tyrphostin reduces the organ injury in haemorrhagic shock: role of inducible nitric oxide synthase

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.


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: [email protected] (C. Thiemermann).

0300-9572/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/S0300-9572(03)00156-4

mailto:[email protected]

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.


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.


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.


(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



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



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.


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.


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.





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.





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.


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].


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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