Camila Scorticati, Juan P.
Prestifilippo, Francisco X. Eizayaga, Salvador Romay,
María A Fernández, Abraham Lemberg, Juan C. Perazzo, Cátedra de
Facultad de Farmaciay Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina
Jos L. Castro, Cátedra de Farmacología,
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina
Supported by Grant # TB 56 from the University of Buenos
Correspondence to: Professor. Dr.
J. C. Perazzo, Cátedra de
Junín 956, 5° Piso, (1113), Ciudad
Autónoma de Buenos Aires,
Fax: +54-11-4964 8268
Received: 2003-09-23 Accepted:
AIM: To study the blood-brain
barrier integrity, brain edema, animal behavior and ammonia plasma levels
in prehepatic portal hypertensive rats with and without acute liver
METHODS: Adults male Wistar rats were
divided into four groups. Group I: sham operation; II: Prehepatic portal
hypertension, produced by partial portal vein ligation; III: Acetaminophen
intoxication and IV: Prehepatic portal hypertension plus acetaminophen.
Acetaminophen was administered to produce acute hepatic injury. Portal
pressure, liver serum enzymes and ammonia plasma levels were determined.
Brain cortex water content was registered and trypan blue was utilized to
study blood brain barrier integrity. Reflexes and behavioral tests were
RESULTS: Portal hypertension was
significantly elevated in groups II and IV. Liver enzymes and ammonia
plasma levels were increased in groups II, III and IV. Prehepatic portal
hypertension (group II), acetaminophen intoxication (group III) and both
(group IV) had changes in the blood brain-barrier integrity (trypan blue)
and hyperammonemia. Cortical edema was present in rats with acute hepatic
injury in groups III and IV. Behavioral test (rota rod) was altered in
CONCLUSION: These results suggest the
possibility of another pathway for cortical edema production because blood
brain barrier was altered (vasogenic) and hyperammonemia was registered
(cytotoxic). Group IV, with behavioral altered test, can be considered as
a model for study at an early stage of portal-systemic
Scorticati C, Prestifilippo JP,
Eizayaga FX, Castro JL, Romay S,
Fernández MA, Lemberg A, Perazzo JC. Hyperammonemia, brain edema
and blood-brain barrier alterations in prehepatic portal hypertensive rats
and paracetamol intoxication. World J Gastroenterol
2004; 10(9): 1321-1324
hypertension (PH) is a major syndrome that frequently accompanies chronic
liver diseases such as cirrhosis. Prehepatic PH creates a splanchnic
hyperdynamic circulation and hyperemia with increased splanchnic
resistance and production of collateral vessels that drive splanchnic
blood flow to systemic circulation.
Portal-systemic encephalopathy (PSE), the most commonly encountered
form of hepatic encephalopathy (HE), describes a wide spectrum of
reversible and irreversible neuropsychiatric abnormalities that appear as
a complication in patients with acute or chronic liver disease. HE is
usually found as an overt form or in a mild form with lesser
neuropsychiatric symptoms, which can be misdiagnosed.
Acetaminophen (APAP) is a non-steroid anti-inflammatory drug
(NSAID), frequently used in adults and children. At high concentration of
APAP, the gluthation-dependent conjugation system generates a toxic
metabolite that binds covalently to cellular proteins and macromolecules
followed by cell destruction[3,4].
Finally, ammonia originated from the gut protein metabolism has
been implicated as an important factor in the production of HE. In chronic
liver disease, ammonia evades liver catabolism to urea through portal
systemic shunts and collaterals, and reaches the brain in high blood
previous experiments we documented different aspects of the relationship
between prehepatic PH in rats and central nervous system: alterations in
uptake and release of norepinephrine and modification and tyrosine
hydroxylase activity in discrete regional mesencephalic
nucleus[6,7]. Furthermore, we considered that PH rats underwent
a subclinical HE.
The aim of this
experiment was to study the blood-brain barrier (BBB) integrity, brain
cortical edema, ammonia plasma levels and behavior in rats with different
MATERIALS AND METHODS
Male Wistar rats (240-260 g of
body mass, 12 h of light cycle: 8 a.m.-8 p.m.) were used and animal
welfare was in accordance with guidelines of the Faculty of Pharmacy and
Biochemistry of Buenos Aires and approved by ethical committee according
to Helsinki’s Declaration. Animals were placed in individual cages and
allowed to recover from surgery. Rats were fed standard laboratory chow
and water ad libitum.
Group II (chronic PH): Portal hypertensive (PH) animals were
obtained by calibrated stenosis of the portal vein according to Chojkier
et al.. Rats were lightly anesthetized with ether and
then midline abdominal incision was made. The portal vein was located and
isolated from surrounding tissues. A ligature of 3.0 silk suture was
placed around the vein, and snuggly tied to a 20-gauge blunt-end needle
placed along the side of the portal vein. The needle was subsequently
removed to yield a calibrated stenosis of portal vein, after which the
abdomen was sutured. Operations were performed at 2 p.m. to obey circadian
rhythm and fourteen days later the animals developed PH.
Group III (acute intoxication): Acetaminophen was injected i.p. at
a dose of 750 mg/kg.d per rat on the 13th and 14th
day, considering as the start
day or day zero, the day of
the surgical procedure of group II.
(chronic PH plus acute intoxication): Rats underwent partial portal vein
ligation as in group II and then received acetaminophen the same dose as
in group III.
Group I: Shamly
operated rats underwent the same experimental procedure like group II,
except that portal vein was isolated but not stenosed.
All groups received a subcutaneous injection (25 mL/Kg of body
mass) of a 50 g/L dextrose, 4.5 g/L saline solution, containing 20 mEq/L
of potassium chloride, every 12 h after the first injection of APAP, as a
supportive therapy to prevent hypoglycemia and renal
failure. Rats were sacrificed on d 14, 6 to 8 h after the
last acetaminophen injection.
experiment were performed in four groups of animals (I: SHAM, II: HP, III:
APAP and IV: HP+APAP, n=6/8 per group).
Rats were anesthetized with
sodium pentobarbital (40 mg/kg, ip). The spleen was cannulated with a
polyethylene cannula (PE 50) filled with heparinized saline solution (25
U/mL) to measure portal pressure by Statham Gould P23ID pressure
transducer (Statham, Hato Rey, Puerto Rico) coupled to a Grass 79 D
polygraph (Grass Instruments, Quincy, MA).
samples were obtained by abdominal aortic artery puncture for the
determination of biochemical parameters. Plasma aminotransferase activity
was determined using standardized and optimized commercial
Boheringer-Manenhein kits (Germany). Ammoniac Enzyimatique UV kits
(Biomerieux-France) were used to determine plasma ammonia concentration.
Plasma levels of urea, glucose and creatinine were measured by current
biochemical colorimetric UV methods (Wiener, Argentine).
optical microscopy (HROM): sections of liver were fixed in buffered
formalin and embedded in paraplast. Routine stains were used:
hematoxylin-eosin, PAS and Masson’s trichrome for light microscopy and
toluidine blue stain for HROM.
Rats were sacrificed by exsanguination
under complete ether anesthesia, their brains cortex water content was
measured to quantify possible brain edema according to
One cerebral hemisphere was quickly removed after rats were
sacrificed and stored at 4 °C to be processed within 30 min. The hemisphere was cut
into coronal slices and eight samples taken from the cerebral cortex,
approximately 10 mg in weight, was placed in a bromobenzene-kerosene
density gradient column to measure brain water. The column
was previously calibrated with varying concentrations of potassium sulfate
to ensure a linear relation (>0.997) between the K2 SO4 and readings in
the density gradient. The equilibration point of the samples was read at 2
min and averaged, conversion from specific gravity to brain water was done
as previously reported.
Trypan blue transcardial perfusion
Rats were perfused with trypan blue (TB) solution and
then fixed in paraformaldehyde.
TB solution (5 g/L) was made by dissolving 1 g of TB in 200 mL of
PBS with gentle heat. The solution was allowed to cool room temperature
and then added to the filtrate, then the solution was placed on ice and
used immediately. The temperature of TB solution was 10-12 °C at the time of perfusion. Rats were anesthetized with
ethyl urethane (1 mg/kg) and perfused transcardially with 200 mL of TB
solution, followed by 300 mL
of ice-cold paraformaldehyde (20 g/L in PBS), the low rate of perfusate
was maintained at 25 mL/min. Brains were dissected and post-fixed
overnight in 0.3 Kg/L sucrose for 2 d. Subsequently, brains were frozen
in powdered dry ice and stored at -80 °C until processed for microscopic studies. Slices of
brain hippocampus were obtained with cryostat in sections of 300 microns
according to Watson and Paxinos (Hippocampus fig 24, bregma 4.8,
interneuron 4.2). Hippocampal slices were evaluated under light microscopy
and the results were expressed as positive (+) or negative (-) for TB
staining. Medial eminence and choroids plexus staining were used as
control of TB appropriated perfusion.
Pharmacological and behavioral
Corneal, pain-response and right reflexes were
performed. Automated open field (Animex) and rota-rod tests (to study
motor coordination) were realized. Rota-rod speed was fixed at 25
turns/min. Rats were trained 48 h before the experiment until they fell
down less than 3 times in 5 min. The number of falls during the experiment
and time elapsed to the first fall were recorded[12,13].
were expressed as mean±SE. Statistical analysis was performed by means of
repeated measurement analysis of variance (ANOVA) followed by
Newman-Keuls‘s test. Dunn’s test was used for non-parametric data, P
values less than 0.05 were considered statistically significant.
pressure (PP) was significant higher in groups II and IV
(aP<0.01) when compared to control group. There was
no significant difference when compared PP of group I (Sham) with group
III (APAP) (Table 1).
Portal pressure (PP) and plasma aminotranferase
PP was significantly increased in
groups II and IV (aP<0.05 vs control and APAP group).
AST plasma levels were significantly increased in groups II, III and IV
(bP<0.001 vs sham group). ALT was significantly increased in
groups III and IV (aP<0.05 and
bP<0.001 vs sham group, respectively).
levels were significant increased in groups II, III and IV when compared
with group I (bP<0.001). ALT plasma levels were significant
increased in groups III and IV when compared with control group (Table 1).
levels of urea, glucose and creatinine were normal in all groups of rats
(data not shown).
blood concentrations (mmol/L) showed significant increase in PH rats, APAP
group and PH plus APAP (P<0.05 and P<0.001
respectively) (Figure 1).
microscopy and HROM showed normal histology in liver parenchyma in group
I, minimal focal necrosis in group II; diffuse hemorrhagic necrosis in
group III and focal hemorrhage and confluent necrosis in group IV. (Figure
cortical water content (H2O/ g brain weight %) showed
significant increase in rats injected with APAP (P<0.001) and in
those with PH+APAP (P<0.01) (Figure 3).
II, III and IV trypan blue was positive in the perivascular space in the
hippocampus area (Figure 4).
reflexes studied including the Animex were normal for all groups except
for rota-rod test that showed in group IV a significant difference at the
time elapsed to the first fall (not in number of falls), when compared to
shamly operated animals (Table 2).
Figure 1(PDF) Significantly increased ammonia
plasma levels in all groups of rats aP<0.05 and
bP<0.01 when compared with sham group.
Figure 2 Liver Microscopy. A: Shows a normal liver
histology corresponding to a rat in sham group (HE, 100×), B: Minimal focal necrosis in
group II (HE, asterisk, 500×), C: Diffuse hemorrhagic
necrosis in group III (HE, arrows, 400×), D: Focal hemorrhagic
confluent necrosis in group IV (HE, asterisk, 400×).
increased brain water content in groups III and IV
dP<0.01, bP<0.001 when compared
with sham group.
Figure 4 Trypan blue and BBB. A: Trypan blue dye in
hippocampus of a sham rat. No breakdown of BBB was observed. Trypan blue was
only positive in vascular space (100×), B: Trypan blue dye diffusion
due to the breakdown of BBB as an example of what was observed in
hippocampus in groups II, III and IV (100×).
Table 2 Rota rod test: First fall
1 : expressed in seconds. Rota rod
test was significantly different in group IV compared with all the others
(aP<0.05 Dunnett’s test).
Experimental prehepatic portal hypertension produces a
hyperdynamic redistribution of splanchnic circulation and minimal liver
damage. It could be considered that prehepatic PH is a subclinic PSE
stage. In this study APAP was used to produce liver injury
because of its aggressive effect on liver parenchyma[3,4]. It
was administrated to two groups that reflected different situations,
namely an acute form of liver injury (group III) and a chronic liver PH
group plus an acute liver intoxication.
pressure (PP) was significantly increased in group II, as it was described
elsewhere and in group IV. PP in group III (APAP) had normal
values. This agreed with acute intoxications and specifically with acute
APAP intoxication. Group IV and group II had similar PP
values, and it might be due to a chronic PH induced shunt (groups II and
reflected hepatocyte damage. When liver parenchyma was injured, the
enzymes increased as it could be seen in early stages of PSE. Plasma
enzyme levels were higher in group III than those in APAP induced acute
liver damage. In addition, liver histopathology was concordant with these
data as a diffuse hemorrhagic necrosis was observed in group
plasma level data showed normal values in group I and significantly
increased values in groups II, III and IV. The values registered were
remarkably lower than those observed in different models of acute liver
failure (ALF). It is important to point out that in group II
hyperammonemia was produced by opened shunts due to the splanchnic blood
flow redistribution. It is also interesting that in this
experimental model the hepatic blood flow was reduced, but not completely
shunted as in other models (e.g. porto-caval shunts, PCS). Ammonia was
markedly increased as the major responsible molecule related to
HE had several pathways, lately the cGMP-NO-glutamate that
leads to cytotoxic brain edema characterized by swelling of astrocytes and
their processes, raised intracranial pressure and as a consequence, to
brain herniation. This was the main cause of mortality in
ALF and it could be predicted by increased arterial ammonia plasma
levels. Besides this, PSE could lead to increased nNOS
activity, associated with L-arginine uptake in a PCS
model of PH, brain edema was not correlated with ammonia increase.
Furthermore brain edema was documented in group III and ammonia level was
lower than that in group II and no cerebral edema was observed. Besides
this, brain edema was lower in group IV than in group III and the highest
recorded ammonia was recorded. What is the major mechanism involved in the
development of brain edema in this experiment? One of the pathways of
brain edema is the vasogenic and if that mechanism was involved in groups
III and IV, why in group II BBB integrity was broken-down and ammonia
plasma levels were increased, and no cortical brain edema was documented?
Although a mixed mechanism (vasogenic and cytotoxic) was proposed for
brain edema in ALF, in this experiment no brain edema was
present in the chronic group without acute hepatocyte injury. In the
chronic group plus acute liver injury brain edema was registered and the
cytotoxic mechanism could be mostly responsible. Under these experimental
conditions another mechanism of brain edema might involve aquaporins. The
aquaporin-type water channels are integral membrane proteins that have
been found to function as conduits for osmotically driven transport of
water across cell plasma membranes. In this experiment BBB
integrity was altered and thus endothelial cells were involved. AQP4
presented in both, endothelia and astrocytes might play an active role in
regulation of astrocyte swelling and the presence of brain
altered integrity of the BBB at the hippocampus areas involved in learning
and memory consolidation in groups II, III and IV. Furthermore, the only
group with an altered behavioral test, at first fall in rota rod test, was
group IV. These might be related with the observation in the only group
with PH, elevated enzymes, hepatocyte injury, hyperammonemia, brain edema,
positive TB and altered rota rod test. Dixit demonstrated
that in rats with galactosamine-induced ALF BBB breakdown evidenced by TB
dye infusion studies began in grade III coma. Despite this, under the
present experimental conditions no clinical evidence of coma could be
registered in any group. Therefore, group IV can be considered as an
experimental model of subclinic PSE.
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