Immunohistochemical
analysis
of
distribution
of
hepatic
gangliosides
following a partial hepatectomy and exposure to different hyperbaric oxygen
treatments
Tina Tičinović Kurir1, Tatijana Zemunik3promjeniti brojeve!, Ivica Grković4, Vedrana
Čikeš Čulić2, Nadan Petri5, Anita Markotić2*,
1Department
of
Pathophysiology,
Split
University
Hospital,
Split,
Croatia;
Departments of 2Biochemistry, 3Biology, and 4Anatomy, Histology, and Embryology,
Split University Medical School, Split, Croatia, 5Undersea and Hyperbaric Medicine,
Naval Medicine Institute of the Croatian Navy, Split, Croatia; 6Institute for Medical
Physics and Biophysics, University of Münster, Germany
Short title:
Immunohistochemistry of various liver gangliosides in the rat
*Corresponding
author
Anita Markotić
Split University School of Medicine
Šoltanska 2
21000 Split
CROATIA
Telephone: 385.21.557938
Telefax: 385.21.557625
E-mail: markotic@bsb.mefst.hr
1
Background/Aims: It has been shown that gangliosides play an important
functional role in the liver growth regulation. In this study we performed
immunohistochemical analysis of liver gangliosides on weanling rats following a 15%
partial hepatectomy and different pre- and post-operative hyperbaric oxygenation
treatments.
Methods: Frozen sections of liver tissues were analysed with confocal
microscopy
after
staining
with
five
antibodies,
specific
for
gangliosides
GM3(Neu5Ac), GM3(Neu5Gc), nLc4Cer, GM2(Neu5Ac), and anti-GalNAc-GM1b,
with or without permeabilization.
Results: All antibodies were capable of labelling of regenerated hepatocytes,
predominantly at the cell surface level. The strongest reactivity was observed for
GM3(Neu5Gc), equally in all groups and in all animals. Following a permeabilization
with organic solvent the anti GM3(Neu5Ac) antibody displayed different labeling
pattern in pre-operative oxygenated animals; the staining was detected in the
perinuclear area of hepatocytes. The same group showed the highest expression of
nLc4 glycoantigen. GM2 and GalNAc-GM1b gangliosides were clearly detected in
permeabilized hepatocytes of pre-operative oxygenated animals while same
glycoantigens were weakly labeled in all other groups.
Conclusion: Alternation in the expression of gangliosides following a partial
hepatectomy suggests that gangliosides play an important in liver growth regulation
and may be utilised as markers for the liver recovery after partial hepatectomy.
Key words: partial hepatectomy, hyperbaric oxygenation, liver regeneration,
gangliosides, immunostaining
2
1. Introduction
Gangliosides,
sialic
acid-containing
glycosphingolipids
(GSLs),
mainly
associated with plasma membranes, are of interest because of their suggested
involvement in cell surface- regulated phenomena (1). GM3, the most prominent and
widely distributed ganglioside in mammalian cells, has been shown to exert a variety
of biological activates, being primarily involved in cell adhesion and signal
transduction events (2). Recent data point to an important role of GM3 in cell growth
regulation (3,4), T cell activation (5), signal transduction (6) and insulin signaling (7).
The ganglioside concentration and composition bound to plasma membranes
become altered under various physiological (8-10) and pathological conditions
(11,12).
Liver regeneration is a highly controlled process of proliferation of both
parenchymal and nonparenchymal cells (17). It occurs as a response to variety of
liver tissue damages, including partial hepatectomy (PH). PH causes mitochondrial
oxidative stress (12) and the remnant liver tissue demands an increased amount of
oxygen for mitochondrial oxidative phosphorylation (13). During rat liver regeneration
following a partial hepatectomy removing about 70% of the liver tissue, the
ganglioside content and distribution has been reported to undergo significant
changes (15,16). Resections of less than 40% of tissue in adult animals elicit little
DNA synthesis, suggesting that the functional deficit produced by removal of a
relatively small amount of tissue is not sufficient to trigger significant regeneration
(17). In contrast to adult rats, this threshold is not apparent in weanling animals, in
which the proportionality between the amount of tissue removed and the level of DNA
synthesis is maintained even in small resections (17).
3
Hyperbaric
oxygenation
(HBO)
protects
hepatocytes
against
carbon
tetrachloride–induced injury (18) and improves regeneration of the remnant liver
tissue following the portal vein embolization (19).
Recently, we described beneficial effects of HBO treatment in experiments
applying different protocols of HBO pretreatment on animals receiving a minimal
(15%) hepatectomy (20). Using the same animal experimental model, we also
performed
detailed
structural
characterization
of
major
GM3(Neu5Ac)
and
GM3(Neu5Gc) as well as of less abundant neolacto-series gangliosides of rat liver 54
h after PH. This was achived by using nano electrospray ionization quadropole timeof-flight (ESI-QTOF) mass spectrometry in combination with high-performance thinlayer chromatography (HPTLC) immunostaining (22). Minor GM2, GM1- and GM1btype gangliosides were identified by immunostaining using a panel of GSL-specific
antibodies (21).
In view of the above, the aim of the present study was to use
immunohistochemical methods to detect changes of ganglioside expression on
hepatocytes of animals expossed to different oxygen treatments (pre- and postoperative hyperbaric oxygen pressure) following a partial hepatectomy.
2. Materials and methods
2.1. Antibodies
The polyclonal chicken anti-GM3(Neu5Ac), anti-GM3(Neu5Gc), and antinLc4Cer antibodies have been characterized in previous papers (22,23). The antinLc4Cer antibody recognizes the Galβ1-4GlcNAc-residue and thus reacts with
4
nLc6Cer and nLc8Cer as well. The polyclonal chicken anti-GM2(Neu5Ac) antibody
was produced according to the method of Kasai et al. (24).
The specificity of the chicken polyclonal anti-GalNAc-GM1b antibody has been reported
earlier (25).
2.2. Rats
Experiments were performed on one-month-old male Wistar rats weighing
between 70 and 100 g. All rats were raised under controlled conditions (temperature,
221oC; light schedule: 14 hours of light and 10 hours of dark) at the Split University
Medical School Animal Facility. Laboratory food and tap water were supplied ad
libitum. Animals were bred and maintained according to the Guide for Care and Use
of Laboratory Animals (Institute of Laboratory Animals Resources, Commission on
Life Sciences, National Research Council, 1996) and the experimental protocol has
been approved by the Split University Medical School Ethics Committee.
2.3. Partial hepatectomy
Three unoperated animals which were not exposed to the hyperbaric
oxygenation (HBO) treatment served as the control group. Nine rats underwent the
left middle lobectomy, which resulted with a removal of around 15 % of the liver
tissue. This quick operative procedure was performed under diethylether anesthesia
and caused a minimal distress to animals. Operated animals were divided into three
experimental groups (each containing 3 animals). First group recovered under the
normal ambient conditions of oxygen pressure following the operation (defined as
nonHBO group). The second group included 3 animals pretreated with hyperbaric
oxygen before the operation, after which they recovered under the normal ambient
5
conditions (preHBO group). The third group included animals which received the
hyperbaric oxygen after the operation (postHBO group). The preHBO group was
treated at day 2 (48 h), day 1 (26 h), and day 0 (4 h) before PH with HBO (100% O 2,
45 min/day at 2 atm). The postHBO group was treated at day 0 (4 h), day 1 (26 h),
and day 2 (48 h) after PH with HBO (100% O2, 45 min/day at 2 atm). The pressure of
2 atm (=202650 Pa) was selected as the HBO regimen because this pressure is
within the safety range of HBO protocols in humans (19). The HBO-exposure took
place in a Comex hyperbaric chamber (Comex, Marseilles, France). The oxygen and
carbon dioxide concentrations in the chamber during HBO-exposure were controlled
by a Servomex Oxygen Analyzer 570A (Servomex, Houston, TX, USA) and by a
Carbon dioxide Gas Analyzer Infrared (Industries Inc., Santa Barbara, CA, USA). All
animals were sacrificed exactly 54h following the operation.
2.4. Sampling
For the tissue collection all animals were re-anaesthetised as described above
and perfused transcardiacally with heparinised saline (1000 U/L) followed by 300 ml
of Zamboni’s fixative (2% paraformaldehyde and 15% picric acid in 0.01 M
phosphate buffered saline (PBS) at pH 7.4. Livers of all animals were removed and
postfixed in the same fixative for 2-5 hours at 4, then transferred to 0.01M PBS.
Following an overnight cryoprotection in 20% sucrose, livers were sectioned using a
cryotome (Shandon AS620SME). Liver sections (8 µm thick) from each animal were
stained with four different polyclonal chicken primary antibodies [GM3(Neu5Ac),
GM3(Neu5Gc), nLc4Cer, GM2(Neu5Ac), and GalNAc-GM1], diluted with 0.1 M PBS
supplemented with 0,5% BSA and 0.02% NaN3 for 24 hours. Nonspecific antibody
6
binding was blocked by pre-incubating the sections with 1% bovine serum albumin
(BSA) in 0.1 M PBS, pH 7.4, for 1 hour.
Primary antisera were visualised with a species-specific secondary antibody, affinitypurified
donkey
anti-chicken
IgY
(IgG)(cat.
no.
703-095-155,
Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania, USA) coupled with
Texas-Red diluted 1:150 with PBS.
Tissues were examined on a fluorescent
microscope (Olympus BX 51) fitted a confocal device (UltraView by PerkinElmer,
England) and filters that allowed visualization of Texas Red. A Spot Insight QE
cooled, charged-coupled device camera (Vision Systems, GmbH) and Spot
Advanced for Windows imaging software (Vision Systems, GmbH) were used to
record images.
To enable antibody to enter within the cell a separate group of section were
pretreated with methanol and then with chloroform/methanol (1:1, v/v), each for 10
min, before the immunostaining (26). After organic solvent treatment, sections were
air-dried and stained according to the same protocol as described for non-extracted
sections.
A number of control experiments were performed where the primary antibody was
omitted from the diluent mixture, and secondary antibodies were applied as normal.
This resulted in no staining of neither the cell membranes nor nuclei.
2.6. Data analysis
Antibody staining was evaluated under a confocal microscope and staining was
graded as – for no staining, + for a weak positive staining which included a part of the
membrane, ++ for a moderate staining detected on more than half of the membrane
7
perimeter , +++ for strong staining which included the whole hepatocyte membrane,
and ++++ for very intense staining over the entire membrane perimeter.
3. Results
This study presents a comparison of distribution patterns for various gangliosides
and related glycoconjugates in rat livers on a ‘partial hepatectomy model’ combined
with
oxygen
pressure
treatments
Immunohistochemical detection
of
both
five
pre-
antibodies
and
post-operatively.
specific for
gangliosides
GM3(Neu5Ac), GM3(Neu5Gc), nLc4Cer, GM2(Neu5Ac), and GalNAc-GM1b, with (A)
or without organic solvent pretreatment (B) were compared and representative areas
of immunoreactivity are presented on five plates.
When detectabele, the strongest immunolabeling was always confined to the surface
of hepatocites and displayed typical membrane-bound reactivity, which can clearly be
seen on large magnification images (all figures). Using criteria described above, the
comparative semiquantitative ‘density score’ was produced for every section
analysed by means of screening the whole section (see table 2) and a representative
area was captured using constant confocal microscopy settings (figures 1-5).
Of
all
five
antibodies
analyzed,
the
GM3(Neu5Gc)
displayed
strongest
immunoreactivity (four +) regardless of the experimental protocol (figure 2). In
addition, methanol and chloroform/methanol pretreatment did not appear to have any
effect on the intensity of the staining.
8
Contrary to the above finding, both Anti-GM2(Neu5Ac) and Anti-nLc4Cer antibody
displayed weak to moderate immunoreactivity with slightly increased signal following
the methanol and chloroform/methanol pretreatment. The It is also worth noticing that
the immunoreactivity was stronger in preHBO then in postHBO groups for these two
antibodies.
Anti GM3(Neu5Ac) antibody displayed strong reactivity in the preHBO and nonHBO
animals with weak binding detected within the perinuclear space following
permeabilization with organic solvents in all livers except for the preHBO liver where
the staining was of the same intensity regardless of the organic solvent pretreatment
(Fig.1B).
Finally, the Anti-GalNAc-GM1b weakly labeled the HBO liver tissue and more intense
the pre HBO liver (Fig.5A). Strong reactivity was seen in the hepatocytes of all
groups after lipid extraction (Fig.5B).
4. Discussion
Significant changes in the ganglioside content and distribution during rat liver
regeneration after PH have been reported (15,16). Recently, we have described the
altered expression of predominant GM3 and less abundant neolacto-series, GM1and GM1b-type gangliosides in the liver of rats, under various experimental models
related to the oxygen pressure before or after PH (21). The enhanced expression of
the major gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) in preHBO correlated with
the significant increase in the starting process of the liver mass restitution
(determined as liver weight/body weight ratio) in the preHBO treated rats (20).
Animals in this group did not display signs of liver dysfunction and this was evaluated
by alanine transaminase and aspartate transaminase activities (Table 1). Liver lipid
9
peroxides concentration was the lowest in the preHBO group and the light
microscopy findings revealed that the lobules composition of preHBO group tissues
was similar if not the same as in control tissues 20.
The aim of this study was to use the immunohistochemical approach to detect
patterns of expression of hepatic gangliosides from the weanling rats that underwent
the same oxygen treatment protocols following PH, as we described earlier (20,21).
Observations
of
permeabilized cells
revealed
an
intracellular
staining
of
GM3(Neu5Ac) molecules in preHBO treated rats. Charlene et al. postulated that
newly synthesized GM3, in both resting and activated cells, can be associated with
the Golgi apparatus (27). Synthesis of the simplest ganglioside GM3 is the first
committed step in the formation of gangliosides. Analysis of the sub-Golgi distribution
of GM3 synthase activity by biochemical fractionation has indicated that the enzyme
is enriched in the cis-compartment (28,29,30). Reported differences in sub-Golgi
localization (31) of GM3 synthese activity may reflect Golgi organization that is
specific to cell-type or cell maturity and may also be complicated by methodological
consideration. Confocal immunohistochemical localization, used in this study,
provided an alternate approach that allows assessment of glycoantigen distribution,
located in its normal tissue environment. Intense anti-GM3(Neu5Ac) staining around
hepatocyte nuclei in preHBO treated rats corresponds to recent observations by
Giraudo et al (33). Using multiple color fluorescence imaging techniques in CHO-K1
cells they found
GM3 synthase located in early compartments of the secretory
pathway, including ER membranes. The outer nuclear membrane is continuous with
the endoplasmic reticulum (34). Although glycosphingolipid synthesis is generally
regarded as confined to the Golgi apparatus and ER, the fact that cytidine 5'monophosphate N-acetylneuraminic acid synthetase has been purified from rat liver
10
nuclei (35) suggests that the nucleus may have some capacity of this kind as well.
Keeping in mind that GM3 precursors ceramide and sphingosine accumulated in
nuclei may induce the apoptosis of hepatocytes in vivo, enhanced GM3 expression
around the nuclei could have important implications. Namely, if GM3 molecules are
more intensively expressed, their precursors ceramide and sphingosine would be
present in a smaller amount. Therefore, the apoptosis during liver regeneration would
be less intense, resulting in more rapid liver regeneration (36). Furthermore,
sphingomyelin shares ceramide and sphingosine precursor with GM3 ganglioside.
Increased amount of GM3 could mean deceased amount of sphingomyelin.
Degradation of sphingomyelin induces a decrease of cholesterol in nuclear
membrane of rat hepatocytes after partial hepatectomy (35). Changes in cholesterol
content modify the nuclear membranes fluidity and, as consequence, mRNA
transport and cell function (35).
Strong reactivity was observed for GM3(Neu5Gc), without the difference in
intesity between the groups examined, but with difference in GM3(Neu5Gc)
distribution in liver tissue. Confocal observations of permeabilized cells of preHBO
group revealed GM3(Neu5Gc) staining that form a ring, roughly concentric with the
central vein (Figure 2B, panel preHBO). GM3 is known as a marker of
glycosphingolipid enriched microdomains or lipid rafts (5). T-cell activation leads to
the redistribution and clustering of membrane as well as of intracellular kinase-rich
raft microdomains with GM3 being a component of a multimolecular signaling (5).
Recently, sphingolipid-enriched microdomains were described in the hepatocyte
membrane (37,38). Therefore, thick layers stained with anti-GM3(Neu5Gc) in
permeabilized hepatocytes of preHBO rats could represent redistributed and
clustered lipid rafts. In contrast to anti-GM3(Neu5Ac), anti-GM3(Neu5Gc) staining
11
did not label the area around hepatocyte nuclei. Some reports suggested that NeuGc
was produced from NeuAc through enzymatic hydroxylation of the N-acetyl residue
of free NeuAc, CMP-NeuAc, or glycoconjugate-linked NeuAc (39,40). Kawano et. al
have purified CMP-NeuAc hydroxylase from the cytosolic fraction of mouse liver and
demonstrated that the enzyme is highly specific to CMP-NeuAc and does not use
free NeuAc or NeuAc-containing GM3 as a substrate (41). We could speculate that
GM3(Neu5Gc) was absent from the area around hepatocyte nuclei, due to
potentional shortage of availability of CMP-NeuAc hydroxylase around nuclei.
Significant enhance in the gangiosode nLc4Cer expression was found in pre
HBO liver rat after the lipid extraction compared to other groups. A number of studies
using mouse embryo cells, brain cells and lymphocytes have provided evidence that
cell surface glycoconjugates containing neolacto-series carbohydrate chains are
involved in intercellular adhesion as well as cell-matrix interactions (42,43,44).
Sulfoglucuronylneolactoglycolipids,
which
are
specifically
expressed
in
the
mammalian nervous system, are known to bind to laminin and mediate cell-matrix
interactions (45). Also, the levels of three neolacto-GSLs, nLc4, nLc6 and nLc8 are
elevated during corneal epithelial cell migration and wound healing (46). The most
intense staining with anti-nLc4Cer, which was found in preHBO group, after
permeabilization, could indicate that preHBO treatment stimulated hepatocytes
migration following injury.
Extracellular signals that mediate regenerative process of the liver tissue
following PH act initially at the plasma membrane level, where binding of receptors to
endocrine and paracrine agents and to elements of the extracellular matrix, transport
of nutritiens, and interactions with neighboring cells occur. Since liver regeneration
appears to involve a large number of hormone-receptor systems and cellular
12
changes (i.e., oncogene expression, TGF- expression), modulation of the
ganglioside content of hepatocyte plasma membrane could provide means for
upregulating cellular responsiveness to a variety of factors.
Pre-operative HBO treatment led to enhanced expression of gangliosides.
Thus, the pre-operative HBO treatment is the procedure of choice for managing
some clinical settings where ischemic-reperfusion injury and energy depletion occur,
such as liver transplantation and hepatic resectional surgery. Possible functional
roles of gangliosides in the regulation of liver growth and reconstitution after PH open
the ability to influence liver regeneration on the level of the ganglioside metabolism.
Acknowledgements
Data shown resulted in the framework of the project no. 0216013 (A. Markotić)
sponsored by the Ministry of Science, Education and Sports, Republic of Croatia.
ZAHVALA JOHANNESU!
Abbreviations
Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; GSLs,
glycosphingolipids; HBO, hyperbaric oxigenation; PH, partial hepatectomy; ESIQTOF, electrospray ionisation quadropole time-of-flight; HPTLC, high-performance
thin-layer chromatography; FTC, fluoresceinisothiocyanat; PBS, phosphate buffered
saline; BSA, bovine serum albumin, TGF, tumour growth factor . The designation
of the GSLs follows the IUPAC-IUB recommendations (47) and the nomenclature of
Svennerholm
(48).
Lactosylceramide
13
or
LacCer,
Galß1-4Glcß1-1Cer;
gangliotriaosylceramide
gangliotetraosylceramide
or
or
Gg3Cer,
Gg4Cer,
GalNAcß1-4Galß1-4Glcß1-1Cer;
Galß1-3GalNAcß1-4Galß1-4Glcß1-1Cer;
neolactotetraosylceramide or nLc4Cer or nLc4, Galß1-4GlcNAcß1-3Galß1-4Glcß11Cer; neolactohexaosylceramide or nLc6Cer or nLc6, Galß1-4GlcNAcß1-3Galß14GlcNAcß1-3Galß1-4Glcß1-1Cer;
GM3,
II3Neu5Ac-LacCer;
GM2,
II3Neu5Ac-
Gg3Cer; GM1, II3Neu5Ac-Gg4Cer; GM1b, IV3Neu5Ac-Gg4Cer; GalNAc-GM1b,
IV3Neu5Ac-Gg5Cer;
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20
Table 1. Total wet weights and liver regeneration-related factors of rat livers from nonoperated and partially hepatectomized rats after different oxygen treatments.
Variable / No.a
1. controld 2. nonHBOe 3. preHBOf
Total wet weightb (g)
44.43
Liver wet weight/body weightc 0.0697 ±
(g/g)
0.0013
4. postHBOg
38.54
41.05
39.67
0.0517 ±
0.0016
0.0618 ±
0.0084
0.056 ±
0.0014
Lipid peroxides (nM/g MDA)h 110.1±4.3 318.1 ± 24.6 119.7 ± 4.9
288.3 ± 12.4
Albumin plasma level (g/l)h
40.4±0.3
33.5 ± 0.7
20.0 ± 2.4
25.3 ± 0.4
ALT activity (U/l)h
23.3 ± 4.2 53.3 ± 4.4
30.3 ± 5.1
67.8 ± 9.6
AST activity (U/l)h
43.3 ± 5.4 137.5 ± 6.3
101.1 ±12.1 218.4 ± 40.6
a
numbering of ganglioside fractions from differently treated rats
b
total wet weights of livers obtained from 10 male rats
c
ratio of the wet weight of the remnant liver lobes to the body weight calculated from
excised livers after partial hepatectomy and 54 hours recovery under different oxygen
conditions; p<0.05 vs control assessed by Kruskal-Wallis test followed by Dunn’s test
d
non-operated and non-oxygenated rats
e
partially hepatectomized and recovered under normal ambient conditions
f
treated with oxygen before operation and recovered under normal ambient conditions
g
treated with oxygen after operation and recovered under hyperbaric oxygen conditions
haccording
to Kurir et al. (2004); MDA: malonyldialdehyde, ALT: alanine transaminase,
AST: aspartate transaminase
21
Table 2. Immunohistochemical analysis of rat hepatic gangliosides.
Control
nonHBO
Antibody
preHBO
postHBO
Chloroform/methanol extraction
No
Yes
No
Yes
No
Yes
No
Yes
Anti-GM3(Neu5Ac)
-
+
+++
+
+++
+++
++
+
Anti-GM3(Neu5Gc)
++++
++++
++++
++++
++++
++++
++++
++++
Anti-nLc4Cer
++
++
+
++
+
+++
+
+
Anti-GM2(Neu5Ac)
+
+
-
+
+
+
-
+
Anti-GalNAc-GM1b
-
+++
+
+++
++
+++
-
+++
-, no staining; +, weak positiv staining; ++, moderate intesitivity staining; +++, strong staining; ++++,
very strong staining
22
Figure 1
Immunofluorescence analysis of liver ganglioside GM3(Neu5Ac) without (A) and with
pretreatment with organic solvents (B) from different treated rats after partial
hepatectomy. Anti GM3 (Neu5Ac) antibody strongly stained the pre HBO and non
HBO liver (A), with weak binding was presented at the intracellular space after lipid
extraction in all livers except for the pre HBO liver where the staining was more
intense (B).
Figure 2
Immunofluorescence analysis of liver ganglioside GM3(Neu5Gc) before (A) and after
pretreatment with organic solvents (B) from different treated rats after partial
hepatectomy. In all livers analysed very strong binding of GM3 (Neu5Gc) was
localized at the cell surface (A), the most prominent staining around blood vassels.
Lipid extraction did not affect staining in all livers (B).
Figure 3
Immunofluorescence analysis of liver ganglioside nLc4Cer before (A) and after
pretreatment with organic solvents (B) from different treated rats after partial
hepatectomy. Anti nLc4Cer antibody weakly stained the hepatocytes in all groups
(A). The same positivity was visibled after pretreatment with organic solvents, most
intesity was present in pre HBO liver (B).
Figure 4
Immunofluorescence analysis of liver ganglioside GM2 before (A) and after
pretreatment with organic solvents (B) from different treated rats after partial
hepatectomy. Weak staining with anti GM2 antibody was present only in the control
23
at pre HBO liver (A), after chloroform/methanol pretreatment weak labeling was
found in all livers.
Figure 5
Immunofluorescence analysis of liver ganglioside GalNAc-GM1b before (A) and after
pretreatment with organic solvents (B) from different treated rats after partial
hepatectomy. Anti- GalNAc-GM1b weakly labeled in the nonHBO and more intesive
in the pre HBO liver. Strong reactivity was seen in the hepatocytes of all groups after
lipid extraction(B).
24