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

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 fluidpercussion 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 i 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 Chapter 6: Role of NOS inhibitors in rats following combined fluid-percussion brain injury and prolonged hemorrhagic shock. Paper Chapter 7: Discussions Paper 1-5 ii 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: 881889, 2004. iii Figure Legend Figure 2.1 Untreated rats showed significant decrease in percentage 17 survival when compared to sham-operated rats. Figure 2.2 Untreated rats showed significant decrease in mean arterial 18 blood pressure when compared with sham-operated rats. Figure 2.3 Untreated rats showed significant decrease in mean heart 18 rate when compared with sham-operated rats. Figure 2.4 Isolated prolonged hemorrhagic shock untreated rat aortic 19 strip showed significant decrease in the amount of contraction when compared with sham-operated rats. Figure 2.5 Isolated prolonged hemorrhagic shock untreated rat aortic 19 strip showed significant decrease in the amount of contraction when compared with sham-operated rats. Figure 2.6 Hemotoxylin and eosin stain of kidney, liver, lung and 21 stomach of sham-operated rats and saline-treated rats. Figure 3.1 Survival percentages in different groups of rats that 32 survived beyond 72 hours. 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 40 TTC staining at different time points (24, 48, 72 hours) after prolonged hemorrhagic shock in rat sections of salinetreated rats. Figure 3.8 Total lesion volumes of different group of rats. 41 Figure 3.9 Rotameric performance in different groups of rats. 42 Figure 4.1 MABP in different groups of rats 54 iv Figure4.2A,B Sham-operated & Normal saline + prolonged hemorrhagic 55,56 shock showing iNOS & COX-2 bands. Figure 4.3A Cortical nitrate/nitrite levels at 24, 48 and 72 hours in 58 different groups of rats. Figure 4.3B Cortical PGE2 levels at 24, 48 and 72 hours in different 58 groups of rats. Figure 4.3C Plasma nitrate/nitrite levels at 24, 48 and 72 hours in 59 different groups of rats. Figure 4.3D Plasma PGE2 levels at 24, 48 and 72 hours in different 59 groups of rats. Figure 4.3E Plasma creatinine levels at 24, 48 and 72 hours in different 60 groups of rats. Figure 4.3F Plasma GOT levels at 24, 48 and 72 hours in different 60 groups of rats. 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- 67 operated rats, normalsaline + prolonged hemorrhagic shock, NS-398 + prolonged hemorrhagic shock & AG + prolonged hemorrhagic shock rats. Figure 5.1 Survival percentages in different groups of rats that 74 survived beyond 72 hours. Figure 5.2 MABP of different groups of rats. Figure 5.3 Isolated prolonged hemorrhagic shock rat 76 aortic strip 77 Isolated prolonged hemorrhagic shock rat aortic strip 77 treated with ANGII. Figure 5.4 shocked treated with noradrenaline. Figure 5.6 Creatinine levels in different groups of rats. 80 Figure 5.7 GOT levels in different groups of rats. 81 v 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 94 groups of rats. Figure 6.4 L-NAME-, AG- and saline-treated rats did not show any 95 significant difference in their nitrate/nitrite levels when compared to sham-operated rats before FPI,HS, FPI+HS. Figure 6.5 During FPI+HS, all treated groups showed significant 96 difference in nitrate/nitrite levels when compared with all treated rats in FPI and HS. Figure 6.6 AG and L-NAME treated rats showed significantly lower 97 nitrate/nitrite levels when compared to saline-treated rats after FPI,HS, FPI+HS. Figure 6.7 Neurons in the cerebral cortex of the brain in sham- 98 operated, saline-, L-NAME- and AG-treated (FPI+HS), (FPI), (HS) rats. Figure 6.8 Light micrographs showing apoptotic cortical neurons in saline-, L-NAME- and AG-treated (FPI+HS), (FPI), (HS) rats. vi 99-100 Table legend Table 2.1 Semi-quantitative analysis of major organ injury of different 22 groups of rats. 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 35 groups of rats. Table 4.1 The different treatment groups are tabulated below. It comprises 47 of sham-operated and prolonged hemorrhagic shock groups. Table 4.2 The number of rats surviving at different time points (24, 48, 72 53 hours). Table 5.1 Semi-quantitative analysis of major organ injury of different 82 groups of rats. Table 6.1 NISSL and TUNEL scores for the different groups of rats in FPI, HS, FPI/HS vii 100 LIST OF ABBREVIATIONS AG Aminoguanidine COX Cyclo-oxygenase CO Carbon monoxide CNS Central nervous system DAO Diamine oxidase DHS Delayed hemorrhagic shock DNA Deoxynucleic acid eNOS Endothelial nitric oxide synthase FPI Fluid percussion injury GIT Gastrointestinal tract GOT Glutamic oxaloacetic transaminase HS Hemorrhagic shock HTS Hypertonic saline BBB iNOS Inducible nitric oxide synthase L-NAME N G -nitro-L-arginine methyl ester MODS Multiple organ dysfunction syndrome PPP PPP BBB NSAIDs Non-steroidal anti-inflammatory drugs NO Nitric oxide NS Normal saline nNOS Neuronal nitric oxide synthase NS-398 (N-[2-(Cyclohexyloxy)-4nitrophenyl]methanesulfonamide PGE BBB BBB Prostaglandin E BBB BBB TBI Traumatic brain injury TUNEL in situ terminal transferase d-UTP nick-end labelling viii 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 ix 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-Larginine methyl ester (L-NAME) and a non-selective inhibitor and S-Nitroso-Nacetylpenicillamine (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. x 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 salinetreated prolonged hemorrhagic shock rat. The histological examinations that showed xi 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 ANGIItreated 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 xii nitrate/nitrite level compared to S-Nitroso-N-acetylpenicillamine (SNAP, an NO donor) treatment, with or without ANG II. GOT level and creatinine levels in AG + ANGII treated animals were reduced compared to both NG-nitro-L-arginine methyl ester (LNAME), a non-selective inhibitor and SNAP-treated hemorrhagic shock rats. Histological examinations also showed a reduction of organ damages and higher survival rates in AG with ANGII-treated prolonged hemorrhagic shock rats when compared with L-NAME and SNAP with or without ANGII-treated prolonged hemorrhagic shock rats. Finally we investigated the role of NO in combined fluid percussion injury and hemorrhagic shock (FPI+HS). Nitrate/nitrite levels were attenuated in AG-treated rats when compared with saline-treated FPI+HS, FPI or HS rats. Immunohistochemical analysis showed a marked number of iNOS immunopositive cells in the cerebral cortex ipsilateral to the injury in saline-treated FPI+HS, FPI or HS rats. Histological findings showed increased numbers of hyperchromatic and shrunken cortical neurons in salinetreated FPI+HS, FPI or HS rats. Decreased MABP and percentage cerebral tissue perfusion (CTF) were shown in saline-treated FPI+HS, FPI or HS rats. Aminoguanidine (AG) significantly decreased the number of hyperchromatic and shrunken cortical neurons when compared with L-NAME- or saline-treated with FPI+HS, FPI or HS rats. In brief, the significant findings of this thesis were that Prolonged hemorrhagic shock worsens physiological parameters, in vitro vascular hyporesponsiveness, organ damages and mortality rate. xiii The reduction in cardiovascular responses, increased organ damages and high mortality rate were attenuated with AG treatment. This may suggest the detrimental role NO play in prolong hemorrhagic shock. The selective NOS inhibitor, AG, might be a potential therapeutic agent in prolonged hemorrhagic shock. It is suggested that NO might be involved in neurological deficit. These findings suggest that NO formation via iNOS activation may contribute to organ damage (brain) and that the iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following prolonged conscious hemorrhagic shock (result is similar to previous study in anesthetized hemorrhagic shock rats). Our present findings suggest that NO produced by iNOS might result in organ damages. This in turn might lead to COX-2 up-regulation and increases the production of reactive oxygen species and toxic prostanoids such as PGE2. NOmediated organ damage might be one way where toxic products of COX-2 might further contribute to NO’s deleterious effect in the later stages of prolonged hemorrhagic shock. The NOS inhibitor, AG, may be of interest as a therapeutic agent in attenuating organ damages when compared with the COX-2 inhibitor, NS-398. Our experiments showed that inhibition of excessive NO formation that occurred during prolonged hemorrhagic shock, had augmented organ damages and induced vascular hyporeactivity of ANGII following prolonged hemorrhagic shock. A xiv greater beneficial effect was achieved via treatment of NOS inhibitor, AG, and ANGII combination. Upregulation of NO might worsen physiological parameters, neuronal cell survival following fluid percussion injury and hemorrhagic shock. It is therefore suggested that treatment of AG via inhibition of iNOS might contribute to improved physiological parameters and neuronal cell survival following fluid percussion injury and hemorrhagic shock. xv Aims of this thesis is to investigate 1. Pathophysiology of prolonged hemorrhagic shock. 2. Role of nitric oxide (NO) in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids, NO donor and NOS inhibitors. 3. Role of nitric oxide (NO) and PGE2 in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids, NOS and COX-2 inhibitors. 4. Role of angiotensin II in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids and NOS inhibitors. 5. Role of NOS inhibitors in a combined model of prolonged hemorrhagic shock and fluid percussion injury. xvi [...]... this thesis is to investigate 1 Pathophysiology of prolonged hemorrhagic shock 2 Role of nitric oxide (NO) in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids, NO donor and NOS inhibitors 3 Role of nitric oxide (NO) and PGE2 in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids, NOS and COX-2 inhibitors 4 Role of angiotensin II in prolonged. .. 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,... 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... 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. .. stages of prolonged hemorrhagic shock The NOS inhibitor, AG, may be of interest as a therapeutic agent in attenuating organ damages when compared with the COX-2 inhibitor, NS-398 Our experiments showed that inhibition of excessive NO formation that occurred during prolonged hemorrhagic shock, had augmented organ damages and induced vascular hyporeactivity of ANGII following prolonged hemorrhagic shock. .. higher in normal salinetreated prolonged hemorrhagic shock rat The histological examinations that showed xi 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. .. NOS inhibitor, AG, might be a potential therapeutic agent in prolonged hemorrhagic shock It is suggested that NO might be involved in neurological deficit These findings suggest that NO formation via iNOS activation may contribute to organ damage (brain) and that the iNOS inhibitor, AG, may be of interest as a therapeutic agent for neurological recovery following prolonged conscious hemorrhagic shock. .. prolonged hemorrhagic shock rats Finally we investigated the role of NO in combined fluid percussion injury and hemorrhagic shock (FPI+HS) Nitrate/nitrite levels were attenuated in AG-treated rats when compared with saline-treated FPI+HS, FPI or HS rats Immunohistochemical analysis showed a marked number of iNOS immunopositive cells in the cerebral cortex ipsilateral to the injury in saline-treated... treatment of NOS inhibitor, AG, and ANGII combination Upregulation of NO might worsen physiological parameters, neuronal cell survival following fluid percussion injury and hemorrhagic shock It is therefore suggested that treatment of AG via inhibition of iNOS might contribute to improved physiological parameters and neuronal cell survival following fluid percussion injury and hemorrhagic shock xv Aims of. .. 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 . 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. following fluid percus sion injury and hemorrhagic shock. xvi A ims of this thesis is to investigate 1. Pathophysiology of prolong ed hemorrhagic shock. 2. Role of nitric oxide (NO) in. (NO) in prolonged hemorrhagic shocked rats and therapeutic effects of conservative fluids, NO donor and NOS inhibitors . 3. Role of nitric oxide (NO) and PGE 2 in prolonged hemorrhagic shocked