes did
not exhibit any PAS staining. Nine dogs had well-differentiated HCCA.
Mitochondrial alterations were the most striking ultrastructural changes,
with pleomorphic and enlarged mitochondria. In the STs of the present
study, VH is a diffuse liver disease characterized by ballooned hepatocytes
without glycogen content and exhibiting mitochondrial modifications. The
two major hepatic complications described in the present cohort are fibrosis
(30%) and neoplasia (21%).
Hépatopathie vacuolaire chez 43 Scottish Terriers français:
morphologique
Keywords: Vacuolation, liver, Scottish Terrier, histology
étude
Les Scottish Terriers (ST) sont souvent atteints d’hépatopathie vacuolaire
(HV), en association avec une élévation de l’activité des phosphatases
alkalines (PAL), laquelle reflète possiblement une défaillance d’origine
génétique affectant la stéroïdogénèse. Cette défaillance pourrait également
expliquer le développement de carcinome hépatocellulaire (CHC) chez cette
race. Les objectifs de la présente étude étaient d’évaluer les lésions hépatiques
dans une cohorte de ST français atteints de HV en utilisant l’histologie et la
microscopie électronique à transmission (MET) dans le but de caractériser
les modifications cellulaires et la pathogénèse de cette affection. Quarantetrois ST ont été inclus sur la base de leur race, d’une élévation de l’activité
plasmatique de leurs PAL et de la présence d’hépatopathie vacuolaire à
l’histologie. Pour chaque chien, un bilan biochimique et hématologique
sanguin complet a été réalisé, ainsi qu’un examen échographique de
l’abdomen et des biopsies hépatiques. Globalement, 54.6% des chiens
montraient une vacuolisation modérée à sévère. L’inflammation était
absente à minimale dans 90% des cas et une fibrose extensive a été observée
chez 30% des chiens. Aucune systématisation ou distribution zonale n’a été
notée concernant la localisation et la répartition des hépatocytes vacuolisés.
Ces derniers ne prenaient pas la coloration PAS. Neuf chiens présentaient
un CHC. Les modifications ultrastructurales les plus marquantes étaient les
alterations mitochondriales, avec des mitochondries pléomorphiques et de
taille élargie. Chez les STs de cette étude, l’hépatopathie vacuolaire se définit
comme une affection hépatique diffuse caractérisée par des hépatocytes
ballonisés, sans contenu glycogénique et montrant des modifications
mitochondriales. Les deux complications majeures observées au niveau du
foie sont la fibrose (30%) et l’apparition de néoplasie (21%).
Mots-clés : Vacuolisation, foie, Scottish Terrier, histologie
Introduction
Scottish Terriers (STs) are known to develop vacuolar
hepatopathy (VH) associated with elevated alkaline
phosphatase (ALP) activity [3]. VH usually reflects
hepatocellular cytosolic glycogen accumulation, confirmed
with Periodic Acid Schiff (PAS) staining and easily
differentiated from lipid vacuolation [4]. Hepatocytes are
prominently swollen with clear and wispy cytoplasm and
central nucleus [6, 4]. Distribution can be diffuse, zonal or
involve individual cells. Discrete vacuoles are not observed
unless concurrent lipid vacuolation exists, which actually
makes the term “vacuolar” hepatopathy inappropriate [6, 4].
The origin of this hepatic disorder is currently unclear, and
its pathogenesis remains undetermined [9, 20]. In a study
conducted by Zimmerman et al., the authors hypothesized
that increased ALP activity in STs is most likely attributable to
non-clinical hyperadrenocorticism [34]. In a separate study,
Sepesy et al. focused on disorders potentially associated
with VH, and their results suggest an association between
hepatic vacuolation, neoplasia, hepatobiliary diseases,
and physiologic stress [28]. In a recent report, Center et
al. concluded that high ALP activity and VH in STs likely
reflects a genetic defect affecting steroidogenesis through
an overproduction of androgenic hormones [3]. This
result might also explain the appearance of hepatocellular
carcinoma (HCCA) in these dogs.
We have described VH in STs in previous reports and
have hypothesized that it could be a familial disease in this
breed, with a heritable mode of transmission. If the light of
this hypothesis, VH in STs might be independent of steroid
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VACUOLAR HEPATOPATHY IN 43 SCOTTISH TERRIER DOGS
exposure and other endocrinian causes of elevated ALP
activity, such as hyperadrenocorticism, diabetes mellitus
or hypothyroidism [14]. In another report, we suggested a
possible association between VH, hepatic fibrosis and HCCA
[15]. On the basis of these findings, the aims of the present
study were to describe histopathological characteristics of
French STs presenting VH.
Materials and methods
ANIMALS
Dogs were enrolled both retrospectively and prospectively
on the basis of their breed (they had to be purebred STs), elevated
plasma ALP activity and the presence of VH in histological
samples. They were also part of another study focusing on
clinical, blood biochemical and hepatic histological data in
French STs [23]. STs with VH were selected among these
dogs to be included in the present cohort. In the previous
study, dogs were grouped according to their ALP activity
(normal, elevated, very elevated) and associations between
clinical, biochemical and histological criteria were looked
for. Forty-three pure-bred STs were included in the present
study: 22 females (14 spayed) and 21 males (4 castrated). Ages
ranged from 2 to 13 years (mean: 8.4). Twenty-two dogs were
enrolled retrospectively and 21 were enrolled prospectively.
Diagnosis of VH was based on the presence on liver biopsies
of enlarged and swollen hepatocytes (so-called “vacuolated
hepatocytes” in the present study as we describe features of
a disease named “vacuolar hepatopathy”, although the term
might be inappropriate) with clear cytoplasm, whatever their
distribution (diffuse, zonal or individual).
BIOLOGICAL ANALYSIS
In the present cohort, ALP activity was between 161
and 8255 units/liter (U/L) with a mean of 1820 U/L. Dosage
of ALP activity included all the iso-enzymes (bone, liver,
intestine…) and was determined based on the laboratory
reference interval (Idexx Alfort) for which the upper limit
was set at 140U/L, based on internal studies conducted
on a cohort of 100 healthy dogs receiving no medication,
preliminary examined by a veterinarian.
The dogs of the present cohort presented to the Clinique
des Cerisioz (Lyon, France) for different reasons (annual
blood testing, digestive troubles, PUPD, lethargy, and/
or dysorexia/anorexia). For each dog, a complete physical
examination was performed and a detailed medical history
was recorded. Previous medications and treatments
that could have influenced hepatic metabolism (such as
glucocorticoids or phenobarbital) lead to the exclusion
of the dog. Each dog underwent serum biochemistry and
hematology testing, abdominal ultrasounds and liver
biopsies. Biochemistry panel included plasma or serum ALP,
ALT, AST, cholesterol, GLDH, LDH, bilirubin, basal cortisol,
basal bile acids, GGT, Total T4 and triglycerides [23].
Liver biopsies were performed on the basis of plasma ALP
Revue Méd. Vét., 2015, 166, 7-8, 176-184
177
activity above 140U/L and presence of hepatic parenchymal
modifications by ultrasound examination. Ultrasonographic
criteria that raised the suspicion of vacuolar hepatopathy
were hyperechoic, heterogenous and/or nodular hepatic
parenchyma, with or without hepatomegaly. When
hyperadrenocorticism, hypothyroidism and/or hepatic
failure were suspected, based on compatible clinical signs
(polyphagia, PUPD, abdominal ptosis, dermatologic changes,
weight gain or loss…), hematological and/or biochemical
results (elevated liver enzymes activity, stress formula…)
and ultrasound examination (bilateral adrenal enlargement,
hepatomegaly, hepatic parenchymal modifications…),
further examination was performed (ACTH stimulation
test, low-dose dexamethasone suppression test, pre- and
post-prandial serum bile acid analysis, thyroid hormone
measurements, and/or additional imaging procedures)
[8]. Dogs were excluded from the study if their medical
history included medications that could have modified the
biochemical results (especially plasma ALP activity) and/
or liver histology and if their blood tests were compatible
with conditions known to modify hepatic metabolism
and histology (such as hyperadrenocorticism or diabetes
mellitus).
METHODS
Histological procedures
Liver biopsies were made by the same ECVIM-CA
diplomate, using a TruCut® (Coveto, France) biopsy needlea
(ultrasound guided) or realized surgically under laparotomy
or coelioscopy (wedge biopsies). Biopsies were taken in
area where changes in the hepatic parenchyma were most
visible (either at ultrasound or directly visualized during
laparotomy/coelioscopy). At least two samples were taken
each time. All liver biopsies (either using a biopsy needle or
wedge biopsies) were fixed in alcohol, formalin, and acetic
acid, and embedded in paraffin (Paraplast X-TRA, SigmaAldrich, USA). Sections (3μm thickness) were deparaffinized,
rehydrated and stained with HES for conventional histology,
picrosirius red for analyzing fibrosis, PAS for analyzing
glycogen, and Perl’s Prussian Blue for analyzing iron
deposits. Copper overload was examined using rhodanin
staining. Because of its cross-reactivity with canine species,
immunohistochemical staining using specific antibodies
against smooth muscle cells’ alpha-actin (Envision HRP
K4000 kit, ref M0851, clone number 1A4, Dako, Denmark)
was performed in order to identify activated hepatic stellate
cells (responsible for fibrogenesis)[32].
Transmission electron microscopy procedures
Ten samples were fixed in 2% glutaraldehyde–0.1M
Na-cacodylate/HCl buffer at a pH of 7.4 for 1 hour at 4°C,
rinsed in 0.1M Na-cacodylate/HCl buffer; and post-fixed in
1% OsO4–0.15 M Na-cacodylate/HCl buffer at a pH of 7.4
for 1 hour at 4°C. Dehydration was performed using graded
concentrations of ethanol, and samples were embedded in
178
PEYRON (C.) AND COLLABORATORS
Epon (EMbed 812 kit, Hatfield, UK). After polymerization
and evaluation of areas of interest by semi-thin sections
stained with toluidine blue, tissue was cut in ultra-thin
sections, mounted on copper mesh grids, contrasted with
uranyl acetate in methanol and lead citrate and examined
with a transmission electron microscope (JEOL 1200EX,
Peabody, USA) [11, 17].
Semi-quantitative evaluation of lesions
To ensure diagnostic consistency, histological sections
(3µm thickness) were all read by the same blind observer,
a hepatopathologist specializing in comparative pathology
(MC). For each slide, the entire surface was read and each
criterion was scored in a semi-quantitative way (vacuolation,
activity, fibrosis). Two separate readings were done for each
slide in order to refine the scores.
Hepatocellular vacuolation was scored from G1 to
G3 (G1, or mild = <25% vacuolated hepatocytes; G2, or
moderate = 26-50% vacuolated hepatocytes; G3, or severe =
>50% vacuolated hepatocytes), according to the procedure
in Center et al. [2]. Zonal location of vacuolated hepatocytes
was also examined. The extent and pattern of fibrosis was
evaluated using the METAVIR scoring system, in agreement
with WSAVA guidelines on canine liver pathology [5, 26].
Fibrosis was scored from F0 to F4 (F0 = no fibrosis; F1 =
portal fibrosis; F2 = moderate or minimally extensive
fibrosis, starting to extend from portal areas to other zones of
the hepatic lobule; F3 = extensive fibrosis, present in all the
different zones of the hepatic lobule; F4 = cirrhosis, i.e., severe
fibrosis with nodular regeneration). Hepatocellular apoptosis,
necrosis, and hepatic inflammation (collectively referred
to as activity) were scored from A0 to A3 (A0 = piecemeal
Vacuolation
Fibrosis
and lobular necrosis absent; A1 = minimal piecemeal
necrosis associated with absent to severe lobular necrosis,
or absent piecemeal necrosis associated with moderate to
severe lobular necrosis; A2 = moderate piecemeal necrosis
associated with absent to severe lobular necrosis; A3 = severe
piecemeal necrosis associated with absent to severe lobular
necrosis) using the METAVIR scoring system (Table I). Preneoplastic and neoplastic lesions (cell dysplasia, HCCA)
were also looked for and identified [33]. The same procedure
was performed for non-cirrhotic architectural modifications,
such as nodular regenerative hyperplasia (NRH) [10].
RESULTS
LIVER BIOPSIES
Fifty-five liver biopsies were performed: 39 using the
biopsy needle and 16 wedge biopsies. The number of biopsies
does not correspond to the number of dogs, as some dogs
had both wedge biopsies and biopsies performed using the
biopsy needle. For dogs that had both types of biopsies,
samples were evaluated together as one single entity.
HISTOPATHOLOGIC FINDINGS
Vacuolated hepatocytes were swollen and clear, with
small nuclei transposed to the periphery of the cell (Figure
1.A1). No inflammatory cells were present. Nineteen dogs
(44%) showed mild vacuolation (G1), and 24 dogs (56%)
exhibited moderate to severe vacuolation (12 dogs G2 and
12 dogs G3) (Table 2). No systematization was observed
regarding the location of the vacuolized hepatocytes, with no
zonal distribution. Vacuolated hepatocytes did not exhibit
any PAS staining, in contrast with adjacent non-vacuolated
Activity
Lobular necrosis
Absent/Minimal
G1: mild (<25%)
G2: moderate (2650%)
G3: severe (>51%)
F0: absent
F1: portal fibrosis
F2: minimally extensive
fibrosis
F3: extensive fibrosis
F4: cirrhosis
Piecemeal necrosis
Absent
Minimal
Moderate
Severe
A0
A1
A2
A3
Moderate
A1
A1
A2
A3
Severe
A1
A1
A2
A3
Table I: Semi-quantitative evaluation of vacuolation, fibrosis and activity
Vacuolation
Activity (piecemeal,
lobular necrosis, portal
inflammation)
Fibrosis
Grade
G1
G2
G3
Dogs nb (%)
19 (44%)
12 (28%)
12 (28%)
Grade
A0
A1
A2
A3
Dogs nb (%)
22 (51%)
16 (38%)
4 (9%)
1 (2%)
Grade
F0
F1
F2
F3
F4
Dogs nb (%)
12 (28%)
7 (16%)
10 (23%)
8 (19%)
6 (14%)
Table II: Number and percentage of dogs with hepatocellular vacuolation, activity, and fibrosis.
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VACUOLAR HEPATOPATHY IN 43 SCOTTISH TERRIER DOGS
179
Figures 1: Histological liver changes in vacuolar hepatopathy. A; after Hematoxylin-Eosin-Safran (HES) staining. A1; Vacuolar hepatopathy: Swelling of hepatocytes with vacuolated and reticular cytoplasm (arrow) (X40). A2; Example of activity within canine vacuolar hepatopathy, including piecemeal necrosis
(arrows) and portal lymphoplasmacytic inflammation (X20). B; after picrosirius red staining. B1; Extensive portal, periportal, and bridging fibrosis within
the liver in canine vacuolar hepatopathy (arrows) (X10). B2; Complete disturbance of the hepatic architecture by fibrosis and formation of regenerative
nodules (*) within the liver in canine vacuolar hepatopathy, indicative of cirrhosis (X10). Note some residual portal tracts (PT).
hepatocytes (Figure 2).
Liver fibrosis was present in portal and periportal areas
(Figure 1.B1) and cirrhosis was noticed in 6 animals (Figure
1.B2). Nineteen (44%) dogs showed no significant fibrosis
(F0+F1), 10 dogs (23%) had minimal fibrosis (F2), 8 dogs
(19%) had extensive fibrosis (F3) (Table 2).
ADDITIONAL HISTOLOGIC FINDINGS
Figure 2: Vacuolated versus normal hepatocytes. Note the absence of
staining in the vacuolated cells (arrow), contrasting with normal hepatocytes (arrow heads). PAS (X40).
Three dogs had a replacement of the normal hepatic
parenchyma by variably sized nodules that contained
alternating areas of atrophy and hypertrophy, with minimal
to no fibrosis, resembling human nodular regenerative
hyperplasia (Figure 3.A). Three additional dogs had
histologic changes consistent with a pre-neoplastic lesion
identified as small cell dysplasia (Figure 3.B1). Nine dogs had
well-differentiated hepatocellular carcinoma (Figure 3.B2), 4
of which also had significant fibrosis (F3/F4).
IRON AND COPPER
Regarding activity (i.e., inflammation and necrosis),
piecemeal/lobular necrosis and chronic portal inflammation
(Figure 1.A2) were observed in approximately 50% of the
dogs: 16 dogs (38%) had minimal activity, and only five dogs
(11%) showed moderate to severe activity (Table 2).
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The iron overload commonly observed in canine
macrophages was present. No copper overload was noted.
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PEYRON (C.) AND COLLABORATORS
Figures 3: Precancerous and cancerous lesions in Vacuolar Hepatopathy.
A; after picrosirius red staining. Formation of nodules formed by two
or more cell-thick hepatocyte cords that lack normal lobular architecture, resembling nodular regenerative hyperplasia in humans (X10).
Note the lack of extensive fibrosis and the presence of small remaining
parenchymal areas with atrophic hepatocytes (arrow). B; after HES
staining. B1; Presence of small cell dysplasia in the upper right (SCD),
consisting of basophilic hepatocytes arranged in one to two cell-thick
cords (X20). Note vacuolated hepatocytes (VH) on the lower left.
B2; Hepatocellular carcinoma (HCC), characterized by hepatocytes
irregularly arranged in cords of varying thickness and the presence of
gland-like spaces (X10).
IMMUNOHISTOCHEMICAL FINDINGS
The expression of smooth muscle a-actin was increased
around ballooned hepatocytes (Figure 4.A) and in fibrotic
areas in dogs with moderate to severe fibrosis (Figure 4.B).
ELECTRON MICROSCOPY FINDINGS
Hepatocytes with ultrastructural changes were usually
scattered among normal adjacent cells (Figure 5.A1).
No canalicular or nuclear changes were observed within
hepatocytes ultrastructurally. Hepatocytes had optically
empty areas in their cytoplasm, pushing aside cytoplasmic
organelles and making them appear clustered around the
nucleus or close to cytoplasmic membrane. The cytosol
contained large clear spaces with few organelles and lacked
normal glycogen rosettes (Figure 5.A2). The lack of glycogen
within affected hepatocytes was confirmed through PAS
staining, without any evidence of cytoplasmic glycogen
rosettes (Figure 2).
Figures 4: Activation of stellate cells labeled by immunohistochemistry (PO) using alpha smooth muscle actin antibody. A; Normal (black arrows) and
ballooned hepatocytes (white arrows). Ballooned hepatocytes are surrounded by activated stellate cells immunoreactive to alpha smooth muscle actin
(arrow heads), forming a delicate continuous network (X20). B; Abundant network of activated stellate cells immunoreactive to alpha smooth muscle actin
(arrows) surrounding regenerative nodules (*) in a dog with cirrhosis (X5). Note the residual portal tract (PT).
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VACUOLAR HEPATOPATHY IN 43 SCOTTISH TERRIER DOGS
181
Figures 5: Ultrastructural hepatocyte lesions by transmission electron microscopy (TEM). A; Ballooned hepatocytes. A1; A ballooned hepatocyte (BH) and
adjacent normal hepatocytes (H) and normal mitochondria (black arrows). A2; Detail of a ballooned hepatocyte. Note the clear areas within the cytosol (*)
with absence of glycogen rosettes, dense bodies, and nucleus (N). B; detail of mitochondrial alterations. B1; Higher magnification of ballooned hepatocyte
containing several mitochondria of varying shapes and sizes (M). B2; Higher magnification of a giant mitochondria (GM) containing crystalline inclusions
(C), denser matrix, and matrix inclusions (white arrows).
Mitochondrial alterations were the most striking
ultrastructural changes (Figure 5.B1), with pleomorphic and
enlarged mitochondria showing highly disorganized matrix
substance and loss of matrix dense bodies (Figure 5.B2), with
abnormally long and thick crests and black crystallin internal
inclusions.
Discussion
The objective of the present study was to assess the
hepatic lesions in a cohort of French STs with VH using
histological techniques and TEM in order to characterize
cellular changes and attempt to identify the pathogenesis of
this disease, in the hope that understanding of the observed
Revue Méd. Vét., 2015, 166, 7-8, 176-184
ultrastructural changes might highlight the mechanisms
underlying hepatocytic modifications.
Canine VH, first described by Center et al. in 1996 [2], is
cytologically characterized by enlarged hepatocytes, rarified
cytosol and increased hepatocyte fragility [4]. The term VH
is used inconsistently, but usually reflects hepatocellular
cytosolic glycogen accumulation, visible microscopically as
tiny rosettes lacking membrane confinement [4]. As described
by Center et al., VH is characterized by discrete vacuoles,
unless concurrent lipid vacuolation exists, particularly in STs
with VH (mixture of glycogen and lipids) [4]. In the present
study, vacuolation was mild (G1) in 45.4% of the population
182
studied, while 54.6% of the dogs showed moderate to severe
vacuolation (G2-G3). No zonal distribution was observed
for vacuolated cells. In several papers, the clear aspect of
the affected hepatocytes was described as being glycogen
[1, 4, 7, 24]. Interestingly, in our studies, the glycogen cell
content was evaluated for all liver samples using PAS staining
and was not shown to be increased in affected cells. Indeed,
no rosette-like features were observed in the cytoplasm
of altered hepatocytes, unlike in the adjacent normal
hepatocytes. This observation was confirmed by TEM, which
showed a ballooned aspect of the affected hepatocytes, with
a reticulated cytoplasm instead of vacuoles, making the term
“vacuolar” potentially unsuitable in the present study. The
difference observed between our study and previous reports
may be explained by differences in both methodologies but
as glycogen was objectivated in normal adjacent hepatocytes,
it seems reasonable to assume that glycogen would also have
been detected in ballooned hepatocytes if present. In the
same way, previous studies reported that the nucleus of the
hepatocytes was central in VH [4, 6] while in the present
study, the nucleus was displaced to the cell periphery. These
observations raise the question of whether VH in STs from
the US and STs from France is the same disease or not.
In light of the observations of the present study, VH
in French STs can be described as a diffuse liver disease
characterized by ballooned hepatocytes without glycogen
content and exhibiting mitochondrial modifications. The two
most frequent associated lesions reported in the present study
were fibrosis and neoplasia, leading the authors to consider
VH as a potential cause. Although almost 90% of the dogs
exhibited no or minimal activity (apoptosis, necrosis, and/
or inflammation), extensive fibrosis was observed in 30%
of the animals. Three dogs had architectural abnormalities
consistent with nodular regenerative hyperplasia (NRH),
characterized by an abnormal distribution of vessels leading
to parenchymal reorganization in nodules without any
fibrotic process [10]. Other concurrent hepatic lesions in
this study were small cell dysplasia (3 dogs) and cirrhosis
(6 dogs), which are thought to be pre-cancerous lesions
[7, 25, 33]. HCCA was observed in 9 dogs (21%) and was
associated with cirrhosis in 4 of them. This last observation is
in agreement with findings described by Center et al., even if
the percentage of dogs with VH and HCCA is slightly lower
(30% in Center’s study versus 21% in the present study) [4].
To corroborate Center’s observations, in a previous study,
we found a significant association (p<0.05) between ALP
activity and vacuolation of liver parenchyma [23] which is
also in agreement with a previous report conducted on 336
dogs with histologically confirmed VH [4, 28]. In the same
previous study, we also found significant association between
ALP activity and presence of HCCA, with ALP activity being
strongly correlated with HCCA. These results support the
association between ALP activity, VH and HCCA.
Several studies have implicated glucocorticoid
metabolism in the development of the disease, but little is
PEYRON (C.) AND COLLABORATORS
actually known about the real mechanism involved [24,
28, 34]. It is interesting to note that in the present study,
vacuolated hepatocytes showed common features with
hepatocytes of dogs presenting hyperadrenocorticism (which
is a dysfunction in the glucocorticoid metabolism), such as
cytoplasmic and mitochondrial changes. Similarly to what is
seen in canine hyperadrenocorticism, the cytosolic optically
empty areas seemed to push aside cytoplasmic organelles,
making them appear clustered around the nucleus or close to
cytoplasmic membrane, and some mitochondria were giant
and of irregular form, with abnormally long and thick crests
and black crystallin internal inclusions.
In human pathology, mitochondrial disorders are
observed in hereditary diseases termed “mitochondrial
cytopathies”[1, 19]. Liver tissue can be predominantly affected,
leading to hepatic failure through development of fibrosis.
The common pathogenesis in this spectrum of diseases is
a defect in the mitochondrial respiratory chain, leading to
oxidative stress. Mitochondrial injuries described in these
mitochondriopathies share similarities with those observed
in the canine liver samples studied using TEM. Interestingly,
Human Non Alcoholic Steato-Hepatitis (NASH) shares some
features with vacuolar hepatopathy as described in the present
study [29,30]. Murine models demonstrated that a decrease
in hepatic fatty acid oxidation associated with a decrease in
mitochondrial enzyme activity precedes the development of
NASH [12,27]. These observations suggest that a disruption
in mitochondrial ultrastructure and metabolism could be
responsible for the development of VH. Regarding the shape
of hepatocytes in VH, modifications of the cytoskeletal
intermediate filaments K8 and K18 in response to oxidative
stress could be responsible for the ballooning of the cells as
demonstrated in murine models [13,21].
Paradis et al. demonstrated an association between NASH
and HCCA, raising the hypothesis that NASH could pave the
way for an alternative manner of carcinogenesis, allowing
HCCA to develop in non-cirrhotic hepatic parenchyma,
contrary to what is found in most human liver diseases. [22,
25]. This process could be another similarity between NASH
and VH, as in the present study, among the 9 dogs that
developed HCCA, 5 exhibited no signs of cirrhosis.
The present study has several limitations. Liver biopsies
were not performed in the same manner for all dogs (either
needle biopsies or wedge biopsies) and this might have been
a source of bias in the interpretation of liver histology. Wedge
biopsies provide a more accurate view of most liver disorders
than needle biopsies but needle biopsies are sufficient for
many approaches, since samples from more than one liver
lobe are provided [6]. However, to prevent such a bias, groups
could have been made separating dogs according to the way
biopsies had been realized (needle or wedge biopsies). For
dogs that had both types of biopsies, samples were evaluated
as a single entity while it might have been interesting to make
comparisons in order to see if the way biopsies were made
had an incidence on histological results. For cost reasons,
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VACUOLAR HEPATOPATHY IN 43 SCOTTISH TERRIER DOGS
only 10 cases were studied using TEM, which is not much
given the total number of dogs (43). Studying all patients
with TEM would have provided a larger view of the hepatic
lesions. The fact that histological samples were read by a
non-veterinarian, non-boarded pathologist can be seen as
a limitation; however, in the present study, the pathologist
who read the slides is an international reference on human
liver histology and is also very adept at analyzing canine
histological samples, having worked on numerous studies
involving comparative pathology.[16,18, 31] Regarding the
way hepatic lesions were graded, the METAVIR scoring
system was used to characterize activity and fibrosis in the
present study. This scoring system was initially built for
the evaluation of human chronic hepatitis and is routinely
used in human liver histology. We carefully considered the
WSAVA guidelines regarding the evaluation of liver biopsies
and the features of the METAVIR scoring system fit all the
criteria and recommendations listed by the liver study group
[5].
In the present study, VH in French STs was characterized
by different grades of hepatocyte vacuolation and fibrosis.
Hepatic lesions showed little inflammation and/or necrosis.
Interestingly, vacuolation was not associated with glycogenic
content, contrary to what was described in previous reports.
These observations as well as our TEM findings tend to
describe the affected hepatocytes as “ballooned” rather than
vacuolated cells. The similarities between human NASH
and VH characteristics described in the present study are
striking, and using NASH as a model for canine VH may be
helpful in investigating the etiology, prevention and possible
treatments of this disease. Many molecular patterns remain
to be investigated in order to understand the underlying
mechanisms that trigger the development of VH, especially
the role of keratin filaments and mitochondrial abnormalities.
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