Dose- and Time-Dependent Oval Cell Reaction in
Acetaminophen-Induced Murine Liver Injury
Alexander V. Kofman,1 Glyn Morgan,2 Adam Kirschenbaum,1 Jon Osbeck,1 Mehboob Hussain,1
Scott Swenson,1 and Neil D. Theise1,3
We examined the response of murine oval cells, that is, the putative liver progenitor cells, to
acetaminophen. Female C57BL/6J mice were injected intraperitoneally with varying doses
of N-acetyl-paraaminophen (APAP) (250, 500, 750, and 1,000 mg/kg of weight) and sacrificed at 3, 6, 9, 24, and 48 hours. In preliminary studies, we showed that anticytokeratin
antibodies detected A6-positive cells with a sensitivity and specificity of greater than 99%.
The oval cell reaction was quantified, on immunostaining for biliary-type cytokeratins, as
both number and density of oval cells per portal tract, analyzed by size of portal tract.
Acetaminophen injury was followed by periportal oval cell accumulation displaying a moderate degree of morphological homogeneity. Oval cell response was biphasic, not temporally
correlating with the single wave of injury seen histologically. Increases in oval cells were
largely confined to the smallest portal tracts, in keeping with their primary derivation from
the canals of Hering, and increased in a dose-dependent fashion. The timing of the two peaks
of the oval cell reaction also changed with increasing dose, the first becoming earlier and the
second later. In conclusion, our studies indicate a marked oval cell activation during the
height of hepatic injury. Oval cells appear to be resistant to acetaminophen injury. The close
fidelity of mechanism and histology of acetaminophen injury between mouse and human
livers makes it a useful model for investigating liver regeneration and the participation of
stem/progenitor cells in that process. (HEPATOLOGY 2005;41:1252-1261.)
E
xperimental models currently available to study
hepatic progenitor cells (HPCs) in animals include
liver injury caused by partial hepatectomy, various
chemical hepatotoxins, irradiation, toxic diets, and generation of transgenic (urokinase-type plasminogen activator, hepatitis B surface antigen, suicide genes) and
knockout (fumaryl acetoacetate hydroxylase– deficient)
mice.1-6 Nonetheless, except for partial hepatectomy,
Abbreviations: HPC, hepatic progenitor cell; APAP, N-acetyl-paraaminophen;
OVc, oval cell; PBS, phosphate-buffered saline; CK, cytokeratin; FITC, fluorescein
isothiocyanate; PT, portal tract; BD, bile duct; PV, portal vein; NAPQI, N-acetylp-benzoquinone imine; SCF, stem cell factor.
From the 1Department of Medicine, Division of Digestive Diseases, Liver & Stem
Cell Research Laboratory, Beth Israel Medical Center, New York, NY; 2Department of Surgery, New York University School of Medicine, New York, NY; and
3Departments of Medicine and Pathology, Albert Einstein College of Medicine,
New York, NY.
Received November 22, 2004; accepted March 7, 2005.
Supported by NIH grant 5 R01 DK58559-04 and Singer/Hellman Research
Grant (Beth Israel Medical Center, New York, NY).
Address reprint requests to: Neil D. Theise, Division of Digestive Diseases, First
Avenue at 16th St, Beth Israel Medical Center, New York, NY 10003. E-mail:
[email protected]; fax: 212-420-4373.
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.20696
Potential conflict of interest: Nothing to report.
1252
none of the above-mentioned models shows close fidelity
to the pathological alterations found in human livers.
However, acetaminophen (N-acetyl-paraaminophen,
tylenol, paracetamol, APAP) is a predictable hepatotoxin
that reproducibly demonstrates close fidelity of histological injury between animals and humans.7-10 Because
APAP is the most frequent cause of fulminant liver failure
in both the United States and the United Kingdom, with
a mortality rate of approximately 90%, exploration of
HPC functioning in this model may have direct clinical
applicability.11-13
The main loss of liver mass after high-dose APAP ingestion occurs because of hepatocyte necrosis in the centrilobular areas. Appearance of necrosis is preceded by
hepatic microvascular injury and congestion.14,15 In the
massive injury characteristic of high-dose APAP intoxication, hepatocytes may be unable to accomplish full parenchymal reconstitution, and, thus, regeneration must
include activation and hepatocyte differentiation of
HPCs. In animal models of liver regeneration and hepatocarcinogenesis, these HPCs are referred to as oval cells
(OVc), which constitute a heterogeneous cell population,
characterized by an ovoid nucleus, small size, and scant
basophilic cytoplasm.16 They express phenotypical mark-
HEPATOLOGY, Vol. 41, No. 6, 2005
ers of both the biliary epithelium (cytokeratins CK7,
CK19), and hepatocyte lineages (␣-fetoprotein, albumin).17-20 OVc cytoplasm is also strongly immunoreactive for rat oval cell marker OV-6, targeting an epitope
shared by CK14 and 19.21 Monoclonal antibody A6, developed by Valentina Faktor and colleagues, targets an
uncharacterized protein that is also widely considered a
reliable marker of OVc.22,23
In mice and in humans, HPC proliferations derive
largely from the canals of Hering.23,24 We first demonstrated the derivation of HPCs from the canals of Hering
in humans in the setting of APAP-induced massive human liver necrosis.25 However, the time- and dose-dependent response of local HPCs to the massive APAPinduced liver injury has not yet been carefully
documented. Moreover, methods of detection and reliable quantification of OVc in murine livers have not been
well established.
Here we used the murine model of APAP-induced injury to study time course, dose–response, and distribution
of the HPC reaction secondary to APAP-induced liver
injury. The results show that in the earliest period after
APAP challenge the HPC reaction is time- and dose-dependent, biphasic in sublethal doses, and prevails in the
smallest, most proximal portions of the biliary tree. These
data support the concept of the contribution of HPCs to
hepatocellular regeneration.
Materials and Methods
Animals and APAP Treatment. All animal experiments were carried out after obtaining permission from
the Division of Laboratory Animal Resources, New York
University School of Medicine. Female C57BL/6J mice
(Taconic, Germantown, NJ) each weighing approximately 20 g were provided free access to chow and water
for at least 1 week before study. Before APAP administration, animals were fasted for 8 hours with free access to
water. Fresh suspensions of APAP (Sigma Chemical Co.,
St. Louis, MO) were prepared in warm (40°C) phosphate-buffered saline (PBS), pH ⫽ 7.2, and given in a
volume of 1 mL intraperitoneally at doses of 250, 500,
750, and 1,000 mg/kg body weight. Control littermate
mice received vehicle (PBS at the same volume) only.
Mice from each group were killed for organ retrieval at 3,
6, 9, 24, and 48 hours after acetaminophen administration.
In an additional experiment, mice received injections
of APAP at doses of 750 and 1,000 mg/kg body weight, as
well as injections of PBS and sham injections (without
administration of any fluid), and then were sacrificed after
1 and 2 hours.
KOFMAN ET AL.
1253
Tissue Preparation and Histological Grading of
Hepatic Injury. Liver tissues for permanent sections
were fixed in 10% phosphate-buffered formalin for 4
hours, embedded in paraffin, sectioned at 4-␮–thick sections, and stained with hematoxylin-eosin. Hepatic injury
was evaluated as a percentage of necrotic tissue in the 5
randomly selected areas at a magnification of ⫻40. Separate samples of liver were snap frozen in liquid nitrogen
and stored at ⫺20°C. Five-micrometer cryostat sections
of these were prepared on charged slides and air dried for
immunofluorescent staining.
Detection of OVc by Immunofluorescence and Immunoperoxidase Staining. Evaluation of murine OVc
reactions is hampered by the lack of commercially available antibodies against OVc differentiation markers.
OV-6, a widely accepted human and rat OVc marker, is
known to target a shared epitope of cytokeratins 14 and
19; yet anti–OV-6 antibodies (R&D Systems, Minneapolis, MN; catalogue no. MAB2020) failed to detect OVc
in mouse tissues (data not shown). Another widely accepted marker of OVc found predominantly in the epithelia is recognized by rat monoclonal antibody A6.22,23
The A6 epitope has been shown to be less detectable in
formalin-fixed tissues26; therefore, we selected commercially available antibodies that target wide-spectrum cytokeratins, including murine biliary-type cytokeratins:
rabbit anti-cow cytokeratin polyclonal antibodies (catalogue no. Z0622, DAKO, Carpinteria, CA). These polyclonal antibodies were raised against bovine epidermal
keratin units of 58, 56, and 52 kd, among others, and
cross-reacted with a wide range of cytokeratins (CKs),
including the high–molecular weight CKs present in
cholangiocytes (DakoCytomation, Carpinteria, CA; data
sheet).
In the preliminary studies, to validate the use of these
anti-CK antibodies, we immunofluorescently doublestained frozen liver tissue from normal and APAP-treated
mice at various times after APAP injection. The frozen
sections were air dried and incubated at 37°C for 2 hours
with the rabbit polyclonal anti-CK antibodies and with
rat monoclonal A6 antibodies. After a PBS wash, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit
antibodies (Molecular Probes, Eugene, OR; catalogue no.
F2775), and Cy5-conjugated goat antirat antibodies (Abcam, Cambridge, MA; catalogue no. ab6565) were then
layered on the tissue and incubated for another 2 hours at
room temperature. The slides were then counterstained
with 4⬘,6-diamidine-2-phenylindole and coverslipped
with Vectashield mounting medium (Vector Laboratories, Burlingame, CA; catalogue no. H1200). Immunofluorescence was detected using the 4⬘,6-diamidine-2phenylindole, FITC, and Cy5 filters of an Olympus
1254
KOFMAN ET AL.
(Melville, NY) fluorescent microscope and captured,
pseudocolored, and combined using imaging software
IPLab 3.9 (Scanalytics Inc., Fairfax, VA) as previously
described.27 Immunoperoxidase with the same anti-CK
antibodies for OVc light-microscopic detection on all experimental tissues was then performed. Formalin-fixed
liver sections were deparaffinized, and endogenous peroxidase was quenched with 3% H2O2 for 15 minutes. Slides
were then treated with Proteinase K 100 ␮L (DAKO) for
10 minutes at room temperature, thoroughly rinsed,
blocked with serum-free protein in PBS (DAKO Catalized Signal Amplification System) for 20 minutes, and
incubated with rabbit anti-CK antibodies previously described for 2 hours at 37°C. After washing with PBS, the
secondary antibody (anti-rabbit biotinylated immunoglobulins) (DAKO) was applied. Antigen localization
occurred after repeat washing with avidin-biotin horseradish peroxidase macromoleclar complex (ABC reagent,
VECTASTAIN, Vector Laboratories) according to the
manufacturer’s instructions and counterstained with
Mayer’s hematoxylin (0.1%; Sigma Chemical Co.).
Evaluation of OVc Reaction. In each fixed liver tissue sample, we examined up to 25 portal tracts (PTs) of
the appropriate size (see below) with visible bile duct(s)
(BD) and portal vein (PV) and counted OVc, defined as
densely cytokeratin-positive cells with oval/cuboidal morphology and high nuclear to cytoplasmic ratio. Care was
taken to identify those cells that did not border on lumina
and were therefore not part of the preexisting normal BD.
Intermediate hepatocyte-like cells, that is, cells with hepatocyte-like morphology but cytokeratin-positive staining, were rarely present in the regenerating mouse livers
and were not included in quantifications.
In normal mouse liver, the PV occupies nearly the
entire PT area, with scant stroma. Thus, to evaluate distribution of OVc according to PT size, we used the easily
definable wall of the PV as a surrogate marker for PT size.
Because the largest portal tracts in any section (one or
two) exhibited extensive branching, cross-sectional measurements could not be assessed; thus, these largest units
were also excluded from analysis.
The longest and shortest diameters of the sectioned PV
were measured using an eyepiece with measuring grid at
the magnification 40⫻. At this magnification, one scale
unit of the measuring grid corresponds to 2.5 ␮m. The
PV area was calculated according to the formula S (␮m2)
⫽ 2.52␲ab/4, where a and b were the major and minor PV
diameters expressed in the eyepiece scale units. Thus, we
calculated not only the number of OVc per portal tract,
but also the density (D) of OVc per portal tract, using the
formula D ⫽ N/ab, where N was the number of OVc per
HEPATOLOGY, June 2005
portal tract, and a and b were the major and minor PV
diameters expressed in the eyepiece scale units.
Statistical Analysis. Results were expressed as means
for each group of animals. Parametric (Pearson) correlation and the two-tailed P value were calculated. Results
were considered statistically significant at P ⱕ .05.
Results
Histology of APAP Injury. Progressive centrilobular
vacuolization, congestion, necrosis, and inflammation
could be observed in all APAP-treated animals. Administration of APAP at the highest dose (1,000 mg/kg) resulted in massive destruction of liver tissue (up to 90%100%) and infiltration of blood cells into the space of
Disse with the highest level of destruction at 9 and 24
hours. At 48 hours, despite the presence of significant
residual hemorrhagic areas, well-defined areas of restoration could be identified in surviving animals. The lower
doses of APAP (750, 500, and 250 mg/kg body weight)
also caused significant damage (30%-70%), with the peak
at the 9-hour time point. At every time and at every APAP
dose administrated, viable hepatocytes were concentrated
in the periportal areas around the CK cells, which appeared to form foci of regeneration of the liver tissue (Fig.
1). The 1,000-mg/kg APAP dose represented the median
lethal dose in these experiments.
Validation of Anti-CK Antibodies for Detection of
OVc. A6 antibody stained bile ducts, ductules, and morphologically recognizable OVc, as previously described,22,28 although authors also reported some A6negative epithelial cells in terminal bile ductules. In the
preliminary studies, portal tracts and surrounding tissue
were examined by fluorescence microscopy. Our analysis
included 22 portal tracts and periportal areas, identifying
746 associated OVc. The anti-CK antibody was positive
in all but one A6-positive cell (which appeared morphologically intermediate between OVc and hepatocytes).
Four CK-positive, A6-negative morphological OVc were
also identified. Hepatocytes (1,022) in the captured images were negative with both antibodies. Using A6 as the
standard, anti-CK antibodies detected OVc with a sensitivity and specificity of greater than 99%, validating their
use in murine OVc detection (Fig. 2). Interestingly, in
A6/CK-positive cells, the antigens detected had only partially overlapping subcellular localization.
Expansion of CK-Positive OVc After APAP-Induced Injury. Activation of HPC compartment under
the various pathological conditions is reflected by the expansion of OVc. Systemic administration of APAP at different doses led to accumulation of OVc that appeared
singly or in irregular strings without lumens (Fig. 3). OVc
HEPATOLOGY, Vol. 41, No. 6, 2005
Fig. 1. Histological alterations caused by murine N-acetyl-paraaminophen (APAP)-mediated toxicity; APAP at dose 750 mg/kg body weight,
24 hours after injection. Sections demonstrate a murine zonal lesion
nearly identical to that seen in humans: the centrilobular pattern of
hepatocyte injury, sparing the portal regions. Cytokeratin staining highlights the portal tracts by marking the bile ducts and also reveals the
periportal distribution of the oval cell proliferation. (A) Hematoxylin-eosin
staining (original magnification ⫻2). (B) The serial section of the same
sample; cytokeratin staining (original magnification ⫻2). (C) Hematoxylin-eosin staining (original magnification ⫻10). (D) The serial section of
the same sample; cytokeratin staining (original magnification ⫻10).
KOFMAN ET AL.
1255
sometimes appeared to emerge from the BD. OVc displayed a moderate degree of morphological homogeneity:
all of the cells were strongly positive for cytokeratin,
though the intensity of staining varied from very strong
(more characteristic to cholangiocyte-like, “true”oval
cells) to rather weak (in cells with slightly more hepatocyte-like morphology).
Quantification of OVc around the portal tracts with
portal veins of cross-sectional areas less than 5,000 ␮m2
showed a statistically significant increase (P ⬍ .005) in
OVc after APAP treatment compared with PBS- and
sham-treated animals (Fig. 4). For the sham group (n ⫽
85; the number of counted portal tracts) the average number of OVc per portal tract was 8.07 ⫾ 5.02; for PBSinjected animals (n ⫽ 160), 7.60 ⫾ 5.53; and for the
APAP group (n ⫽ 940), 9.66 ⫾ 6.1. Standard errors of
the sample means were 0.54, 0.44, and 0.2 for the sham,
PBS, and APAP groups, respectively. These data for
APAP-injured livers, as well as those from sham and PBSinjected animals, are presented cumulatively, including all
time points and, for APAP-injected mice, all doses.
OVc Reaction Is APAP Dose- and Time-Dependent. Figure 5 summarizes changes in OVc numbers at
different times depending on the APAP dose. OVc response did not correlate well with the development of the
morphological signs of liver injury (i.e., necrosis, hemorrhagic infiltration). Whereas the intensity of liver damage
was proportional to APAP dosage and appeared in a single
wave extending outward from the central vein (Fig. 1),
OVc dynamics were biphasic in response to the intermediate APAP (250-, 500-, 750-mg/kg) doses. Injection of
APAP at the lowest concentration, 250 mg/kg resulted in
a two-wave dynamic, with the highest points at 9 and 48
hours after APAP injection. APAP at both 500 mg/kg and
750 mg/kg resulted in OVc reaction characterized by the
same detectable two-wave time course, but with peaks at 6
and 24 hours. APAP 750 mg/kg caused the highest increase in OVc, at 6 hours.
In a separate experiment, at 1 and 2 hours after intraperitoneal injection of APAP, we also detected an increase
in the amount of OVc compared with sham- and PBSinjected animals. However, the difference between the
groups was statistically insignificant (data not shown).
Within the first 3 hours after PBS injection, the levels of
OVc increased somewhat compared with sham-injected
mice (data not shown).
In summary, in the first 2 days after APAP-induced
liver injury, 2 peaks of OVc appearance characterized the
local progenitor response at doses of 250, 500, and 750
mg/kg. The biphasic nature of these responses is confirmed by statistically significant differences between values of each peak and the trough value between them. The
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KOFMAN ET AL.
HEPATOLOGY, June 2005
Fig. 2. Validation of anti-cytokeratin (anti—CK) antibodies for oval cell detection. Co-localization of anti-CK antibodies (fluorescein-isothiocyanate (FITC)labeled, pseudocolored green) with anti– oval cell
antibody A6 (Cy5-labeled, pseudocolored red) in frozen liver tissues. Co-localization throughout all
branches of the biliary tree results in yellow to orange
overlaps. Substantially uniform co-localization of the
two antibodies confirms the validity of anti-CK antibodies for detection of oval cells. Original magnification, ⫻20.
1,000-mg/kg response did not share this statistically significant biphasic distribution. APAP concentration at 750
mg/kg elicited the highest OVc reaction. No statistically
significant changes were found for the cells constituting
bile ducts (data not shown).
Association of OVc With the Smallest Portal
Tracts. To address the question of whether OVc reaction was uniform around all PTs over their complete
size range, we first calculated the correlation between
the average OVc numbers around the PT and PT sizes
in normal and injured livers (Fig. 6). The Pearson correlation coefficient (r) for these 2 parameters was statistically significant for all groups (r ⫽ 0.41 for sham
group, r ⫽ 0.34 for PBS-injected, and r ⫽ 0.46 for
APAP-treated animals). The correlation levels were approximately the same within all the ranges of PT sizes
divided into 5 size ranges for ease of calculation: less
than 1,000 ␮m2, between 1,000 and 2,000 ␮m2, between 2,000 and 3,000 ␮m2, between 3,000 and 4,000
␮m2, more than 4,000 ␮m2 (Fig. 6A). To characterize
more precisely the relationships between OVc generated around the PT and PT size, we next calculated the
density of OVc around each PT (see Materials and
Methods). In the experimental group (APAP 750 mg/
kg), OVc density significantly (P ⬍ .05) exceeded that
in the PBS group (Fig. 7A). Whereas in PBS-injected
animals density remained basically the same over time,
6 hours after 750 mg/kg APAP injection, density rose
almost 3 times (P ⬍ .02), yet quickly returned to the
previous levels later (Fig. 7B). The scatter analysis of
the density and portal vein size in APAP group showed
a statistically significant negative correlation between
Fig. 3. Expansion of cytokeratin (CK)-positive cells after N-acetyl-paraaminophen (APAP) treatment. (A) Normal liver. 1, portal vein; 2, normal bile
duct. (B) Oval cell reaction after APAP injection (750 mg/kg body weight, 48 hours). Heterogeneous population of expanding CK-positive cells varying
in size and morphology with mostly oval nuclei. 1, portal vein; 2, normal bile duct. (C) Oval cell reaction after APAP injection (250 mg/kg body weight,
9 hours). 1, portal vein; 2, bile duct; 3, intermediate hepatobiliary cells. Original magnification, ⫻20.
HEPATOLOGY, Vol. 41, No. 6, 2005
KOFMAN ET AL.
1257
Fig. 4. Increased number of oval cells (OVc) around portal tracts in
N-acetyl-paraaminophen (APAP)-treated animals. Values in the bars represent the average amount of oval cells per portal tract for each group. The
difference between the APAP group and control groups is statistically significant (P ⬍ .005). See further explanations in the text. PBS, phosphatebuffered saline.
these 2 parameters in all of the groups (r ⫽ ⫺0.60 for
the normal group, r ⫽ ⫺0.37 for PBS-injected, and
r ⫽ ⫺0.31 for APAP-treated animals, see Fig. 8A).
Although OVc were also present in significant amounts
Fig. 6. Distribution of oval cells according to the portal tract size. (A)
Oval cells (OVc) around portal tracts versus portal tract size. (B) Average
oval cells numbers within the various ranges of PT size. r ⫽ Pearson
correlation coefficient. PT, portal tract; PBS, phosphate-buffered saline;
APAP, N-acetyl-paraaminophen.
Fig. 5. Dose- and time-dependent oval cell dynamics. Graphics represent
changes in average amounts of oval cells per small portal tract over time.
Arrows highlight points at which differences between oval cell numbers were
statistically significant (P ⬍ .05); namely, at doses of 250, 500, and 750
mg/kg the peaks differ significantly from the trough, confirming a biphasic
pattern of oval cell response. Although the 1000 mg/kg has the appearance
of a shallow trough, separating the 2 peak values, this was not statistically
significant. The loss of a clear biphasic pattern at that dose may relate to its
high lethality. PBS, phosphate-buffered saline.
within the larger PTs, their density in large PTs was
much lower, and it was much higher in small PTs. In
other words, APAP treatment caused a greater increase
in density in the smallest PTs (Fig. 8B). Interestingly,
intraperitoneal injection of PBS itself was associated
with the higher density compared with normal animals
(P ⬍ .05).
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KOFMAN ET AL.
HEPATOLOGY, June 2005
Fig. 7. Periportal oval cell density. (A) Values represent average
density of oval cells (OVc) around portal tracts for each group. Differences between the groups (Sham vs. phosphate-buffered saline [PBS],
and PBS vs. N-acetyl-paraaminophen [APAP]) are statistically significant
(P ⬍ .05). Data for APAP are cumulative for all animals injected with
APAP at the dose 750 mg/kg body weight, inclusive of all time points in
the experiment. (B) OVc density dynamics over time: at 6 hours after
APAP injection (750 mg/kg body weight), the increase of density is
statistically significant (P ⬍ .05).
Discussion
OVc Reaction and Liver Regeneration. APAP is
metabolized by cytochrome P-450 into the toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI
is normally conjugated in the liver with glutathione to
yield a harmless mercaportal tracturic acid; however,
overdose of APAP depletes hepatic glutathione, and
NAPQI covalently binds to DNA and cysteine residues
on numerous hepatic proteins,7 resulting in the formation
of 3-(cysteine-S-yl) APAP adducts. The first pathological
changes in the liver may be detected within minutes after
APAP injection.29-31
Our findings confirm that APAP-induced liver toxicity in the mouse elicits an HPC response as it does in
humans. The appearance of OVc could first be recognized within hours; this is different from the several
days reported in murine cocaine injury.32 The difference may arise in part because of the nature of the
injury: cocaine yields periportal necrosis. The disruption in stroma, nonparenchymal cells, and perhaps the
canals of Hering and ductules themselves, is likely to
require recovery before the organized OVc reaction can
take place. In APAP injury, the lesion is central and,
thus, the zone wherein OVc reactions take place is
Fig. 8. Distribution of oval cells (OVc) according to their periportal
density. (A) OVc density versus PV size. (B) Average OVc density within
the various ranges of PV size. r ⫽ Pearson correlation coefficient.
Differences within the experimental groups (sham-, PBS-, and APAPtreated animals) between OVc density around small portal tracts
(⬍1,000 ␮m2) and OVc density around the larger PVs are statistically
significant (P ⬍ .005). Differences between the experimental groups of
animals within the same range of small portal tracts (⬍1,000 ␮m2) are
statistically significant (P ⬍ .05). PV, portal vein; PBS, phosphatebuffered saline; APAP, N-acetyl-paraaminophen.
HEPATOLOGY, Vol. 41, No. 6, 2005
anatomically preserved. Our findings are in accordance
with the literature’s data pointing to the rapid biochemical and morphological changes in the liver in
response to APAP; we detected statistically significant
OVc reaction as early as 3 hours after injection of
APAP at the highest dose. It remains unclear whether
the APAP-induced OVc are entirely derived from intrabiliary HPCs or else some engraft from the circulation.33
Biphasic OVc Responses to APAP Injury. The time
course of the OVc response did not directly correlate with
the histologically defined liver damage. This may be explained by the fact that excess NAPQI is probably cyclically reduced back to APAP and again to NAPQI, thus
resulting in the progressively growing liver damage. Also,
whereas OVc are cytochrome P-450 negative and would
have a survival advantage, OVc-derived hepatocytes,
which express P-450, would then become sensitive to residual APAP. This may, in part, explain the second wave
of OVc proliferation in response to APAP.
The biphasic time course of OVc activation after
APAP at 250 mg/kg precisely matches the previously reported biphasic, temporal fluctuations of serum stem cell
factor (SCF) in mice after APAP liver injury.34 SCF is a
transmembrane protein in all epithelia of the liver, enzymatically cleaved from the cell surface during injury.35
The receptor for SCF, c-kit, plays a fundamental role in
stem cell functioning and, in the liver, is expressed by
OVc, the canals of Hering, and the intralobular bile
ducts.25,33 SCF cleavage and release from damaged hepatocytes may lead to the first wave of OVc activation. The
second wave may come with de novo production and secretion of new SCF. Importantly, exogenously administered
SCF rescues mice from lethal APAP administration,34 suggesting the importance of this signaling pathway in HPCmediated hepatic regeneration. The loss of this biphasic
response in the median lethal dose (1,000 mg/kg) may relate
to the significant lethality at this dose level.
Quantification of OVc: Absolute Number Versus
Density per Portal Tract. The methods used to quantify OVc, once detected, also have been historically problematic. It has previously been approached in 3 ways: (1)
semiquantitative grading, (2) quantifying numbers of
cells in randomly selected medium- or high-power fields,
(3) quantification of OVc per portal tract. The first approach is imprecise and interferes with statistical analysis.
Quantification by randomly selected microscopic fields is
inappropriate for phenomena that are not themselves randomly distributed, such as OVc. The third approach rests
on the assumption that OVc proliferation in response to
injury is uniform around all portal tracts. Here we show
that the overwhelming mass of increasing oval cells is
KOFMAN ET AL.
1259
found around the smallest portal tracts (Fig. 8). Following
on this, it then makes sense to limit assessment of OVc
expansion to analysis of those smallest portal tracts, excluding the larger ones.
The effects of such an analysis are clear in comparing
the data presented in Figs. 4 and 6 with those presented in
the leftmost portion of Fig. 7B. Trends with indistinct or
low-level significance when assessed by number of OVc
per portal tract, including portal tracts of every size, become much more statistically meaningful when confined
to the smallest portal tracts, approximately 1,000 ␮m2.
Thus, our method represents a rational and reproducible
system for quantitative evaluation of OVc reactions in
acute liver injury. The data also support our prior contention that the proximal biliary tree is a primary source of
HPCs.24,25
A6 is considered a differentiation marker of both
epithelial and erythroid cell lineages in the developing
mouse and mouse liver.22 The pattern of A6 expression
in the epithelial cells of the mouse fetal liver precisely
represents that of cytokeratin 8 in the developing bile
duct of the rat liver.36 In human tissues, the most
widely used markers of intermediate hepatobiliary
cells, the human OVc equivalent, has been anti-CK
antibodies targeting biliary-type cytokeratins (CK7
and CK19).37 This corresponds to the use of OV-6,
which recognizes a shared epitope of CK14 and CK19.
Thus, we chose to validate and use anticytokeratin antibodies for OVc detection, but focusing on antibodies
that were both commercially available and easily used
to decorate formalin-fixed, paraffin-embedded tissues
for the extensive light microscopic evaluation and
quantification required. The extensive colocalization
of the employed anti-CK antibodies with A6 confirms
that it is a valid tool for detection of murine OVc.
OVc Activation in Response to Intraperitoneal
Fluid Injection. Intraperitoneal PBS injection also
caused a small, but statistically significant, increase in
OVc density compared with sham-treated animals. The
reasons for this observation remain unclear. A direct irritation of the intra-abdominal branches of the vagus nerve
by intraperitoneal fluid or reciprocal activation of the vagal nerve system after the stress caused by the injection
could lead to autonomic-based activation of the hepatic
progenitor cell compartment via muscarinic acetylcholine
receptor.38 Another possibility is that increased intraabdominal fluid pressure after injection affects lymphatic or
blood flow into the liver that, in turn, may be recognized
as a damage signal by some other mechanism and induce
cell proliferation as a part of regenerative response. Because the anatomical relationships of the canals of Hering
to lymphatic drainage or to the finest branches of the
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KOFMAN ET AL.
vessels supplying the liver are, as yet, unknown, these and
other possibilities cannot be excluded. The absence of the
biphasic OVc activation suggests that it is distinct and
separate from the toxic impetus to additional proliferation.
The use of anti-CK antibodies for OVc detection in
formalin-fixed mouse livers, validated here by its extremely high sensitivity and specificity compared with A6
antibody, provides a methodological link between the approaches customarily used by investigators relying on animal experimentation and those exploring HPC
activation in human tissues. Using a mouse model of
APAP hepatic injury, we show that in the first 2 days after
APAP administration the OVc response is both time- and
dose-dependent. OVc proliferate most around the smallest PTs, in keeping with their primary origin in the most
proximal biliary tree, in particular the canals of Hering.
Our analysis of OVc distribution suggests a rational, anatomy-based approach for precise OVc quantification in
injured mouse livers, which can allow for statistically
meaningful comparison of OVc response within and between models of acute injury.
The marked OVc activation during the height of
hepatocyte injury suggests that they are resistant to the
toxic effects of APAP metabolites, perhaps through
minimal presence of cytochrome P450 metabolic pathway in their resting and early proliferative stages. The
OVc activation is biphasic, mirroring previously reported biphasic SCF release, suggesting the importance
of this chemokine for OVc activation. Also, perhaps,
the second wave might reflect a gain of susceptibility to
APAP-toxicity when they begin to differentiate down
an hepatocyte lineage while the drug and its metabolites are still present, leading to a second (though lesser)
wave of hepatocyte injury triggering a further OVc
proliferation.
In summary, in view of its close fidelity with human
APAP-related injury, we suggest that the murine model
of APAP toxicity provides a most useful tool for investigation of hepatocellular regeneration, in particular
from intra-organ and extra-organ stem cell compartments.
Acknowledgment: A6 antibody was supplied to us by
Dr. V. Faktor (Laboratory of Experimental Carcinogenesis, NCI, NIH).
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