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Tyrphostin reduces the organ injury in haemorrhagic shock:role of inducible nitric oxide synthase
Michelle McDonald a, Maha Abdelrahman a, Salvatore Cuzzocrea b,Christoph Thiemermann a,*
a The Department of Experimental Medicine, Nephrology and Critical Care, St. Bartholomew’s and The Royal London School of Medicine and
Dentistry, The William Harvey Research Institute, University of London, Queen Mary, Charterhouse Square, London EC1M 6BQ, UKb Istituto di Farmacologia, Universita di Messina, Torre Biologica-Policlinico Universatario, Via C. Valeria, 98100 Messina, Italy
Received 5 March 2003; received in revised form 3 April 2003; accepted 10 April 2003
Resuscitation 58 (2003) 349�/361
www.elsevier.com/locate/resuscitation
Abstract
We investigate the effects of the tyrosine kinase inhibitor, tyrphostin AG126 on the organ injury and dysfunction (kidney, liver,
pancreas, muscle and or brain) associated with haemorrhagic shock in the anaesthetised rat. Haemorrhage (sufficient to lower mean
arterial blood pressure to 45 mmHg for 90 min) and subsequent resuscitation with the shed blood resulted (within 4 h after
resuscitation) in expression of inducible nitric oxide synthase inhibitor (iNOS), positive staining for nitrotyrosine (liver), renal, liver
and pancreatic injury, and injury to the muscle and brain. Pre-treatment (30 min prior to the onset of haemorrhage) with the
tyrosine kinase inhibitor tyrphostin AG126 reduced the iNOS expression, nitrotyrosine formation, hepatic, brain or muscular
injury, and to a lesser extent, the renal injury caused by haemorrhage and resuscitation. Selective inhibition of iNOS activity with N -
(3-(aminomethyl)benzyl) acetamidine (1400 W, 10 mg kg�1 i.v., 5 min prior to the onset of resuscitation), also attenuated
nitrotyrosine formation, renal dysfunction, liver injury and brain or muscular injury associated with haemorrhagic shock. The
expression of iNOS protein was unaffected by 1400 W. We propose that the activation of tyrosine kinases and the induction of iNOS
contribute to the multiple organ injury caused by severe haemorrhage and resuscitation.
# 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nitric oxide; Haemorrhage; Resuscitation; Multiple organ failure; Liver
Resumo
Investigamos o efeito de um inibidor da cinase da tirosina, a Tirfostina AG126 na lesao e disfuncao de orgaos (rim, fıgado,
pancreas musculos e cerebro) associada a choque hemorragico em ratos anestesiados. Provocou-se hemorragia (suficiente para a
pressao arterial baixar ate aos 45 mmHg durante 90 min) sendo de seguida feita a reanimacao com o sangue removido e 4h depois
pesquisou-se a expressao do inibidor da sintetase do oxido nıtrico induzıvel (iNOS) traduzida pela coloracao para a nitrotirosina
(fıgado), pela lesao renal hepatica e pancreatica e pela lesao dos musculos e do cerebro. O pre-tratamento (30 min antes do inıcio da
hemorragia) com inibidor da cinase da tirosina, a Tirfostina AG126 reduziu a expressao do iNOS, da formacao de nitrotirosina, da
lesao hepatica, cerebral ou muscular e em menor dimensao a lesao causada pela hemorragia e pela reanimacao. A inibicao selectiva
da actividade da iNOS com N -(3-(aminometil)benzil) acetamidina (1 400W, 10 mg Kg�1 iv, 5 min antes do inıcio da reanimacao)
tambem reduz a formacao de nitrotirosina, a disfuncao renal, a lesao do fıgado, cerebro e musculo associada a choque hemorragico.
A expressao do iNOS proteico nao foi afectada por 1 400W. Propusemos que a activacao e da tirosina cinase e a inducao da iNOS
contribuem para a lesao multipla de orgaos consequencia da hemorragia grave e da reanimacao.
# 2003 Elsevier Ireland Ltd. All rights reserved.
Palavras chave: Ordens para nao tentar (DNAR); Envolvimento do doente; Formularios pre impressos
* Corresponding author. Tel.: �/44-207-882-6116; fax: �/44-207-251-1685.
E-mail address: c.thiemermann@qmul.ac.uk (C. Thiemermann).
0300-9572/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/S0300-9572(03)00156-4
Resumen
Investigamos los efectos de la tyrphostin AG126, inhibidor de la tirosina quinasa, sobre la lesion y disfuncion de organos (rinon,
hıgado, pancreas, musculo y o cerebro) asociada con el shock hemorragico en ratas. La hemorragia ( suficiente para bajar la presion
arterial media a 45 mm Hg por 90 minutos) y la subsecuente resucitacion con la sangre extraıda resulto (dentro de 4 hrs despues de la
resucitacion) en expresion de inhibidor inducible de de sintetasa de oxido nıtrico (iNOS), tincion positiva para nitrotirosina
(hıgado), lesion renal, hepatica y pancreatica, y lesion de musculo y cerebro. El pre tratamiento con tyrophostin AG126 inhibidor de
tirosina quinasa (30 minutos previos al inicio de la hemorragia) redujo la expresion de iNOS, la formacion de nitrotirosina, lesion
hepatica cerebral y muscular, y en menor extension, la lesion renal causada por la hemorragia y la resucitacion. La inhibicion
selectiva de la actividad de iNOS con N -(3-(aminometil) benzil) acetamidina (1400 W, 10 mg kg�1 i.v., 5 minutos previos al inicio de
la resucitacion), tambien atenuo la formacion de nitrotirosina, disfuncion renal, lesion hepatica y lesion cerebral y muscular
asociadas con shock hemorragico. La expresion de la proteina iNOS no fue afectada por 1400W. Proponemos que la activacion de
la tirosina quinasa y la induccion de iNOS contribuye a la lesion organica multiple causada por hemorragia severa y resucitacion.
# 2003 Elsevier Ireland Ltd. All rights reserved.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361350
Palabras clave: Oxido nıtrico; Hemorragia; Resucitacion; Falla organica multiple; Hıgado
1. Introduction
Several investigators have demonstrated that the
release of pro-inflammatory cytokines contributes to
the pathophysiology of multiple organ failure following
severe haemorrhage [1�/3]. The molecular mechanisms
involved in the enhanced expression of the pro-inflam-
matory cytokines and of the induction of inducible nitric
oxide synthase (iNOS) include the activation of tyrosine
kinases [4,5]. The tyrphostins AG126 and AG556 are
known to inhibit the activity of tyrosine kinases [6]. Pre-
treatment with tyrphostin AG126 reduces the rise in
serum levels of TNF-a and the mortality caused by
endotoxin in the mouse [5]. The beneficial effects of
tyrphostin AG126 correlate with its potency to block
tyrosine phosphorylation of a p42 MAP kinase protein
substrate in murine macrophages exposed to endotoxin
[5]. Similarly, the tyrphostins AG556 and AG126 reduce
the multiple organ failure in animal models of peritonitis
[7] and endotoxaemia [8]. In addition, the protein
tyrosine kinase inhibitor, genestein, attenuated vascular
hyporeactivity, [9] lung injury, [10] and multiple organ
failure [8] caused by endotoxin in the rat [11]. We have
reported recently that the dose of tyrphostin AG126
used attenuates the phosphorylation of tyrosine residues
in the rat in vivo [12]. However, the evidence for the role
of tyrosine kinases in the multiple organ failure follow-
ing severe haemorrhage is limited [13].
Early studies using electron spin resonance techniques
were the first to demonstrate the association between
haemorrhagic shock and enhanced formation of NO
[14]. In 1993, we reported that an enhanced formation of
NO by INOS contributes to the delayed circulatory
failure associated with haemorrhagic shock in the rat
[15]. Smail and colleagues (1998) report that a delayed
(peak occurs 4 h after resuscitation), but prolonged,
increase in nitrite/nitrate (NO2�/NO3
�) levels occurs
after trauma-haemorrhage and that the gut and the
liver are the main organs responsible for the increase in
NO production [16]. Non-specific NOS inhibitors may
cause excessive vasoconstriction, and enhance the in-
cidence of both microvascular thrombosis and neutro-phil adhesion to the endothelium. Therefore, agents
such as L-NAME, which are more potent inhibitors of
endothelial cNOS than iNOS activity have the potential
to worsen oxygen delivery and exacerbate organ injury
in animal models of haemorrhagic shock [17]. Thus,
agents that either inhibit the expression of iNOS protein
(i.e. tyrphostin AG126) or agents which, selectively
inhibit the activity of iNOS may produce favourableresults.
In order to assess the contribution of tyrosine kinase
towards the development of multiple organ failure
following haemorrhagic shock, this study investigates
the effects of the tyrosine kinase inhibitor, tyrphostin
AG126, on the organ injury/dysfunction caused by
severe haemorrhage and resuscitation in the anaesthe-
tised rat. To gain further understanding into themechanism(s) of action, we have investigated the effects
of tyrphostin AG126 on the expression of iNOS and the
formation of nitrotyrosine in the liver. We found that
tyrphostin AG126 reduced the expression of iNOS,
nitrotyrosine formation and the multiple organ injury
in rats subjected to severe haemorrhage. To identify the
exact role of iNOS, we have investigated the effects of a
selective inhibitor of iNOS activity, 1400 W on multipleorgan injury following haemorrhagic shock.
2. Materials and methods
2.1. Surgical procedure
The experiments described in this article were per-
formed with adherence to National Institute of Health
guidelines on the use of experimental animals. All
experiments were performed with adherence with the
Home Office Guidance on the Operation of the Animals
(Scientific Procedures) Act 1986, published by HMSO,
London [18].
This study was carried out on 65 male Wistar rats
(Tuck, Rayleigh, Essex, UK) weighing 220�/340 g
receiving a standard diet and water ad libitum. All
animals were anaesthetised with thiopentone sodium
(120 mg kg�1 i.p.) and anaesthesia was maintained by
supplementary injections of thiopentone sodium as
required. The trachea was cannulated to facilitate
respiration and rectal temperature was maintained at
37 8C with a homeothermic blanket. The right femoral
artery was catheterised and connected to a pressure
transducer (MLT 1050, AD Instruments Ltd, Hastings,
UK) for the measurement of mean arterial blood
pressure (MAP) and heart rate (HR) which were
displayed on a data acquisition system (Powerlab†
Version 4.0.4, AD Instruments, Hastings, UK) installed
on a Dell Dimension 4100 personal computer. The right
carotid artery was cannulated to facilitate blood with-
drawal (see below). The jugular vein was cannulated for
the administration of drugs. The bladder was also
cannulated for the collection of urine. Upon completion
of the surgical procedure, cardiovascular variables were
allowed to stabilise for 20 min. Then, blood was
withdrawn from the catheter placed in the carotid artery
to achieve a fall in MAP to 45 mmHg within 10 min.
Thereafter, MAP was maintained at 45 mmHg for a
total period of 90 min by either withdrawal (during the
compensation period) or re-injection of blood. The
amount of shed blood re-injected during the 90 min
period of haemorrhage did not exceed 10% of the total
volume of the blood withdrawn. At 90 min after the
beginning of haemorrhage, the shed blood was re-
injected into the animal. The volume of blood with-
drawn in rats subjected to haemorrhage was similar in
all groups studied and ranged from 8.19/0.4 to 9.19/0.4
ml (Table 1, P �/0.05).
2.2. Experimental design
Eight experimental groups were used for the study:
1) At 30 min prior to haemorrhage, animals were pre-
treated with the vehicle (DMSO 50% v/v, 1 ml kg�1
i.p., n�/9; HS DMSO).2) At 30 min prior to haemorrhage, animals were pre-
treated with tyrphostin AG126 (5 mg kg�1 i.p., n�/
9; HS Tyrphostin).
3) Rats subjected to the same surgical procedure, but
which were not subjected to haemorrhagic shock
and pre-treated with DMSO, vehicle for tyrphostin
AG126 (50% v/v, 1 ml kg�1 i.p., n�/8; Sham
DMSO).4) Rats subjected to the surgical procedure, but which
were not subjected to haemorrhagic shock and pre-
treated with tyrphostin AG126 (5 mg kg�1 i.p. n�/
7; Sham Tyrphostin).
5) At 5 min prior to resuscitation with the shed blood,
control rats were treated with vehicle (saline, 1 ml
kg�1 i.v., n�/10; HS Saline).
6) At 5 min prior to resuscitation with the shed blood,animals were treated with the selective iNOS
inhibitor 1400 W (10 mg kg�1 i.v., n�/10; HS
1400 W).
7) Rats were subjected to the same surgical procedure
without causing haemorrhage, and were treated
with vehicle (saline, 1 ml kg�1 i.v., n�/8; Sham
Saline).
8) Rats were subjected to the same surgical procedurewithout causing haemorrhage, but received 1400 W
(10 mg kg�1 i.v., n�/4; Sham 1400 W).
The dosage of tyrphostin AG126 was chosen as this
dose of tyrphostin AG126 has been shown to prevent
the organ injury caused by endotoxin in the rat [8] and
the phosphorylation of tyrosine residues in rats with
experimental colitis [12]. Similarly, the dosage of 1400 W
was chosen as a similar dose of 1400 W has been shown
to attenuate the circulatory failure and the rise in plasmalevels of nitrite/nitrate caused by endotoxin in the rat
[19].
2.3. Quantification of organ function and injury
Four hours after resuscitation (end of the experi-
ment), 1.5 ml of blood was collected into a serum gel
tube (Sarstedt, Germany) from the catheter placed in the
right carotid artery. The blood sample was centrifuged
(1610�/g for 3 min at room temperature) to separate
serum. All serum samples were analysed within 24 h by a
contract laboratory for veterinary clinical chemistry(Vetlab Services, Sussex, UK). The following marker
enzymes were measured in the serum as biochemical
indicators of multiple organ injury/dysfunction: Liver
Table 1
The volume of blood withdrawn during the haemorrhagic phase
Group Blood volume withdrawn (ml) n
Haemorrhagic shock saline 8.59/0.2 14
Haemorrhagic shock 1400 W 8.19/0.4 10
Haemorrhagic shock DMSO 8.69/0.4 9
Haemorrhagic shock tyrphostin 9.19/0.4 9
Rats subjected to haemorrhagic shock were treated with DMSO (HS
DMSO, n�/9), tyrphostin AG126 (HS Tyrphostin AG126 n�/9),
saline (HS Saline, n�/10), or 1400 W (HS 1400 W, n�/10). Please note
that there were no significant differences between any of the experi-
mental groups studied.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361 351
injury was assessed by measuring the rise in serum levels
of alanine aminotransferase (ALT, a specific marker for
hepatic parenchymal injury) and aspartate aminotrans-
ferase (AST, a non-specific marker for hepatic injury)[20,21]. Renal dysfunction was assessed by measuring
the rises in serum levels of creatinine (an indicator of
reduced glomerular filtration rate, and hence, renal
failure) and urea (an indicator of impaired excretory
function of the kidney and/or increased catabolism) [22].
In addition, the serum level of lipase was determined as
an indicator of pancreatic injury [8]. Finally, the increase
in the serum levels of creatine kinase (CK) wasdetermined as an indicator for the development of
muscle (skeletal or cardiac) or brain injury.
2.4. Light microscopy
Organ (lung, liver, kidney) biopsies were taken at the
end of the experiment. The biopsies were fixed in
buffered formaldehyde solution (10% in phosphate
buffered saline) at room temperature, dehydrated bygraded ethanol and embedded in Paraplast (Sherwood
Medical, Mahwah, NJ). Sections (thickness 7 mm) were
deparaffinised with xylene, stained with trichromic Van
Gieson and studied using light microscopy (Dialux 22
Leitz).
2.5. Immunohistochemical localisation of nitrotyrosine
Tyrosine nitration, an index of the nitrosylation of
proteins by peroxynitrite and/or other free radicals, was
determined by immunohistochemistry as previously
described [23]. At the end of the experiment, the livers
were fixed in 10% buffered formaldehyde and 8 mm
sections were prepared from paraffin embedded tissues.
After deparaffinisation, endogenous peroxidase was
quenched with 0.3% H2O2 in 60% methanol for 30min. The sections were permeabilised with 0.1% Triton
X-100 in phosphate buffered saline (PBS) for 20 min.
Non-specific adsorption was minimised by incubating
the section in 2% normal goat serum in phosphate
buffered saline for 20 min. Endogenous biotin or avidin
binding sites were blocked by sequential incubation for
15 min with avidin and biotin. The sections were then
incubated overnight with 1:1000 dilution of primaryanti-nitrotyrosine antibody or with control solutions.
Controls included buffer alone or non-specific purified
rabbit IgG. Specific labelling was detected with a biotin-
conjugated goat anti-rabbit IgG and avidin�/biotin
peroxidase complex.
2.6. Immunohistochemical localisation of iNOS
The expression of iNOS protein was evaluated by
immunohistochemistry in the liver of all animals (sub-
jected to sham operation, or subjected to haemorrhage
and resuscitation and treated with either saline, 1400 W,
DMSO, or Tyrphostin AG126) as previously described
[23]. At the end of the resuscitation period, the livers
were fixed in 10% (w/v) buffered formaldehyde and 8 mm
sections were prepared from paraffin embedded tissues.
After deparaffinisation, endogenous peroxidase was
quenched with 0.3% (v/v) H2O2 in 60% (v/v) methanol
for 30 min. The sections were permeabilised with 0.1%
(w/v) Triton X-100 in PBS for 20 min. Non-specific
adsorption was minimised by incubating the section in
2% normal goat serum in PBS for 20 min. Endogenous
biotin or avidin binding sites were blocked by sequential
incubation for 15 min with avidin and biotin. The
sections were then incubated overnight with anti-iNOS
antibody (1:1000 in PBS, v/v). Controls included buffer
alone or non-specific purified rabbit IgG. Specific
labelling was detected with a biotin-conjugated goat
anti-rabbit IgG and avidin�/biotin peroxidase
complex.
Fig. 1. Serum levels of (A) urea and (B) creatinine. Group 1: Sham
operation, no haemorrhage and treated with dimethylsulphoxide
(Sham DMSO, n�/8). Group 2: Sham operation, no haemorrhage
and treated with Tyrphostin AG126 (Sham Tyrphostin, n�/7). Group
3: Operation, haemorrhagic shock (HS), and treated with DMSO (HS
DMSO, n�/9). Group 4: Operation, haemorrhagic shock (HS), and
treated with Tyrphostin AG126 (HS Tyrphostin, n�/9) *, P B/0.05
when compared with HS DMSO by ANOVA followed by Dunnett’s
post hoc test.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361352
2.7. Materials
Thiopentone sodium (Intraval Sodium†) was ob-tained from Rhone Merieux Ltd. (Harlow, Essex,
UK). All stock solutions were prepared in non-pyro-
genic saline (0.9% NaCl; Baxter Healthcare Ltd.,
Thetford, Norfolk, UK). 1400 W was obtained from
Alexis (Nottingham, UK). Biotin blocking kit, biotin-
conjugated goat anti-rabbit IgG, primary anti-nitrotyr-
osine and avidin-biotin peroxidase complex were ob-
tained from DBA (Milan, Italy). Unless otherwisestated, all compounds were obtained from Sigma-
Aldrich Company Ltd. (Poole, Dorset, UK).
2.8. Statistical evaluation
All data are presented as mean9/S.E.M. of n ob-
servations, where n represents the number of animals or
blood samples studied. For comparison of two groups
data was analysed via Student’s unpaired t-test. Forcomparison of three or more groups, data was analysed
by 1-factorial ANOVA, followed by a Dunnett’s test. A
P -value of less than 0.05 was considered to be statisti-
cally significant when compared with HS Saline or HS
DMSO. All statistical analyses were performed with the
aid of a GRAPH PAD PRISM statistical package (Version
3.03).
3. Results
3.1. Effects of the tyrosine kinase inhibitor, tyrphostin
AG126 on the multiple organ dysfunction syndrome
caused by severe haemorrhage
3.1.1. Effects on the renal injury/dysfunction
In sham-operated rats, administration of tyrphostin
AG126 did not result in any significant alterations in the
serum levels of urea and creatinine (P �/0.05, Fig. 1).
When compared with sham-operated rats (ShamDMSO, n�/8), haemorrhage followed by resuscitation
resulted in significant rises in the serum levels of urea
and creatinine, demonstrating the development of renal
dysfunction (HS DMSO, n�/9, P B/0.05, Fig. 1).
Treatment of rats subjected to haemorrhage and resus-
citation with tyrphostin AG126 did not affect the rise in
the serum levels of creatinine (HS Tyrphostin, n�/9,
P �/0.05, Fig. 1a), but significantly reduced the rise inthe serum levels of urea (n�/9, P B/0.05, Fig. 1b).
3.1.2. Effects on the liver injury
In sham-operated rats, administration of tyrphostinAG126 did not result in any significant alterations in the
serum levels of AST and ALT (P �/0.05, Fig. 2). When
compared with sham-operated rats (Sham DMSO, n�/
8), haemorrhage followed by resuscitation resulted in
significant rises in the serum levels of AST and ALT,
demonstrating the development of hepatocellular injury
Fig. 2. Serum levels of (A) AST and (B) ALT. Group 1: Sham
operation, no haemorrhage and treated with dimethylsulphoxide
(Sham DMSO, n�/8). Group 2: Sham operation, no haemorrhage
and treated with Tyrphostin AG126 (Sham Tyrphostin, n�/7). Group
3: Operation, haemorrhagic shock (HS), and treated with DMSO (HS
DMSO, n�/9). Group 4: Operation, haemorrhagic shock (HS), and
treated with Tyrphostin AG126 (HS Tyrphostin, n�/9) *, P B/0.05
when compared with HS DMSO by ANOVA followed by Dunnett’s
post hoc test.
Fig. 3. Serum levels of lipase. Group 1: Sham operation, no
haemorrhage and treated with dimethylsulphoxide (Sham DMSO,
n�/8). Group 2: Sham operation, no haemorrhage and treated with
Tyrphostin AG126 (Sham Tyrphostin, n�/7). Group 3: Operation,
haemorrhagic shock (HS), and treated with DMSO (HS DMSO, n�/
9). Group 4: Operation, haemorrhagic shock (HS), and treated with
Tyrphostin AG126 (HS Tyrphostin, n�/9) *, P B/0.05 when compared
with HS DMSO by ANOVA followed by Dunnett’s post hoc test.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361 353
(HS DMSO, n�/9, P B/0.05, Fig. 2). Treatment of rats
subjected to haemorrhage and resuscitation with tyr-
phostin AG126 significantly reduced the rise in the
serum levels of AST and ALT (HS Tyrphostin, n�/9,P B/0.05, Fig. 2).
3.1.3. Effects on pancreatic injury
In sham-operated rats, administration of tyrphostin
AG126 did not result in any significant alterations in the
serum levels of lipase (P �/0.05, Fig. 3). When compared
with sham-operated rats (Sham DMSO, n�/8), haemor-
rhage followed by resuscitation resulted in a substantial
rise in the serum levels of lipase, demonstrating the
development of pancreatic injury. However, due to theextent of variation observed in the group of rats
subjected to severe haemorrhage and treated with
DMSO, the observed difference was not statistically
significant (HS DMSO, n�/9, P �/0.05, Fig. 3). Treat-
ment of rats subjected to haemorrhage and resuscitation
with tyrphostin AG126 reduced the rise in the serum
levels of lipase (HS Tyrphostin, n�/9, P �/0.05, Fig. 3).
3.1.4. Effects on the serum levels of CK
In sham-operated rats, administration of tyrphostin
AG126 did not result in any significant alterations in the
serum levels of CK (P �/0.05, Fig. 4). When compared
with sham-operated rats (Sham DMSO, n�/8), haemor-
rhage followed by resuscitation resulted in a substantial
rise in the serum levels of CK, demonstrating the
development of injury to the brain, skeletal muscle
and/or cardiac muscle (HS DMSO, n�/9, P B/0.05, Fig.4). Treatment of rats subjected to haemorrhage and
resuscitation with the tyrosine kinase inhibitor, tyrphos-
tin AG126, significantly reduced the rise in the serum
levels of CK caused by haemorrhage (HS Tyrphostin,
n�/9, P B/0.05, Fig. 4).
3.2. Effects of the selective iNOS inhibitor, 1400 W on
the multiple organ dysfunction syndrome caused by severe
haemorrhage
3.2.1. Effects on the renal injury/dysfunction
In sham-operated rats, administration of 1400 W
caused a small increase in the serum levels of urea and
creatinine, which was not statistically significant (P �/
0.05, Fig. 5). When compared with sham-operated rats
(Sham Saline, n�/8), haemorrhage followed by resusci-
tation resulted in significant rises in the serum levels of
urea and creatinine, demonstrating the development of
renal dysfunction (HS Saline, n�/10, P B/0.05, Fig. 5).
Treatment of rats subjected to haemorrhage and resus-
citation with 1400 W did not affect the rise in the serum
levels of urea (HS 1400 W, n�/10, P �/0.05, Fig. 5a),
but significantly reduced the rise in the serum levels of
creatinine (n�/10, P B/0.05, Fig. 5b).
Fig. 4. Serum levels of CK. Group 1: Sham operation, no haemor-
rhage and treated with dimethylsulphoxide (Sham DMSO, n�/8).
Group 2: Sham operation, no haemorrhage and treated with Tyrphos-
tin AG126 (Sham Tyrphostin, n�/7). Group 3: Operation, haemor-
rhagic shock (HS), and treated with DMSO (HS DMSO, n�/9).
Group 4: Operation, haemorrhagic shock (HS), and treated with
Tyrphostin AG126 (HS Tyrphostin, n�/9) *, P B/0.05 when compared
with HS DMSO by ANOVA followed by Dunnett’s post hoc test.
Fig. 5. Serum levels of (A) urea and (B) creatinine. Group 1: Sham
operation, no haemorrhage and treated with saline (Sham Saline, n�/
8). Group 2: Sham operation, no haemorrhage and treated with 1400
W (Sham 1400 W, n�/4). Group 3: Operation, haemorrhagic shock
(HS), and treated with saline (HS Saline, n�/10). Group 4: Operation,
haemorrhagic shock (HS), and treated with 1400 W (HS 1400 W, n�/
10). *, P B/0.05 when compared with HS Saline by ANOVA followed
by Dunnett’s post hoc test.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361354
3.2.2. Effects on the liver injury
In sham-operated rats, administration of 1400 W did
not result in any significant alterations in the serum
levels of AST and ALT (P �/0.05, Fig. 6). When
compared with sham-operated rats (Sham Saline, n�/
8), haemorrhage followed by resuscitation resulted in
significant rises in the serum levels of AST and ALT,
demonstrating the development of hepatocellular injury
(HS Saline, n�/10, P B/0.05, Fig. 6). Treatment of rats
subjected to haemorrhage and resuscitation with 1400
W abolished the rise in AST and ALT caused by
haemorrhage and resuscitation (HS 1400 W, n�/10,
P B/0.05, Fig. 6).
3.2.3. Effects on pancreatic injury
In sham-operated rats, administration of 1400 W
caused a small increase in the serum levels of lipase,
which was not statistically significant (P �/0.05, Fig.
7).When compared with sham-operated rats (Sham
Saline, n�/8), haemorrhage followed by resuscitationresulted in significant rises in the serum levels of lipase,
demonstrating the development of pancreatic injury (HS
Saline, n�/10, P B/0.05, Fig. 7). Treatment of rats
subjected to haemorrhage and resuscitation with the
selective iNOS inhibitor, 1400 W reduced the pancreatic
injury caused by haemorrhage (Fig. 7). However,
because of the degree of variability observed within
the control (HS Saline) group, this reduction was not
statistically significant (HS 1400 W, n�/10, P �/0.05,
Fig. 7).
3.2.4. Effects on the serum levels of CK
In sham-operated rats, administration of 1400 W didnot result in any significant alterations in the serum
levels of CK (P �/0.05, Fig. 8). When compared with
sham-operated rats (Sham Saline, n�/8), haemorrhage
Fig. 6. Serum levels of (A) AST and (B) ALT. Group 1: Sham
operation, no haemorrhage and treated with saline (Sham Saline, n�/
8). Group 2: Sham operation, no haemorrhage and treated with 1400
W (Sham 1400 W, n�/4). Group 3: Operation, haemorrhagic shock
(HS), and treated with saline (HS Saline, n�/10). Group 4: Operation,
haemorrhagic shock (HS), and treated with 1400 W (HS 1400 W, n�/
10). *, P B/0.05 when compared with HS Saline by ANOVA followed
by Dunnett’s post hoc test.
Fig. 7. Serum levels of lipase. Group 1: Sham operation, no
haemorrhage and treated with saline (Sham Saline, n�/8). Group 2:
Sham operation, no haemorrhage and treated with 1400 W (Sham
1400 W, n�/4). Group 3: Operation, haemorrhagic shock (HS), and
treated with saline (HS Saline, n�/10). Group 4: Operation, haemor-
rhagic shock (HS), and treated with 1400 W (HS 1400 W, n�/10). *,
P B/0.05 when compared with HS Saline by ANOVA followed by
Dunnett’s post hoc test.
Fig. 8. Serum levels of CK. Group 1: Sham operation, no haemor-
rhage and treated with saline (Sham Saline, n�/8). Group 2: Sham
operation, no haemorrhage and treated with 1400 W (Sham 1400 W,
n�/4). Group 3: Operation, haemorrhagic shock (HS), and treated
with saline (HS Saline, n�/10). Group 4: Operation, haemorrhagic
shock (HS), and treated with 1400 W (HS 1400 W, n�/10). *, P B/0.05
when compared with HS Saline by ANOVA followed by Dunnett’s
post hoc test.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361 355
followed by resuscitation resulted in significant rises in
the serum levels of CK, demonstrating the development
of injury to the brain, skeletal muscle and/or cardiac
muscle (HS Saline, n�/10, P B/0.05, Fig. 8). Treatment
of rats subjected to haemorrhage and resuscitation with
the selective iNOS inhibitor, 1400 W, significantly
reduced the rise in the serum levels of CK caused by
haemorrhage (P B/0.05, Fig. 8).
Fig. 9. Representative histological sections of a liver obtained from (A) rats subjected to the surgical procedure without causing a haemorrhage
(sham-operation), (B) rats subjected to haemorrhage and resuscitation with shed blood (for 4 h), (C) rats subjected to haemorrhage and resuscitation,
which were pre-treated with the tyrosine kinase inhibitor, tyrphostin AG126, and (D) rats subjected to haemorrhage and resuscitation and treated
with the selective iNOS inhibitor, 1400 W. When compared with sham-operated animals (A), haemorrhage and resuscitation results in substantial
morphological alterations (degradation of the nuclear membrane). Please note that these pathological alterations associated with haemorrhage and
resuscitation was largely attenuated via treatment with tyrphostin AG126 or 1400 W.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361356
Fig. 10. Representative histological sections of a liver obtained from (A) rats subjected to the surgical procedure without causing a haemorrhage
(sham-operation), (B) rats subjected to haemorrhage and resuscitation with shed blood (for 4 h), (C) rats subjected to haemorrhage and resuscitation,
which were pre-treated with the tyrosine kinase inhibitor, tyrphostin AG126, and (D) rats subjected to haemorrhage and resuscitation and treated
with the selective iNOS inhibitor, 1400 W. When compared with sham-operated animals (A), haemorrhage and resuscitation results in positive
(brown) staining for nitrotyrosine (determined by immunohistochemistry), indicating the nitrosylation of proteins within the liver. Nitrotyrosine
formation associated with haemorrhage and resuscitation was largely attenuated via treatment with tyrphostin AG126 or 1400 W.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361 357
Fig. 11. Representative histological sections of a liver obtained from (A) rats subjected to the surgical procedure without causing a haemorrhage
(sham-operation), (B) rats subjected to haemorrhage and resuscitation with shed blood (for 4 h), (C) rats subjected to haemorrhage and resuscitation,
which were pre-treated with the tyrosine kinase inhibitor, tyrphostin AG126, and (D) rats subjected to haemorrhage and resuscitation and treated
with the selective iNOS inhibitor, 1400 W. When compared with sham-operated animals (A), haemorrhage and resuscitation results in positive
(brown) staining for iNOS protein (determined by immunohistochemistry), indicating the expression of iNOS within the liver. Increased expression
(upregulation) of iNOS associated with haemorrhage and resuscitation was largely attenuated via treatment with tyrphostin AG126 but not by 1400
W.
M. McDonald et al. / Resuscitation 58 (2003) 349�/361358
3.3. Effects of 1400 W and tyrphostin AG126 on the
formation of nitrotyrosine and the expression of iNOS
protein in the liver of rats subjected to haemorrhage and
resuscitation
When compared with livers obtained from sham-
operated rats, which had not been subjected to haemor-
rhage and resuscitation, the livers of rats subjected to
haemorrhage and resuscitation exhibited morphologic
abnormalities consistent with shock-induced organ in-
jury (Fig. 9a, b). The degree of liver injury (necrosis) was
markedly reduced in rats subjected to haemorrhage andresuscitation and treated with either tyrphostin AG126
(Fig. 9c) or 1400 W (Fig. 9d). The livers of rats subjected
to haemorrhage and resuscitation also demonstrated a
marked staining for nitrotyrosine formation (Fig. 10b)
and iNOS protein (Fig. 11b). Pre-treatment with the
tyrosine kinase inhibitor, tyrphostin AG126 attenuated
both the staining for nitrotyrosine (Fig. 10c) and iNOS
protein (Fig. 11c). Treatment of rats with 1400 Wattenuated the formation of nitrotyrosine (Fig. 10d)
but did not affect the expression of iNOS protein (Fig.
11d).
4. Discussion
We demonstrate here that haemorrhage for 90 min
followed by resuscitation with shed blood (for 4 h) in therat results in a substantial increase in the serum levels of
urea and creatinine indicating the development of acute
renal dysfunction. Haemorrhage and resuscitation also
caused an increase in the serum levels of the transami-
nases AST and ALT, indicating the development of
hepatocellular injury. Histological examination con-
firmed that the model of haemorrhagic shock used
here caused a substantial degree of tissue injury to theliver. In addition, the rodent model of haemorrhagic
shock used here, leads to increased expression of iNOS
protein and increased nitrotyrosine formation (indica-
tive of the formation of peroxynitrite) in the liver. Pre-
treatment of rats with the tyrosine kinase inhibitor
tyrphostin AG126 attenuated (i) the rise in the serum
levels of urea, (ii) the liver injury and the muscular
(skeletal or cardiac) and/or brain injury caused byhaemorrhage and resuscitation. In addition, tyrphostin
AG126 substantially reduced nitrotyrosine formation
and expression of iNOS protein in the livers of rats
subjected to haemorrhagic shock.
We propose that the beneficial effects of tyrphostin
AG126 in this rodent model of haemorrhagic shock are
due to the ability of this compound to inhibit the
activation of protein tyrosine kinases. Our findingssupport a recent report by Jarrar and colleagues, who
have shown that the depressed cardiovascular function,
rise in serum IL-6 levels and phosphorylation of protein
kinases were attenuated by tyrphostin AG556 in a
rodent model of trauma-haemorrhage and resuscitation
[13].
Our study demonstrates that tyrphostin AG126
attenuates the expression of iNOS protein and nitrotyr-
osine formation in the liver of rats subjected to severe
haemorrhage. The signal transduction events leading to
the induction of iNOS activity in cells challenged with
LPS involves the activation of tyrosine kinases [4,24,25].
The expression of iNOS caused (i) by lipoteichoic acid in
macrophages [26], (ii) by IL-1 in pancreatic b-cells of the
rat [27], and (iii) by LPS and interferon-g in C6 glioma
cells [28] involves the activation of tyrosine kinases as
well as the activation of NF-kB. We have recently
reported that the model of haemorrhage and resuscita-
tion used here leads to the activation of the transcription
factor NF-kB and that inhibition of the activation of
NF-kB (with calpain inhibitor I) attenuates the organ
injury caused by haemorrhagic shock in the rat [29].
There is good evidence that (i) tyrosine phosphorylation
itself plays an important role in the activation of NF-kB
and (ii) that tyrosine kinase inhibitors diminish the
activation of NF-kB. For instance, many chemically
distinct inhibitors of tyrosine kinase attenuate the
translocation of NF-kB in rat cells activated with pro-
inflammatory cytokines or LPS [27,28,30]. Moreover,
pre-treatment with the tyrosine kinase inhibitor, genes-
tein reduces the activation of NF-kB and the acute lung
injury caused by intratracheal administration of endo-
toxin in the rat [10]. Thus, it is possible that tyrphostin
AG126 prevents the activation of NF-kB, the expression
of iNOS protein and, hence, multiple organ failure
following severe haemorrhage and resuscitation.
To assess the contribution of NO (from iNOS)
towards the development of multiple organ failure, we
investigated the effects of the selective iNOS inhibitor
1400 W in rats subjected to severe haemorrhage. Indeed,
direct inhibition of iNOS activity (and not the expres-
sion) produced salutary effects similar to those achieved
with tyrphostin AG126: Treatment with 1400 W re-
duced the renal dysfunction, the hepatocellular injury
and the brain and/or muscular injury associated with
severe haemorrhage and resuscitation (this study).
In addition, the iNOS upregulation following
haemorrhage and resuscitation contributes to the in-
flammatory response leading to multiple organ
failure [31]. Infusion of the iNOS inhibitor,
L-NIL increases heat shock protein expression
(HSP-72 and HSP-32) and reduces the liver
injury associated with severe haemorrhage [32],
while removal of NO via inhibition of iNOS
or the nitric oxide scavenger NOX, improved survival
and reduced the NO levels, organ PMN infiltration and
lung injury in animal models of haemorrhagic shock
[33�/35].
M. McDonald et al. / Resuscitation 58 (2003) 349�/361 359
5. Conclusions
We propose that the reduction of the multiple organ
injury and dysfunction associated with severe haemor-rhage and resuscitation by the tyrosine kinase inhibitor,
tyrphostin AG126 is at least in part, due to the ability of
this compound to inhibit the expression of iNOS protein
and activity.
Acknowledgements
M.A. is a recipient of a Ph.D. studentship provided by
the Department of Experimental Medicine and Ne-phrology (G1A4).
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