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Regular Articles |
From the Departments of Pathology*
and Cell
Biology,
University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distinct presence of biliary epithelium, hepatocytes, and stellate cells allows use of these cultures to identify and study factors essential for the phenotypic maturation of these histological elements. Substances such as dexamethasone (nonmetabolizable corticosteroid), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) cannot be easily studied in mouse genetic models of gene deletion. Homozygous deletion of HGF, or its receptor, c-met, results in embryonic lethality. Livers have been described as smaller than normal, with small immature hepatocytes. No other details of histological abnormalities have been provided and it is not clear whether the hepatic abnormalities are secondary to the placental defects observed in the embryos.5-7 Homozygous deletions of EGF receptor or its multiple ligands have had no impact on hepatic development, probably because of functional overlap with other receptors of the same family.8,9 Given the difficulty of performing adrenalectomy in rodent embryos and the multiplicity of sites synthesizing corticosteroids in embryonic development, evaluation of the role of corticosteroids in hepatic tissue development by in vivo tissue ablation or genetic models is practically impossible. The same applies to other cytokines and hormones, such as insulin, triiodothyronine, and so forth. The model described in this article results in standard, reproducible, and stereotypic histology, described above. Although this histology is not the same as the typical liver histology, the reproducibility of the in vitro structures as well as the easily observable defects in cellular phenotype and histology on elimination or addition of specific components allow another opportunity to study the role of different molecules in hepatic tissue formation. The results of these studies and their implications for hepatic biology are presented below.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Fischer 344 rats from Charles River (Wilmington, MA) were used for the studies described. All animals were treated according to protocols approved by the animal care institutional review board
Materials
EGF was obtained from Collaborative Biomedical (Waltham,
MA). Collagenase for hepatocyte isolation was obtained from
Boehringer Mannheim (Mannheim, Germany). Vitrogen (Celtrix Labs., Palo
Alto, CA) was used for collagen coating of roller bottles. General
reagents were obtained from Sigma Chemical Co. (St. Louis, MO). EGF was
purchased from BD Pharmingen (San Diego, CA). HGF used for these
studies was the 
5 variant and was kindly donated by Snow Brand Co.
(Toshigi, Japan). Antibodies were obtained from the following sources:
proliferating cell nuclear antigen (PCNA) from Signet Laboratories
(Dedham, MA); Ki-67 from Santa Cruz Biologicals (Santa Cruz. CA);
desmin, cytokeratin 19, HEPPAR, and factor VIII from DAKO Corp
(Carpinteria, CA).
Immunohistochemistry
Tissues from the cultures were harvested and fixed in 10% formalin. Tissues were paraffin-embedded, sectioned at 4 to 5 µm, and affixed to charged slides (Superfrost/Plus; Fisher Scientific, Pittsburgh, PA). Immunohistochemistry was performed using the Vectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA). PCNA antibody was used at a concentration of 1:100 on sections that were microwaved in citrate buffer. Ki-67 antibody was used at a concentration of 1:200 and sections were heated under pressure in citrate buffer. Desmin antibody was used at a concentration of 1:100. Cytokeratin 19 antibody was used at 1:10 in sections microwaved in citrate buffer. HEPPAR antibody was used at a concentration of 1:25 in sections microwaved in citrate buffer. Factor VIII antibody was used at 1:400 sections that were treated with pepsin. Secondary antibodies used for this project were goat anti-rabbit, goat anti-mouse, and donkey anti-goat (Chemicon, Temecula, CA) all used at a 1:500 dilution.
Isolation and Culture of Hepatic Cell Populations
Hepatocytes
Rat hepatocytes were isolated by an adaptation of Seglen’s calcium two-step collagenase perfusion technique10 as previously described from our laboratory.4 Hepatocytes isolated from collagenase perfusion of rat liver were added at a concentration of 210,000,000 hepatocytes per 250 ml of medium. As previously described, these preparations are known to contain contaminant small numbers of other hepatic cellular elements, including stellate cells, Kupffer cells, and very few bile duct epithelial cells. The latter typically do not comprise >0.05% of the inoculated cell population.10 By hematoxylin and eosin (H&E) stain of smears of the isolated hepatocyte pellet, small cells arranged in a ductular configuration were occasionally noted. Although precise calculations were difficult to obtain given the random distribution of these clusters, their number seemed to be even less than the range for ductular cell contamination previously described.
Nonparenchymal Cell Fraction
The supernatant of the first low-gravity centrifugation used
to prepare hepatocytes was subjected to a 1000 x g
centrifugation for 3 minutes. This fraction primarily contains
stellate cells, bile duct cells, and endothelial cells. Small
hepatocytes are also present in this fraction, typically comprising
5% of the cells.
Roller Bottle Cultures
Freshly isolated hepatocytes were added to roller bottles (850 cm2 surface) obtained from Falcon (Franklin Lakes, NJ). Each bottle contained 210,000,000 freshly isolated hepatocytes in 250 ml of HGM medium1 supplemented with HGF (20 ng/ml) and EGF (10 ng/m). The bottles were rotated at a rate of 2.5 rotations per minute and kept in an incubator maintained at 37°C, saturated humidity, and 5% CO2.
Composition of the HGM Cell Culture Medium
HGM medium was prepared as previously described.1 Dulbecco’s modified Eagle’s medium powder, HEPES, glutamine, and antibiotics were purchased from Life Technologies, Inc., Grand Island, NY. ITS mixture (insulin, transferrin, selenium) was purchased from Boehringer Mannheim. All other additives were cell-culture grade (Sigma). Unless otherwise indicated for specific experiments, the basal HGM consisted of Dulbecco’s modified Eagle’s medium supplemented with purified bovine albumin (2.0 g/L), glucose (2.0 g/L), galactose (2.0 g/L), ornithine (0.1 g/L), proline (0.030 g/L), nicotinamide (0.305 g/L), ZnCl2 (0.544 mg/L), ZnSO4:7H2O (0.750 mg/L), CuSO4:5H2O (0.20 mg/L), MnSO4 (0.025 mg/L), glutamine (5.0 mmol/L), and dexamethasone (10-7 mol/L). Penicillin and streptomycin were added to the basal HGM at 100 mg/L and 100 µg/L, respectively. The mixed basal HGM was sterilized by filtration through a 0.22-µm low-protein-binding filter system, stored at 4°C, and used within 4 weeks. ITS (1.0 g/L) (rh-insulin 5.0 mg/L, human transferrin 5.0 mg/L, 30% diferric iron saturated, and selenium 5.0 µg/L) was added after filtration immediately before use. The growth factors, as required, were added to HGM fresh at the specified concentrations every time the medium was changed.
Transmission Electron Microscopy
Samples for transmission electron microscopy were washed once in phosphate-buffered saline (PBS) with 1 mmol/L MgCl2, 0.5 mmol/L CaCl2, then fixed overnight at 4°C in 2.5% glutaraldehyde in PBS. Samples were washed three times with PBS then postfixed in 1% OsO4, 1% KFe(CN)6 in PBS for 1 hour at room temperature. Samples were washed three times in PBS, then dehydrated through graded series (30 to 100%) of ethanol. After three changes of 100% ethanol, samples were infiltrated with several changes of Polybed 812 resin (Polysciences, Warrington, PA) at room temperature, with a change overnight at 4°C. Thick sections (300 µm), obtained using a Reichert (Vienna, Austria) ultramicrotome fitted with a diamond knife, were heated onto glass slides, stained with 1% Toluidine blue, and rinsed with water. Ultrathin sections (60 nm) were collected on Formvar-coated (Fullam, Schenectady, NY) grids and stained with 2% uranyl acetate in 50% methanol for 10 minutes, then 1% lead citrate for 7 minutes. Sections were analyzed and photographed on a JEOL JEM 1210 transmission electron microscope at 80 kV.
Analysis of Gene Expression by Northern Blots
Extraction of Total RNA and mRNA from Cultures
Total RNA was extracted by use of RNAzol B (BioTECX,
Houston, TX). RNA extraction from roller-bottle cultures was performed
by mixing 1 volume (pelleted) of scraped tissues with three volumes of
RNAzol. RNA was purified according to the manufacturer’s guidelines.
RNA concentration and purity were determined by routine
spectrophotometry. Size separation of 20 µg of RNA per lane was
completed on denaturing 1% agarose gels and transferring to nylon
membranes (Amersham, Piscataway, NJ) by the capillary method. After
cross-linking under ultraviolet light, membranes were hybridized
overnight with specific complementary DNA (as indicated in Figure 8
) that had been labeled with a
[32P]dCTP using an Amersham random primer kit.
Membranes were subsequently washed under high stringency conditions and
exposed to R film (photographic film) (Eastman-Kodak, Rochester, NY)
for 1 to 3 days. Quantification of the RNA hybridization bands was
performed by laser densitometry.
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Collagen probes were obtained from ATCC (Rockville, MD). Rat albumin probe was obtained from Dr. Mark Zern; transforming growth factor (TGF)-ß1 human probe from Dr. Derynck; Cytochrome P-450 IIB1 (mouse) from Dr. Negishi; collagen IV (mouse) from ATCC.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The surface of the pleated roller bottles was coated with collagen
type I before inoculation of cells, as previously
described.11
The culture medium HGM was supplemented with
HGF and EGF unless otherwise indicated for specific experiments. The
inoculated cells attach to the surface of the culture bottle within
24 hours. Approximately 50% of the hepatocytes enter into apoptosis
in the first 5 days of the culture. The apoptotic cells gradually
disappear from the mix later on as connective tissue develops. By day
18 to 20 of the cultures, the organization of the cellular elements
acquires its typical configuration. Sheets of tissue of gray-brown
coloration cover the surface of the roller bottle, being more prominent
in the grooves of the internal surface. Approximately 2 to 4 g of
tissue can be recovered from a roller bottle at 30 days in culture. The
sheets of tissue were scraped from the surface of the roller bottles,
pelleted, and processed as necessary for histological and biochemical
evaluations. The observed histology is standard and highly
reproducible. Figure 1A
is a low-power
(x20) view of the histological appearance of the many ribbons of
tissue removed by scraping from the roller bottle. A higher power view
(x200) is shown in Figure 1B
. Each ribbon is composed of the same
standard histology. On the surface facing the medium there is a
continual monolayer of cuboidal biliary epithelium. Below the biliary
layer there is a 5 to 10 cell layer composed of hepatocytes embedded in
connective tissue elements. There is a variable amount of connective
tissue separating hepatocytes from the biliary layer, from complete
absence to a thick layer separating the two cell types (as shown in
Figure 1, A and B
). Hepatocytes have a variable nuclear and nucleolar
structure, suggesting different degrees of ploidy. Attached to the
substrate and underlying the hepatocytes and connective tissue is a
layer of endothelial cells. This typical morphology is seen when the
hepatocyte cell fraction from the collagenase perfusion is placed
in culture. When the nonparenchymal cell pellet (containing endothelial
cells, stellate cells, and occasional small hepatocytes) is put in
culture under similar conditions, no growth was observed (data not
shown).
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shows a binucleate hepatocyte. Details of cytoplasmic organization
including mitochondria, rough endoplasmic reticulum, bile canaliculi,
tight junctions, and so forth, are shown in Figure 2B
. Figure 3
shows the cellular ultrastructure of
other cellular elements of the organoid cultures. The biliary
epithelium (Figure 3A)
displays typical cerebriform nuclei and
surface microvilli. A dense network of collagen fibrils underlies the
surface epithelium. Stellate-like cells with small lipid droplets are
shown embedded in the connective tissue matrix in Figure 3B
.
Endothelial cells at the basal layer also display typical subcellular
architecture for the cell type (Figure 3C)
. We did not detect presence
of fenestrated endothelium. Occasional macrophages were also seen.
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Results are shown in Figure 4
. The
superficial biliary epithelial cells were positive for cytokeratin 19,
as expected and they appear as a linear brown staining on low power
(Figure 4A)
. Desmin, typically present in myofibroblasts and stellate
cells, was seen in mesenchymal cells embedded in the connective tissue
matrix and associated with presence of collagen bundles (Figure 4B)
.
HEPPAR antibody12
as well as antibody to cytochrome P-450
IIB1 stained hepatocytes positive, with occasional biliary epithelial
cells also staining positive for the markers (Figure 4, C and E
,
correspondingly). The endothelial cells in the basal surface were
positive for factor VIII (Figure 4D)
. Canaliculi stained positive for
Mg++ ATPase13
(Figure 4F
, see
arrows).
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In the presence of HGF and EGF, most cells (>70% for each type)
stained positive for PCNA (Figure 5A)
.
This indicates that most of the cells in the cultures are in the cell
cycle. The antigen Ki-67 is typically expressed in cells actually in S
phase. Less than 5% of the hepatocytes in the cultures stained
positive for Ki-67 whereas >60% of the biliary epithelial cells were
positive (Figure 5B)
. A higher (>80%) PCNA labeling and a higher
Ki-67 labeling were noted in all systems in which dexamethasone was not
present (see below).
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The results of these studies are shown in Figure 6
(H&E stains) and Figure 7
(cytokeratin 19 stain, as a marker for
the biliary epithelium). The typical histology described above was seen
in cultures maintained in the presence of dexamethasone, HGF, and EGF
(Figures 6A and 7A)
(please note that Figures 1B and 6A
are identical,
for comparison purposes). The histology of the cultures however was
very much affected by selective elimination of these components.
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Combined removal of these two growth factors resulted in elimination of the biliary epithelium in day 20 cultures. Hepatocytes were recognizable but small and remained negative for the HEPPAR and cytochrome P-450 IIB1 antigens (data not shown). Many apoptotic hepatocytes were embedded in the histology of the cultures. No connective tissue development was noted.
Removal of Dexamethasone, Presence of HGF and EGF
There was an overall arrest in phenotypic maturation of
hepatocytes. The cells resembled oval cells seen in rat liver in
vivo. Some immature hepatocytes (<15% of the total) were
positive for HEPPAR and cytochrome P-450 IIB1. Although cytokeratin 19
strongly labeled only the surface epithelium (Figure 7B)
, there was no
clear demarcation between the surface biliary epithelium and the
underlying hepatocytes in H&E stains (Figure 6B)
. There were no
canalicular structures as demonstrable by Mg++
ATPase or electron microscopy (data not shown). Connective tissue was
present. Ki-67 labeling index was 10%.
Removal of Dexamethasone, HGF, and EGF
The surface biliary epithelium was absent (Figure 7D)
.
Hepatocytes (Figure 6D)
appeared immature, similar to those seen in
Figure 6B
. Some immature hepatocytes (<35% of the total) were
positive for HEPPAR and cytochrome P-450 IIB1. Surprisingly, several
mitoses and a high PCNA (>90%) and Ki-67 (25%) labeling index for
hepatocytes were seen in these cultures. Connective tissue was present.
The combined results suggest that dexamethasone is required for the
formation of fully mature, histologically recognizable, hepatocytes,
distinct from the biliary layer. This is more apparent by simple
histological analysis when HGF and EGF are present (compare Figure 6, A and B
). When dexamethasone alone is added, it inhibits cell
proliferation and is associated with smaller atrophic hepatocytes.
Thus, although dexamethasone is a modulator of hepatocyte
differentiation, its effects vary depending on HGF, EGF, and perhaps
other components of the medium. HGF and EGF are required for the
appearance, maintenance, or growth of the biliary epithelium. Addition
of either HGF or EGF alone restored formation of the biliary
epithelium, but not to the full extent as seen when both growth factors
were present. Connective tissue formation also depends on the presence
of HGF and EGF. The mechanisms of this are not clear. As mentioned
above, when the nonparenchymal fraction isolated from collagenase
perfusion of the rat liver was placed in culture in the absence of
hepatocytes, and with the full complement of the HGF medium plus
dexamethasone, HGF, or EGF, no growth of connective tissue elements or
any tissue formation was noted. EGF or HGF alone restored some
connective tissue formation in these cultures. EGF appeared more
efficient in restoring connective tissue formation. The histological
findings paralleled results from analysis of gene expression. Figure 8
demonstrates expression of collagen
type IV in cultures maintained in the presence of no growth factors
(control), EGF alone, HGF alone, and EGF plus HGF. The strongest
expression of collagen IV gene is seen in cultures maintained in the
presence of EGF (alone or in combination with HGF). HGF alone also
increased expression of type IV collagen above the control values at
both day 8 and day 23 in culture, but to a lesser extent than EGF. Both
growth factors however were equally efficient in inducing
expression of TGF-ß. In contrast, there were no apparent differences
related to growth factors for albumin expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The origin of the biliary epithelium in these cultures is intriguing. As mentioned above, studies from other investigators have shown that the original cell inoculum of the hepatocyte fraction isolated from liver perfusion by the two-step collagenase perfusion contains a very small number (<0.05%) of biliary epithelial cell.10 We performed cytokeratin 19 stains on smears of cell isolates directly from collagenase perfusion and the number of cytokeratin 19-positive cells was in the same range as previously described (data not shown). No biliary epithelium is seen in cultures in the absence of EGF and HGF. Each of these two growth factors added alone induced appearance of biliary epithelium to a degree that was much less when the two were combined. This does not imply that these two growth factors are the only regulating molecules for biliary epithelium development. Recent studies by Auth and colleagues14 suggest that additional factors derived from hepatocytes may also be involved. The cellular origin of the biliary epithelium in these cultures is not clear. Two possibilities exist: 1) biliary epithelium is derived from contaminating biliary epithelial cells at the time of cell isolation. Despite the fact that exceedingly small numbers of biliary epithelial cells are present in the original cell isolate, it is possible that these cells are the precursors of the biliary epithelium we see in our cultures. At the published maximum of 0.05% biliary epithelial cells present in the original cell isolation, inoculation of 250,000,000 hepatocytes may include up to 125,000 biliary epithelial cells. Given the loss of hepatocytes in the first 5 to 10 days of the cultures, the percentage of biliary cells may rise if biliary epithelial cells have a selective advantage in survival under the same conditions. 2) Biliary epithelium is derived from hepatocytes undergoing dedifferentiation and redifferentiation. Histological analysis of the cultures at early stages does not demonstrate presence of biliary epithelium. It appears in cultures between 6 to 8 days. The appearance of the biliary epithelium is paralleled by expression of cytokeratin 19. The cells on the surface of the cultures before day 6 look like immature small hepatocyte precursors and are indistinguishable from the other cells within the tissue. The emergence of the cytokeratin 19-positive surface biliary epithelium in these cultures is best explained by the redifferentiation of the immature hepatocytes that happen to be on the surface of the tissue at that time. The possibility that the biliary epithelium in these cultures may be derived from undifferentiated hepatocytes can only be established by selective in vivo tagging of hepatocytes before collagenase perfusion. We have attempted to do such tagging using adenoviral constructs containing ß-galactosidase (data not shown). We discovered however biliary cells were also tagged by the adenovirus (data not shown). Further studies with selective tagging of hepatocytes need to be performed to conclusively test this possibility.
Intriguing is the difference in proliferation rates between biliary epithelium and hepatocytes. The percentage of PCNA-stained nuclei was very high and comparable for both cell types, indicating that the same percentage of cells in both types is in the cell cycle. The percentage of nuclei positive for Ki-67 (indicator of the S phase of the cycle), however, was much larger for the biliary epithelium (60%) versus hepatocytes (5%). This suggests that hepatocytes enter into the cycle in large numbers but become arrested in G1, before entering the S phase. The mechanisms or the implications of this are not clear at this point.
The mesenchymal cells seen in the cultures (stellate cells, endothelial
cells, and so forth) are probably derived from the small percentage of
cellular contaminants present in the pellet of hepatocytes after
collagenase perfusion. The mechanisms leading to localization of the
endothelial cells into the basal portion of the structures are not
clear. Complete round spaces lined by these endothelial cells and
resembling vascular spaces were also seen. It is not clear why there
are so few of these structures and whether they formed from migration
of endothelial cells from the bottom portion of the tissue. Stellate
cells have been shown to be the cell of origin of the fibroblasts seen
in older primary cultures of hepatocytes.3
Our study also
shows that the expansion of the mesenchymal stromal cells is clearly
dependent on the presence of unknown functions provided by hepatocytes.
There was no growth seen when the nonparenchymal cell pellet isolated
from collagenase perfusion was put in culture under similar conditions
(HGM medium with HGF plus EGF in the roller bottles) but in the absence
of hepatocytes. Hepatocytes synthesize fibroblast growth factor-1,
vascular endothelial growth factor, and TGF-
during liver
regeneration after partial hepatectomy.15
Combined
addition of these growth factors in nonparenchymal cell cultures in the
absence of hepatocytes also did not result in growth of connective
tissue (data not shown). The nature of the interactive stimuli between
hepatocytes and the nonparenchymal mesenchymal elements forming the
connective tissue seen in the cultures is not clear and it seems to
involve stimuli not as yet identified. Regardless of the nature of the
factors involved the data underscore that the mature hepatocyte is a
key essential cellular element required for building of hepatic tissue
in culture. Our findings also demonstrate the important role of EGF (or
EGF receptor ligands) for the formation of hepatic histology. EGF was a
more potent inducer of the appearance of the biliary epithelium and the
connective tissue formation than HGF.
In addition to its value as a model for building tissue structures, the
system described also allows an in vitro embryology approach
to study the effects of different growth factors in liver tissue
organization. Obviously any extrapolation from in vitro to
in vivo has innate limitations. On the other hand, several
studies may be performed with this system, which are impossible to
perform in the whole animal (adult or embryo). Although homozygous
deletions of specific genes in mice remain a highly valuable tool to
explore the role of specific signals, they are often of limited
usefulness in conditions resulting in embryonic lethality (such as
deletion of HGF and its receptor5-7,16
). Gene knockout
mice are also not of much help in determining the effects of small
hormones such as corticosteroids and triiodothyronine. Specific
elimination of genes involved in the biosynthesis of these hormones (as
seen in may spontaneously occurring endocrine syndromes in humans)
usually results in accumulation of precursor metabolites upstream from
the enzymatic block, thus complicating interpretation of results.
Equally difficult is the combined elimination of groups of growth
factors (for example HGF plus EGF). The system presented in this study
can be used to provide such information. Previous studies have shown
that HGF and EGF are mitogens for biliary epithelial cells. The results
obtained in our study, however, suggest that these growth factors are
essential for the very appearance and induction of the phenotype of the
biliary epithelium. Although the role of corticosteroids is more
complex, it seems that they are essential for the full phenotypic
maturation of the hepatocytes. It should also be noted that
dexamethasone, although essential for the development of the complete
histology, had an overall negative effect on proliferation of the
epithelial cells. As mentioned above, hepatocytes seem to be essential
for the proper growth of the connective tissue stromal elements,
because no tissue growth was seen when the nonparenchymal fraction was
cultured alone. EGF seems to be a more critical factor in this process
than HGF, as shown by the induction of collagen type IV gene expression
(Figure 8)
. It is not clear whether the effect of HGF and EGF was
direct or mediated through TGF-ß1 or which cellular elements
(epithelial or mesenchymal cells) were the sources of TGF-ß1
production.
The nature of mitogenic stimuli in cultures in which dexamethasone,
HGF, and EGF were omitted is not clear. There was considerable growth
of cells resembling hepatocyte precursors (Figures 5D and 6D)
with
several mitoses easily identifiable. Although most of the cells were
not identifiable as mature hepatocytes, several groups of them were
positive for the HEPPAR antigen. Mesenchymal cells present in the
cultures may produce HGF and proliferating hepatocytes are known to
produce TGF-.17
If this is so, however, the growth
factors produced are not at sufficient levels to induce the appearance
of the biliary epithelium. Insulin and/or diferric iron-saturated
transferrin (standard additives to the HGM medium) may also play a role
in this process.18,19
More studies are required to fully analyze the implications of the results presented in this study. The model however is uniquely amenable for in vitro embryology studies, in which selective blocking agents (antisense RNA, antibodies) to specific components of matrix and growth factors may yield unique information on mechanisms and pathways important for hepatic tissue organization.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants CA30241 and CA35373 (Principal Investigator, George K. Michalopoulos) and CA76541 (Principal Investigator, Donna Beer Stolz).
Accepted for publication August 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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