The role of nitric oxide and prostaglandin e2 in prolonged hemorrhagic shock 1

Preservation of neurological functions by nitric oxide synthase inhibitors in delayed hemorrhagic shock model in conscious rats.. 18 Figure 2.4 Isolated prolonged hemorrhagic shock untre

Publications related to thesis

Chapter 1: Introduction

1 Shirhan Md, Moochhala SM, Kerwin Low SY, Ng KC, Lu J Influence of

selective nitric oxide synthase inhibitor for therapy of refractory hemorrhagic shock Resuscitation 61(2), 221-229, 2004

2 Shirhan Md, Shabbir M Moochhala, Kerwin Low S Y, Sng J, Ng KC, Pamela

Mok, Lu J Preservation of neurological functions by nitric oxide synthase inhibitors in delayed hemorrhagic shock model in conscious rats Life Sciences 76(6), 661-70, 2004

3 Shirhan Md, Moochhala SM, Kerwin SY, Ng KC, Lu J The role of inducible

nitric oxide synthase inhibitor on cyclooxygenase-2 expression in refractory hemorrhagic-shocked rats Journal of Surgical Research 123(2), 206-214, 2005

4 Shirhan Md, Moochhala SM, Kerwin Low SY The role of inducible nitric oxide

synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats Life Sciences 73 (14), 1825-1834, 2003

5 Shirhan Md, Moochhala SM, Ng KC, Kerwin Low SY, Teo AL, Lu J Effects

of aminoguanidine and L-NAME resuscitation in rats following combined fluid-percussion brain injury and severe controlled hemorrhagic shock Journal of

Neurosurgery 101(1), 138-44, 2004

Chapter 2: Pathophysiology of prolonged hemorrhagic shock

Papers 1 & 4

Chapter 3: Prolonged hemorrhagic shock rat model: the role of nitric oxide (NO) and the therapeutics effects of conservative fluids and NOS inhibitors

Papers 1 & 2

Chapter 4: Prolonged hemorrhagic shock model: the role of nitric oxide (NO) and prostaglandin E2 (PGE2) and the therapeutic effects of conservative fluids, NOS and

COX-2 inhibitors

Paper 3

Chapter 5: Prolonged hemorrhagic shock model: the role of NO and angiotensin II and

the therapeutics effects of conservative fluids, NOS donor and inhibitors

Paper 4

Chapter 6: Role of NOS inhibitors in rats following combined fluid-percussion brain injury and prolonged hemorrhagic shock

Paper 5

Chapter 7: Discussions

Paper 1-5

Publications not related to thesis

Lu J, Moochhala S, Shirhan Md, Ng KC, Teo AL, Tan MH, Moore XL, Wong MC,

Ling EA Neuroprotection by aminoguanidine after lateral fluid-percussive brain injury in rats: a combined magnetic resonance imaging, histopathologic and functional study Neuropharmacology 44 (2), 253-63, 2003

Lu J, Moochhala S, Shirhan Md, Ng KC, Tan MH, Teo AL, Ling EA Nitric oxide

induces macrophage apoptosis following traumatic brain injury in rats

Neuroscience Letter 339(2), 147-50, 2003

Moochhala SM, Shirhan Md, Lu J, Teng CH, Greengrass C Neuroprotective Role of

Aminoguanidine in Behavioural Changes Following Blast Injury Journal of Trauma 56 (2), 2004

Shirhan Md, Moochhala SM, Ng PY, Lu J, Ng KC, Teo AL, Yap E, Ng I, Hwang P,

Lim T, Sitoh YY, Rumpel H, Jose R, Ling E Spermine reduces infarction and neurological deficit following a rat model of middle cerebral artery occlusion: A magnetic resonance imaging study Neuroscience124(2), 299-304, 2004

Ng KC, Moochhala SM, Shirhan Md, Yap EL, Low SY, Lu J Preservation of

neurological functions by nitric oxide synthase inhibitors following hemorrhagic shock Neuropharmacology, 44 (2),244-252,2003

Moochhala SM, Lu J, Xing C K, Anuar F, Ng K C, Kerwin Low S Y, Whiteman M,

Shirhan Md Mercaptoethylguandine inhibition of inducible nitric oxide synthase and

cyclooxygenase-2 expressions induced in rats following fluid-percussion brain injury Journal of Trauma 59(2), 2005

Pamela Mok YY, Shirhan Md, Cheong Y P, Wang Z J, Bhatia M, Moochhala SM,

Moore P K Role of hydrogen sulfide in haemorrhagic shock in the rat: protective effect

of inhibitors of hydrogen sulfide biosynthesis British Journal of Pharmacology 143:

881-889, 2004

Figure Legend

Figure 2.1 Untreated rats showed significant decrease in percentage

survival when compared to sham-operated rats

17

Figure 2.2 Untreated rats showed significant decrease in mean arterial

blood pressure when compared with sham-operated rats

18

Figure 2.3 Untreated rats showed significant decrease in mean heart

rate when compared with sham-operated rats

18

Figure 2.4 Isolated prolonged hemorrhagic shock untreated rat aortic

strip showed significant decrease in the amount of contraction when compared with sham-operated rats

19

Figure 2.5 Isolated prolonged hemorrhagic shock untreated rat aortic

strip showed significant decrease in the amount of contraction when compared with sham-operated rats

19

Figure 2.6 Hemotoxylin and eosin stain of kidney, liver, lung and

stomach of sham-operated rats and saline-treated rats

21

Figure 3.1 Survival percentages in different groups of rats that

survived beyond 72 hours

32

Figure 3.2 MABP of different groups of rats 34 Figure 3.3 Nitrate/nitrite in different groups of rats 36 Figure 3.4 Creatinine levels of rat brain in different groups of rats 37 Figure 3.5 GOT levels of rat brain in different groups of rats 38 Figure 3.6 Nitrate/nitrite content of rat brain in different groups of rats 39 Figure 3.7 A schematic representation of histological assessment using

TTC staining at different time points (24, 48, 72 hours) after prolonged hemorrhagic shock in rat sections of

40

Figure 3.8 Total lesion volumes of different group of rats 41 Figure 3.9 Rotameric performance in different groups of rats 42

Figure4.2A,B Sham-operated & Normal saline + prolonged hemorrhagic

shock showing iNOS & COX-2 bands

55,56

Figure 4.3A Cortical nitrate/nitrite levels at 24, 48 and 72 hours in

58

Figure 4.3B Cortical PGE2 levels at 24, 48 and 72 hours in different

58

Figure 4.3C Plasma nitrate/nitrite levels at 24, 48 and 72 hours in

different groups of rats

59

Figure 4.3D Plasma PGE2 levels at 24, 48 and 72 hours in different

59

Figure 4.3E Plasma creatinine levels at 24, 48 and 72 hours in different

groups of rats

60

Figure 4.3F Plasma GOT levels at 24, 48 and 72 hours in different

60

Figure 4.4.1 Hemotoxylin and eosin stain of Kidneys 62 Figure 4.4.2 Hemotoxylin and eosin stain of Lungs 63 Figure 4.4.3 Hemotoxylin and eosin stain of Livers 64 Figure 4.4.4 Hemotoxylin and eosin stain of Cerebral cortex 65 Figure 4.5 Neurons in the cerebral cortex of the brain in

sham-operated rats, normalsaline + prolonged hemorrhagic shock, NS-398 + prolonged hemorrhagic shock & AG + prolonged

67

Figure 5.1 Survival percentages in different groups of rats that

survived beyond 72 hours

74

Figure 5.2 MABP of different groups of rats 76 Figure 5.3 Isolated prolonged hemorrhagic shock rat aortic strip

treated with ANGII

77

Figure 5.4 Isolated prolonged hemorrhagic shock rat aortic strip

shocked treated with noradrenaline

77

Figure 5.6 Creatinine levels in different groups of rats 80 Figure 5.7 GOT levels in different groups of rats 81

Figure 6.1 Percentage survival in different groups of rats 91 Figure 6.2 MABP in different groups of rats 93 Figure 6.3 Percentage change in cerebral tissue perfusion in different

groups of rats

94

Figure 6.4 L-NAME-, AG- and saline-treated rats did not show any

significant difference in their nitrate/nitrite levels when compared to sham-operated rats before FPI,HS, FPI+HS

95

Figure 6.5 During FPI+HS, all treated groups showed significant

difference in nitrate/nitrite levels when compared with all treated rats in FPI and HS

96

Figure 6.6 AG and L-NAME treated rats showed significantly lower

nitrate/nitrite levels when compared to saline-treated rats

97

Figure 6.7 Neurons in the cerebral cortex of the brain in

sham-operated, saline-, L-NAME- and AG-treated (FPI+HS), (FPI), (HS) rats

98

Figure 6.8 Light micrographs showing apoptotic cortical neurons in

saline-, L-NAME- and AG-treated (FPI+HS), (FPI), (HS) rats

99-100

Table legend

Table 2.1 Semi-quantitative analysis of major organ injury of different

groups of rats

22

Table 2.2 Creatinine and GOT levels in different groups of rats 22

Table 3.1 Semi-quantitative analysis of major organ injury of different

groups of rats

35

Table 4.1 The different treatment groups are tabulated below It comprises

of sham-operated and prolonged hemorrhagic shock groups

47

Table 4.2 The number of rats surviving at different time points (24, 48, 72

hours)

53

Table 5.1 Semi-quantitative analysis of major organ injury of different

groups of rats

82

Table 6.1 NISSL and TUNEL scores for the different groups of rats in FPI,

HS, FPI/HS

100

LIST OF ABBREVIATIONS

AG Aminoguanidine

CNS Central nervous system

DAO Diamine oxidase

eNOS Endothelial nitric oxide synthase

BBB

iNOS Inducible nitric oxide synthase

L-NAME NPPP

G

PPP-nitro-L-arginine methyl ester

MODS Multiple organ dysfunction syndrome

BBB NSAIDs Non-steroidal anti-inflammatory drugs

nNOS Neuronal nitric oxide synthase

NS-398

(N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide

PGEBBB2BBB Prostaglandin EBBB 2 BBB

TBI Traumatic brain injury

TUNEL in situ terminal transferase d-UTP nick-end

labelling

ABSTRACT

It is suggested that NO has played a major role in our model of prolonged hemorrhagic shock The detrimental effects of NO might be further worsen when coupled with prostaglandin E2, a known vasodilator, in prolong hemorrhagic shock Nitric oxides being

a vasodilator might be responsible for the loss of vascular hyporesponsiveness in the presence of a potent vasoconstrictor, angiotensin II, in our model of prolonged hemorrhagic shock The deleterious effect of NO is also shown in our combine model of prolonged hemorrhagic shock and fluid percussion injury Our studies showed that the selective inhibitor, AG, maybe beneficial as a potentially useful therapeutic agent in our model of prolonged hemorrhagic shock and the combine model of prolonged hemorrhagic shock and fluid percussion injury

Keywords: prolonged hemorrhagic shock; fluid percussion injury; nitric oxide; prostaglandin E2; aminoguanidine

SUMMARY OF THESIS

In our study of prolonged hemorrhagic shock, the physiological parameters (mean arterial blood pressure & heart rate) in untreated rats showed low blood pressure and cardiac

output respectively A reason for this might be that in our in vitro study where untreated

prolonged hemorrhagic shock aortic strip rats showed vascular hyporesponsiveness towards vasoconstrictors A significant drop in peripheral blood circulation might be a reason in poor perfusion to organs and resulted in organ damages in untreated prolonged hemorrhagic shock rats Therefore, this cumulative effect of decreased blood circulation and perfusion to organs, in untreated prolonged hemorrhagic shock rats showed a

significantly high mortality rate

Hemorrhagic shock is implicated in the induction of inducible nitric oxide synthase that leads to increase production of nitric oxide (NO) AG (selective NOS inhibitor)-treated rats had significantly higher survival rates compared with the conservative fluid, 0.9% normal saline, a selective inducible nitric oxide synthase (iNOS) inhibitor, NG -nitro-L-arginine methyl ester (L-NAME) and a non-selective inhibitor and S-Nitroso-N-acetylpenicillamine (SNAP), a NO donor, 72 hours following prolong hemorrhagic shock A marked increase in MABP level was observed in AG-treated rats when compared with the other treatment groups Histological examinations also showed a reduction of organ damages in AG-treated rats when compared with the other treatment groups Nitrate/nitrite level, glutamic oxalacetic transaminase (GOT) level and creatinine level (an indicator of liver and renal damage respectively) were also significantly improved in AG-treated rats when compared with the other treatment groups

Our previous study (Ng et al., 2003) in anesthetized rats showed increased nitrate/nitrite levels, reduced numbers of degenerating neurons and poor performance in neurological tests in L-NAME or lactate treated-shocked rats Our present study showed similar results

on neurological functions in prolonged hemorrhagic shock conscious rats There was increased brain nitrate/nitrite production 24, 48 and 72 hours after prolonged hemorrhagic shock in saline-treated rats Also, there was an increased brain infarct volume and reduction in cognitive and physical performance evaluated by the rotameric and grip strength tests AG treatment reduced brain nitrate/nitrite levels, brain infarct volume and improved the neurological performance evaluated by the rotameric and grip strength tests while L-NAME did not show protective effect in rats following prolonged conscious hemorrhagic shock rats This result is in line with our previous anesthetized model of hemorrhagic shock

The continual set of experiments was to focus on the effectual relationship between NO and prostaglandin E2 (PGE2), after our first series of experiments showed the important role NO plays in hemorrhagic shock PGE2 is a prostanoid which is up-regulation as a result of an inflammatory response Normal saline-treated prolonged hemorrhagic shock rats served as positive control Semi-quantitative analysis of tissues showed iNOS and COX-2 protein expression was detected in normal saline-treated prolonged hemorrhagic shock rats The levels of brain and plasma nitrate/nitrite and PGE2 were elevated in normal saline-treated prolonged hemorrhagic shock rats Plasma creatinine and GOT (markers for kidney and liver dysfunction), were significantly higher in normal saline-treated prolonged hemorrhagic shock rat The histological examinations that showed

organ damages concurred with the increased levels in creatinine and GOT for normal saline-treated prolonged hemorrhagic shock rats Normal saline-treated hemorrhagic shock rats also showed decrease survival and MABP levels Semi-quantitative analysis of tissues showed iNOS protein was not detected in AG-treated prolonged hemorrhagic shock rats but detected in normal saline- and NS-398-,a known COX-2 inhibitor, treated prolonged hemorrhagic shock rats Tissue COX-2 protein was not detected in AG- and NS-398-treated prolonged hemorrhagic shock rats but detected in normal saline-treated prolonged hemorrhagic shock rats The levels of brain and plasma nitrate/nitrite and PGE2, and plasma creatinine and GOT, were significantly lower in AG-treated prolonged hemorrhagic shock rat group when compared with normal saline-treated prolonged hemorrhagic shock rat group Histological examinations also showed a reduction in organ damage for AG-treated prolonged hemorrhagic shock rats when compared with treated prolonged hemorrhagic shock rats AG-treated prolonged hemorrhagic shock rats significantly increased survival and MABP level when compared with treated prolonged hemorrhagic shock rats

As previously noted, prolonged hemorrhagic shock rats showed a decrease in MABP level, vascular hyporesponsiveness, increase nitrate/nitrite levels, increase organ damages (higher creatinine and GOT levels), and survival rates AG with or without ANGII-treated prolonged hemorrhagic shock rats also showed moderate increase in MABP levels when compared with L-NAME- and SNAP- with or without ANGII-treated prolonged hemorrhagic shock rats The effects of AG treatment on hyporeactivity of ANG II were

reversed in vitro aortic strip prolonged hemorrhagic shock rats Synergy treatment of AG

and ANGII in prolonged hemorrhagic shock rats had significantly decreased