The role of nitric oxide and prostaglandin e2 in prolonged hemorrhagic shock 2

1.6 The role nitric oxide and angiotensin II play in prolonged hemorrhagic shock The renin-angiotensin system is one of the major regulators of arterial blood pressure Oudat et al., 200

CHAPTER 1

Introduction

1.1 Definition of hemorrhagic shock

A variety of definitions of hemorrhagic shock have arisen as more understanding of the mechanisms involved has been developed Shock is “a momentary pause in the act of death” (John Warren 1); “Shock is the manifestation of the rude unhinging of the machinery of life” (Samuel V Gross, 1872) A modern definition of shock would acknowledge that, firstly; shock is inadequate tissue perfusion and inadequate removal of cellular waste products and secondly, that shock is a failure of oxidative metabolism that can involve defects of oxygen (1) delivery, (2) transport or (3) utilization or combinations

of all three The diagnoses of clinical signs of shock are primarily related to organ failure but organ failure is secondary to failure of the cells (Pope et al., 1999)

Shock as described by many authors is a “vicious cycle” They may cascade in a variety

of ways such as the decreased in cardiac output, which leads to a decreasing blood pressure, which on turn leads to decreasing tissue perfusion (Pope et al., 1999)

1.2 Organs involvement in prolonged hemorrhagic shock

The organ sequentially affected in the organ failure induced by shock is the kidney Renal failure may ensue as a consequence of shock and depending on the state of volume resuscitation and other factors may have the following characteristic:

Initial high level of urine output Low pressure in the renal tubules producing sodium retention Renal dysfunction and failure

Renal failure is a complication of severe shock and is associated with a mortality rate of more than 50 percent Vigorous fluid resuscitation has improved the situation by reducing

the incidence of renal failure; early and adequate resuscitation can avoid this dreaded consequence of shock (Pope et al., 1999)

The gastrointestinal consequences of shock include increased acid production and increased permeability of the gastric mucosa The increased permeability allows tissue penetration by acids, bacteria and endotoxins In the past, these complications resulted in the late morbidity from hemorrhagic gastritis, which has a high mortality rate (Pope et al., 1999)

The liver, like all other organs, responds to shock The effect on the liver is not well delineated but does result in major changes in bilirubin, isoenzymes, protein synthesis and perhaps most importantly the reticuloendothelial system Decreased consciousness and changes in neural control mechanisms are the responses of the central nervous system to shock (Pope et al., 1999)

1.3 Physiologic responses to prolonged hemorrhagic shock

Acute hemorrhage produces a decrease in arterial systolic, diastolic and pulse pressures along with an increase in the pulse rate and a decrease in the cardiac stroke volume The cutaneous veins are generally collapsed and fill slowly when compressed centrally (Berne, 1983) The early stages of hemorrhage result in the initiation of a number of feedback mechanisms tend to maintain arterial blood pressure in the presence of a decrease in circulating blood volume and a modest decrease in cardiac output Some of the regulatory mechanisms include cerebral ischemic responses, reabsorption of tissue fluids at the level of capillaries, release of endogenous vasoconstrictor substances such as

In the early stages of moderate hemorrhage, the changes in total renal vascular resistance are slight because intrinsic autoregulatory mechanisms within the kidney tend to maintain renal blood flow The intense splanchnic and renal vasoconstriction may protect the heart and brain but can eventually lead to ischemic injury of the kidney and bowel resulting in kidney failure and further vascular injury and loss of fluids from the vascular compartment into the interstitial space (Pope et al., 1999)

When the arterial pressure falls below 60 mmHg as during serve hemorrhage, hypoxia of the peripheral chemoreceptors in the carotid body results in activation of chemoreceptor reflexes This results in increased of breathing frequency At very low levels of arterial pressure at below 40 mmHg, inadequate cerebral blood flow produces an extremely strong activation of the sympathetic nervous system and intense vasoconstriction in response to cerebral ischemia (Pope et al., 1999)

A number of endogenous vasoconstrictors are released during hemorrhage As a direct response to sympathetic nervous system activation, the release of epinephrine and norepinephrine from the adrenal medulla reinforces the actions of direct sympathetic nervous system innervations of the heart and peripheral circulation Vasopressin, which is

a potent vasoconstrictor, is actively secreted by the posterior pituitary gland in response

to hemorrhage Diminished renal perfusion results in the secretion of rennin from the juxtaglomerular apparatus and the subsequent conversion of angiotensinogen to angiotensin, which is also a powerful vasoconstrictor (Pope et al., 1999)

1.4 The role Nitric Oxide play in prolonged hemorrhagic shock

Excessive production of nitric oxide (NO) as result of inducible nitric oxide synthase (iNOS) induction has been implicated as the most important factor contributing to the pathophysiology of hemorrhagic shock (Szabo et al., 1994; Moncada et al., 1991; Szabo, 1995) The induction of iNOS in turn metabolizes L-arginine, resulting in excessive formation of NO that may contribute to the vascular impairment and multiple organ damage (Hua et al., 1999)

In recent years, numerous efforts and studies have aimed to evaluate the potential of NOS inhibitors in maintaining mean arterial blood pressure (MABP) and increasing the survivability of the shocked animals NOS inhibitors have been shown to be able to maintain a high MABP, after shock was induced, by antagonizing the vasodilatating effects of NO by inhibiting their release (Szabo et al., 1994; Moncada et al., 1991; Szabo, 1995) A high MABP theoretically would maintain curial organ perfusions and would in turn reduced occurrences of organ ischemia

in rats subjected to hemorrhagic shock had significantly increased in control rats citrulline is an indicator of nitric oxide (NO) synthesis Thus these findings indicate that

L-NO production in these areas contributes to the hypotension due to hemorrhage (Goren et al., 2001) Maintenance of NO production by endothelia NOS (eNOS) is important in early stages of ischemia and its inhibition could exacerbate organs injury (Guo Weir

1999, Dawson and Dawson 1996) Patients that have survived severe hemorrhagic shock are known to show neurological changes likely due to brain ischemia (Carrillo et al.,

(iNOS) in the brain (Dalkara et al., 1994; Weir et al., 1999; Liaudet et al., 2000; Szabo and Thiemermann, 1994) has been shown to play an important role in secondary neuronal damage (Iadecola 1995) The neuroprotective properties of selective NOS inhibitors arise from their ability to inhibit the mass release of NO after brain injury (Iadecola 1995; Zhang et al., 1996; Cash et al., 2001; Viktorov, 2000; Higuchi et al., 1998)

1.5 The role nitric oxide and prostaglandin E 2 in prolonged hemorrhagic shock

Hemorrhagic shock produces the bioregulatory molecule nitric oxide (NO) which is generated catalytically by three enzymes (constitutive, neuronal and inducible) collectively termed NO synthase (Teng and Moochhala, 1999) Previous studies have shown that the inflammatory (inducible nitric oxide synthase) iNOS is upregulated in organs such as lungs, livers and kidneys during shock (Thiemermann et al., 1993, Anaya-Prado et al., 2003, Mc Donald et al., 2003) The excessive activation of iNOS results in cardiovascular and organ dysfunction in clinical and experimental setting of inflammatory disease of both septic and nonseptic etiology (Ungureanu-Longrois et al.,

1995, Harbrecht et al., 1992, Petros et al., 1995, Vallance and Moncada, 1993, Grosjean

et al., 1999, Collins et al., 2003, Menezes et al., 2003, Hierholzer et al., 2002, Liu et al.,

2002, Cuzzocrea et al., 2002) The inducible NOS are one of the inflammatory mechanisms that contribute to cerebral damage (Pozzilli et al., 1985, Clark et al., 1995, Chen et al., 1992, 1994, Feuerstein et al., 1998, Iadecola, 1997)

Some investigators have shown that during hemorrhagic shock, cyclooxgenase-2 2) is up-regulation as a result of an inflammatory response (Tsukada et al., 2000, Knoferl

(COX-et al., 2001) Two isoforms of COX have been identified namely COX-1 and COX-2

COX-1 is a constitutive isoform that is expressed in most tissues and is responsible for the physiological production of PGs On the other hand, COX-2 is an inducible isoform that is induced by cytokines, mitogens, and endotoxins in inflammatory cells and is responsible for the elevated production of PG during inflammation (Dubois et al., 1998, Iadecola, 1997) Prostanoids, including prostaglandins (PGs), prostacyclins, and thromboxanes, are synthesized from these enzymatic pathways (Murakami et al., 1997, Vane et al., 1998, Smith et al., 2000) Cerebral damage also enhances the expression of COX-2 (Nogawa et al., 1997, Miettinen et al., 1997, Planas et al., 1995, Collaco-Moraes

et al., 1996, Goodwin et al., 1999) In addition to their role in inflammation, prostanoids have also been shown to modulate vasodilation (Okamoto et al., 1998, Moncada et al., 1993)

The inhibition of NO production that could possibly alter the vasodilatory and

documented Interestingly, studies have shown that pharmacological manipulation of one pathway could result in cross-modulation of the other pathway However the relevance of

these interactions in vivo is controversial The interaction between NO and COX-2 is

likely to play a role in brain diseases associated with inflammation, such as AIDS dementia, multiple sclerosis, brain neoplasm and Alzheimer disease and other pathological conditions such as nephrosis, sepsis or rheumatoid arthritis (Salvemini et al.,

1993, Nogawa et al., 1998)

AG is well known as an iNOS inhibitor but little is known of its ability to inhibit COX-2 up-regulation via NO inhibition in prolong hemorrhagic shock Our experiment might

shed some light on the interaction between NO and COX-2 in Prolong hemorrhagic shock

1.6 The role nitric oxide and angiotensin II play in prolonged hemorrhagic shock

The renin-angiotensin system is one of the major regulators of arterial blood pressure

(Oudat et al., 2003) as it is the most potent pressor substance known (Chesley et al.,

1963) In contrast to ANGII function, under physiological conditions, generation of NO from L-arginine by the constitutive NO synthase (NOS) present in vascular endothelial cells keeps the vasculature in a permanent state of active vasodilatation (Rees et al., 1989)

Studies have shown that during the event of shock, an inducible isoform of NO synthase (iNOS) is expressed, resulting in excessive formation of nitric oxide (NO) that may contribute to the vascular impairment (Ochoa et al., 1991) Animal studies have also suggested that nitric oxide (NO) overproduction may mediate vascular hyporeactivity and decompensation following hemorrhagic shock (Thiemermann et al., 1993 Zingarelli et al., 1992) We hypothesize that reduced sensitivity to angiotensin II is a result of excessive NO formation We also hypothesized that treatment of ANGII with NOS inhibitors, would have a beneficial effect on the blood pressure following prolonged hemorrhagic shock

1.7 The role nitric oxide plays in a combine rat model of prolonged hemorrhagic shock and fluid percussion injury (induction of traumatic brain injury)

Nitric oxide (NO) has been implicated as being an important mediator in a variety of

hemorrhagic shock (Hua and Moochhala, 1999) The role of NO in maintaining homeostasis (Ambrosio et al., 1998) and regulating organ function during traumatic brain injury and hemorrhagic shock is complex The inducible NO synthase (iNOS) has been hypothesized to play a critical role in the pathophysiologic consequences of secondary brain injury and severe hemorrhage During traumatic brain injury (Sinz et al., 1999) and hemorrhagic shock (Szabo and Billiar 1999, Shinoda and Whittle, 2001), there is induction of iNOS The iNOS metabolizes L-arginine resulting in large amounts of nitric oxide (NO) production, which might lead to vascular hypotension and multiple organ damage following hemorrhagic shock (Goren et al., 2001) In the brain, iNOS is produced in large amounts by macrophages and microglia following traumatic brain injury (Shinoda and Whittle, 2001) iNOS-derived NO is acutely detrimental, possibly because of toxic effects of NO metabolites such as peroxynitrite Excessive production of

NO may also be involved in glutamate neurotoxicity and is responsible for neuronal death (Szabo, 1995) It has been shown that prolonged exposure to relatively high concentrations of NO and superoxide ion, produced apoptosis and necrosis in certain

In recent years, the effect of iNOS inhibitors have been studied extensively in various injury and shock models due to their ability to inhibit the excessive release of NO under pathological conditions (Fink 1999, Teng & Moochhala, 1999).One in particular, aminoguanidine (AG), has shown beneficial effects following traumatic brain injury

(Gorlach et al., 2000) or hemorrhagic shock (Teng & Moochhala, 1999)

CHAPTER 2

The pathophysiology of prolonged

hemorrhagic shock (HS)

2.1 Introduction

Hemorrhagic hypotension leads to a decrease in cardiac output and blood pressure (Bond and Johnson, 1985) After prolonged hemorrhage, the ability of the organism to maintain blood pressure and organ perfusion is limited by a progressive loss of responsiveness of the vasculature As a result, blood pressure further declines and becomes refractory to volume substitution which irreversibly leads to death

We investigated the effects of anesthetized rats in fixed pressure model in on survival, mean arterial blood pressure (MABP), heart rate, vascular hyporeactivity, biochemical

analysis and organ damages

2.2 Methods 2.2.1 Animals and reagents

Male Sprague-Dawley rats weighing 300-350 g were used They were acclimatized for at least a week before the experiment and had free access to standard laboratory chow and water Each study was carried out using 8 rats per group The experiment described in this article was performed in adherence to the guidelines of Council for International Organization of Medical Sciences (CIOMS) ethical code for animal experimentation (Howard- Jones, 1995)

Sodium chloride solution (0.9%) was obtained from Sigma Chemical (Sigma, St Louis, MO) Clinical Research Centre (CRC) cocktail consisted of 1 mL of hypnorm (Jansen

Pharmaceutica, Beerse, Belgica), which contained 0.315 mg of fentanyl and 10 mg of

fluanisone, and 1 mL of midazolam (Roche, Basel, Switzerland) which contained 5 mg

heparin sodium (Leo Pharmaceuticals, Ballerup, Denmark) per mL of normal saline Ten percent (10%) neutral buffered formalin consisted of 3.5 g anhydrous sodium dihydrogen phosphate (Sigma) and 6.5 g anhydrous disodium phosphate (Sigma) in 100 mL of 40%

formaldehyde (Merck) and 900 mL of distilled water

2.2.1.2 Animal preparation in rats

The animals were deprived of food for 24 hours before the experiment but allowed free access to water They were anaesthetized with CRC cocktail (0.3 mL/l00 g body weight) intraperitoneally and were maintained under anaesthesia for the duration of the experiment The body temperature was monitored by rectal thermometer and maintained

England) The right carotid artery was exposed by blunt dissection between associated muscles The adjacent vagus nerve was carefully dissected away from the artery A heparinized 24G X 1.90 cm over-the-needle catheter (Terumo Corporation, Tokyo, Japan) was inserted into the carotid artery, and a three-way stopcock was attached in-line for monitoring the mean arterial blood pressure (MABP), withdrawal of blood, and administration of drugs The MABP was monitored using a blood pressure (BP) transducer (BP TRN 050, Kent Scientific, USA) and recorded by a one-channel amplifier (TRN 005, Kent Scientific, USA), which was calibrated with a pressure transducer simulator (BP TRN 051, Kent Scientific, USA) The blood pressure data was recorded

using DBP 001, direct blood pressure data acquisition system

2.2.1.3 Prolonged hemorrhagic shock in rats (fixed pressure)

After a stabilizing period of 15 min, prolong hemorrhagic shock was induced by withdrawing blood in 5 min period until the MABP decreased to 40-45 mmHg, causing a hypotension that lasted for 3 hours The pressure was maintained at this level for 3 hours

by withdrawing or reinfusing of shed blood as required The total amount of blood withdrawn was then recorded Sham-operated animals underwent the same procedure except for the withdrawal of blood After 3 h of hypotension, without reinfusion of shed blood, the rat received intravenously 0.9%sodium chloride solution (injection volume: 1

mL/kg) Sham-operated animals received an equal volume of normal saline (1 mL/kg)

2.2.2 Mean arterial blood pressure (MABP) and survival time in rats

Mean arterial blood pressure was monitored for 60 min after administration of drugs

(this is taken as the resuscitation period) For surviving animals, the catheter was

removed, artery ligated, and incision closed in two layers with chromic catgut (B Braun,

Melsungen AG, Germany) Once the rats recovered from the anesthesia, they were returned to individual cages and allowed access to food and water

2.2.3 Blood collection in rats

Blood samples of 1 mL were collected before shock, 3 hours after prolong hemorrhagic shock, and 60 min after the administration of drugs The blood collected was accounted

in the total hemorrhage volume The withdrawn blood (3 X 1 mL) was replaced by normal saline and shed blood for sham-operated rats and shocked rats, respectively The

blood was centrifuged at 3,000 rpm for 10 min to obtain platelet-rich plasma, and

2.2.4 Biochemical analysis in rats

Creatinine was determined using a Sigma Diagnostic kit, and the creatinine concentration was expressed in SI units ( mol/L) Glutamic oxalacetic transaminase (GOT) was measured using the Sigma Diagnostic kit and the activities were expressed in Sigma –

activities reflect the impairment of kidney and liver respectively

2.2.5 Isolated Rat Aortic Strip in rats

Adult male Sprague Dawley rats were sacrificed after the end of the experiment The descending thoracic aorta was removed, avoiding the fats and connective tissues and placed in a petri dish of oxygenated Krebs solution The whole length of the aorta was cut from the upper part of about 1cm into 2 pieces Each piece was then cut along a close spiral with a width of about 2-3mm One end of the strip was tied with surgical thread to

an L-tube and mounted in a 2ml organ bath filled with Krebs solution, oxygenated with 95% 02 + 5% C02 at 37'C The other end was tied using surgical thread and attached to a

Ugo Basile Isometric force transducer, mechanical force range 0- 10g.The resting tension

on the tissues were adjusted to 3g They were left to stabilize for 10-15min All the

contractions were recorded by an 8-channel MacLab 8S (AD Instruments) coupled to an Apple-Macintosh computer running a data acquisition software program Chart 4.5 (AD

Instruments) The strips were pre-contracted with 0.3pM noradrenaline bitartrate salt (Sigma Chemical Co.) till it plateau and 0.2 M of acetylcholine Iodide (Sigma Co.) was added in the organ bath until full relaxation, then washed The tissues were drip washed for 10min Next 80mM of potassium chloride was used to contract the strips for 10min and washed After l0 min interval, the tissues were exposed to the same dose of potassium chloride again Lastly cummulative doses of angiotensin II, acetate salt (Sigma Chemical Co.) were added to the organ bath for maximum contraction

2.2.6 Histology in rats

Upon the death of animals or 72 hours after the experiment, the heart, lung, liver, stomach, spleen and kidney were obtained and recorded for any macroscopic damage They were fixed in 10% neutral buffered formalin and processed for light microscopy Sections of 5 M thick were cut in a standard fashion, stained with hematoxylin and eosin, and examined for microscopic injuries The sections were quantified and injuries were graded as: - none; +/- occasionally; + slight; ++ moderate; +++ severe by an

examiner blinded to the studies

2.2.7 Statistical analysis

The results were expressed as mean ± standard error of means (SEM) Statistical analysis was performed using the Statistical Package for Social Science for Windows (SPSSWIN) One-way analysis of variance (ANOVA) with Post HOC test (LSD) was performed to

by a single trained “blind” operator to ensure uniformity while handling the slides or

animals respectively

2.3 Results

2.3.1 Effect of prolonged hemorrhagic shock Percentage Survival

All the sham-operated rats survived for more than 72hours while all untreated rats died within 36 hours

Figure 2.1 Untreated rats showed significant decrease in percentage survival when

3hours prolonged hemorrhagic shock vs Sham-operated + 3hours prolonged hemorrhagic

shock)

2.3.2 Effect of prolonged hemorrhagic shock on Mean arterial blood pressure (MABP) & mean heart rate

The volume of blood withdrawn did not differ between groups (p>0.05) In

compared with sham-operated rats

200 225 250 275 300 325 350 375

3 hours prolong hemorrhagic shock

significantly lower compared with sham-operated rats

Figure 2.2 Untreated rats showed significant decrease in mean arterial blood pressure

(Untreated + 3hours prolonged hemorrhagic shock vs Sham-operated + 3hours prolonged

hemorrhagic shock)

Figure 2.3 Untreated rats showed significant decrease in mean heart rate when

3hours prolonged hemorrhagic shock vs Sham-operated + 3hours prolonged hemorrhagic

0 10 20 30 40 50 60 70 80 90 100

2.3.3 Effects of prolonged hemorrhagic shock on Vascular hyporeactivity

In prolonged hemorrhagic shock untreated rats, vascular contractility of the aortic strips towards cumulative dose response of angiotensin II and noradrenaline had reduced by 50% and 34% respectively when compared to sham-operated rats

Figure 2.4 Isolated prolonged hemorrhagic shock untreated rat aortic strip showed

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

shock vs Sham-operated + 3hours prolonged hemorrhagic shock)

Figure 2.5 Isolated prolonged hemorrhagic shock untreated rat aortic strip showed

rats Data shown as Mean SEM * p < 0.05 (Untreated + 3hours prolonged hemorrhagic

shock vs Sham-operated + 3hours prolonged hemorrhagic shock)

2.3.4 Morphological evaluation

The kidneys (Figure 2.6A), livers (Figure 2.6C), lungs (Figure 2.6E) and stomach (Figure 2.6G) in sham-operated rats appeared structurally normal Severe microscopic injury was encountered in various organs of rats following prolonged hemorrhagic shock in untreated rats There was evidence of leakage of blood and tissue damages in the kidneys, livers, lungs and stomachs Significant histopathological alterations were seen in tissue sections of HS kidney that showed loss of cellular infiltration and glomerular edema (Figure 2.6B) The injured liver was also characterized by widening of sinusoids of most hepatocytes (Figure 2.6D) There was evidence of massive cellular infiltration in the lung (Fig 2.6F) Tissue sections of stomach showed extensive exfoliation with necrotic lesions

of surface epithelium (Figure 2.6H)

We observed that in sham-operated rats, the various organs were structurally normal There were severe organ damages observed in untreated rats The severity of organ

damages was graded and summarized in Table 2.1

Figure 2.6 Hemotoxylin and eosin stain of kidney (A), liver (C), lung (E) and stomach (G) of sham-operated rats Leakage of blood and infiltration of lymphocytes (arrow-tips) into extracellular spaces of major organs were observed in untreated rats

Table 2.1 Grading of the severity of organ damages in different groups of rats

operated rats

2.3.5 Creatinine & Glutamic oxalacetic transaminase (GOT) determination (1 h post prolonged hemorrhagic shock)

with non-operated rats However, a marked increase in plasma creatinine level was found

non-operated rat plasma No alteration of GOT activity was evidenced in sham-operated

higher GOT level (148.42 2.36 SF)

Table 2.2 Creatinine and GOT levels in different groups of rats

Creatinine ( mol/L) GOT (SF Units/ml)

2.4 Discussions – Significant results

showed low blood pressure and cardiac output respectively

prolonged hemorrhagic shocked aortic strip rats

3.1 Introduction

Hemorrhagic shock is implicated in the induction of inducible nitric oxide synthase that leads to increase production of nitric oxide (NO) We investigated the effects of NO in two rat models of hemorrhagic shock The fixed pressure model in anesthetized rats on survival, mean arterial blood pressure (MABP), heart rate, vascular hyporeactivity, biochemical analysis and organ damages For the fixed volume model in conscious rats,

we focused on brain lesions and neurologic deficit We wanted to compare whether there was a huge difference in neurological deficit between our previous study (Ng et al., 2003) where we used anesthetized rats and the present one where conscious rats were

experimentalzed

3.2 Methods 3.2.1 Animals and Reagents

Male Sprague-Dawley rats (250-300 g) were used for this study The rats were subjected

to the following experimental procedures: Group 1: Sham-operated (rats underwent the surgical procedure without prolonged hemorrhagic shock): Group 2: Saline + prolonged hemorrhagic shock; Group 3: L-NAME + prolonged hemorrhagic shock; Group 4: AG +

prolonged hemorrhagic shock: Group 6: S-Nitroso-N-acetylpenicillamine (SNAP)(

chloride solution (Sigma) The handling and care of all animals were mentioned in chapter 2

3.2.1.2 Animal preparation in anesthetized rats

The animals were deprived of food for 24 hours before the experiment but allowed free access to water They were anaesthetized with CRC cocktail (0.3 mL/l00 g body weight) intraperitoneally and were maintained under anaesthesia for the duration of the experiment The body temperature was monitored by rectal thermometer and maintained

England) The right carotid artery was exposed by blunt dissection between associated muscles The adjacent vagus nerve was carefully dissected away from the artery A heparinized 24G X 1.90 cm over-the-needle catheter (Terumo Corporation, Tokyo, Japan) was inserted into the carotid artery, and a three-way stopcock was attached in-line for monitoring the mean arterial blood pressure (MABP), withdrawal of blood, and administration of drugs The MABP was monitored using a blood pressure (BP) transducer (BP TRN 050, Kent Scientific, USA) and recorded by a one-channel amplifier (TRN 005, Kent Scientific, USA), which was calibrated with a pressure transducer simulator (BP TRN 051, Kent Scientific, USA) The blood pressure data was recorded

using DBP 001, direct blood pressure data acquisition system

3.2.1.3 Animal preparation in conscious rats

Presurgical preparation of animals, anesthesia used and blood pressure recording system are the same as mentioned above Polyethylene catheters were inserted into the right carotid artery for arterial blood withdrawal/monitoring and the inferior vena cava via the right femoral vein for administration of fluids and drugs After cannulation at the artery, the distal end of the cannula is tunneled under the skin to exteriorize at the nape of the

neck The cannulas were held in place with dental cement and stoppered with a small metal pin Rats were allowed 48 hours to recover before they were subjected to prolong

hemorrhagic shock

3.2.3.1 Prolonged hemorrhagic shock in anesthetized rats (fixed pressure)

Stabilizing period, induction of shock and administration of drugs were described in

chapter 2 Sham-operated animals received an equal volume of normal saline (1 mL/kg)

3.2.3.2 Animal preparation in conscious rats

Presurgical preparation of animals, anesthesia used and blood pressure recording system are the same as mentioned above Polyethylene catheters were inserted into the right carotid artery for arterial blood withdrawal/monitoring and the inferior vena cava via the right femoral vein for administration of fluids and drugs After cannulation at the artery, the distal end of the cannula is tunneled under the skin to exteriorize at the nape of the neck The cannulas were held in place with dental cement and stoppered with a small metal pin Rats were allowed 48 hours to recover before they were subjected to prolonged hemorrhagic shock

3.2.3.3 Prolonged hemorrhagic shock in conscious rats (fixed volume)

The stabilization period and withdrawal of blood are the same as mentioned above The total amount of blood withdrawn was kept constant (volume of blood = 8ml) Surgical

infusion of 0.9%sodium chloride solution (injection volume: 1 mL/kg) are the same as mentioned above

3.2.4 Mean arterial blood pressure (MABP) and survival time in anesthetized rats

Monitoring of Mean arterial blood pressure & administration of drugs were followed in

the protocol mentioned in chapter 2 Recovering rats were returned to individual cages and allowed access to food and water

3.2.5 Blood collection in anesthetized rats

Blood samples of 1 mL were collected before shock, 3 hours after prolonged hemorrhagic shock, and 60 min after the administration of drugs The blood was

analysis

3.2.6 Biochemical analysis in anesthetized rats

The stable end products of NO metabolism, i.e., nitrate and nitrite, were measured using the nitrate/nitrite assay kit based on the Griess reaction (Cayman Chemical, Ann Arbor,

MI Detection limit: 5 M) Creatinine & Glutamic oxalacetic transaminase (GOT) (reflect the impairment of kidney and liver respectively) was measured using the Sigma Diagnostic kit S.I units used were mentioned in chapter 2

3.2.7 Brain nitrate/nitrite in conscious rats

Brain nitrate/nitrite contents were measured at the later stage of ischemia, 24, 48 and 72 h after prolong hemorrhagic shock The rat brains were homogenized in 400 l of distilled

nitrites: 50 l of supernatant were incubated for 1 h in the dark with 20 l of 0.31 M potassium phosphate buffer (pH 7.5), 10 l of 0.86 mM -NADPH (Sigma), 10 l of

0.11 mM FAD in the presence of 20 mU nitrate reductase (Roche Diagnostics, Meylan,

tissue nitric oxide end products, nitrates plus nitrite, was then determined by Griess reaction (Green et al., 1982) Proteins in the supernatant were assayed with bovine serum

albumin as standard (Bradford, 1976) Data expressed as pmol /mg protein

3.2.8 Histology in anesthetized rats

Upon the death of animals or 72 hours after the experiment, all organs were fixed, stained and sectioned as described in chapter 2 The sections were quantified and injuries were graded as: - none; +/- occasionally; + slight; ++ moderate; +++ severe by an

examiner blinded to the studies

3.2.9 2,3,5,-triphenyltetrazolium chloride, TTC in conscious rats

At different time points (24, 48, 72 hours) after prolonged hemorrhagic shock, the

placed in 10% buffered formaldehyde Six serial coronal sections from each brain were cut at 2 mm intervals beginning at 3.7 mm from the bregma using a rodent brain matrix

(Harvard matrix, USA)

The sections were individually photographed digitally and the area of lesion was outlined

by using a computer image analysis system (Image Pro Plus version 4.1; Media Cybernetics, Silver Spring MD) Total lesion volumes, in cubic millimeters, were calculated by the integration of the distance of the chosen sections The presence of cerebral edema was corrected by dividing the total lesion volume by the ratio of the total right hemisphere/left hemisphere volume This method assumed equalized hemisphere before prolong hemorrhagic shock and the absence of significant edema in the left

3.2.10 Neurological Tests in conscious rats

After hemorrhagic shock, the surviving animals underwent physical performances tests (Ng et al., 2003; Lu et al., 2003) Saline resuscitated rats acted as the active comparator Rotameric test were performed using a rotameric device (Columbus Instruments Rotamex 4/8 system, Ohio, USA) to examine the locomotory coordination ability of the animal while being placed on a rotating rod Forelimb grip strength was determined using a grip strength meter (Columbus Instruments, Ohio, USA) The animals were placed on the electronic digital force gauge that measured the peak force exerted by the action of the animal The highest reading of three successive trials over the 3-day monitoring period was taken for each animal

3.2.11 Total lesion volumes in conscious rats

At different time points (24, 48, 72 hours) after prolonged hemorrhagic shock, six serial coronal sections from each brain were cut at 2 mm intervals beginning at 3.7 mm from

the bregma using a rodent brain matrix (Harvard matrix, USA)

The area of lesion was calculated as described in chapter 2

3.2.12 Statistical analysis

The results were expressed as mean ± standard error of means (SEM) Statistical analysis

were conducted as mentioned in chapter 2

0 10 20 30 40 50 60 70 80 90 100

Sham

SNP

All sham-operated animals survived for more than 72 h (100%) In contrast, animals subjected

to prolonged hemorrhagic shock (saline-treated) had a 100% mortality rate (death was within

180 min, <72 h) Administration of AG had significantly increased (p < 0.05) the survival

percentage when compared with L-NAME- and SNAP-treated rats (Figure 3.1)

Figure 3.1 Survival percentages in different groups of rats that survived beyond 72 hours Sham-operated rats survived for more than 72 h (100%) All prolonged hemorrhagic shock rats died within 180 min (<72 hours) thus its data was not shown AG-treated rats showed 52% (>72 h) percentage survival A significant difference was observed (p<0.05) when compared to all treated groups of rats (+denotes p<00.05 for L-NAME- and SNAP-treated rats vs saline-treated rats, * denotes p < 0.05 for AG vs all treated groups of rats)

3.3.2 Effect of prolonged hemorrhagic shock on Mean arterial blood pressure (1 h our post-prolonged hemorrhagic shock) in anesthetized rats

experimental period There was no significant difference in MABP among the rat groups

+

+

prior to prolonged hemorrhagic shock (data not shown) There was no MABP recording post-prolonged hemorrhagic shock in saline-treated rats as the rats died within this prolonged hemorrhagic shock period The MABP for all pre-treatment rat groups was

mmHg, 60 min post-prolonged hemorrhagic shock In contrast, AG treatment gradually

which were significantly lower than all treated groups of rats (Figure 3.2)

Figure 3.2 MABP of different groups of rats (1 hour post-prolong hemorrhagic shock) L-NAME-treated rats restored MABP rapidly at the point of administration AG-treated rats gradually raise the MABP to about the same level as L-NAME 60 min after administration SNAP-treated rats showed significant reduction when compared with all treated groups of rats (* denotes p<0.05 for AG vs SNAP; + denotes p<0.05 for L-NAME

vs all treated groups of rats)

3.3.3 Effect of prolonged hemorrhagic shock on Morphological evaluation in anesthetized rats

The kidneys, livers, lungs and stomach in sham-operated rats appeared structurally normal In saline-treated prolonged hemorrhagic shock rats, severe organ damage were observed All these are described in chapter 2

AG showed minimal organ damages when compared with all treated rat groups The

severity of organ damages was graded and summarized in Table 3.1

Table 3.1 Grading of the severity of organ damages in different groups of rats

Kidneys Livers Lungs Stomachs

3.3.4 Effect of prolonged hemorrhagic shock on Nitrate/nitrite determination (post- prolonged hemorrhagic shock) in anesthetized rats

significant difference (p > 0.05) between sham-operated and non-operated rats The plasma nitrate/nitrite production was significantly increased in saline-treated prolonged

production of nitrate/nitrite when compared with sham-operated rats A lower nitrate/nitrite production was also observed in AG-treated rats but did not show any significant difference (p > 0.05) with sham-operated rats Similarly, SNAP increased the

nitrate/nitrite production (1.34 0.08 M) (Figure 3.3)

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

N

mal

Sha

m-ora d

Sal

L-N

AME

SNP

rats)

3.3.5 Effect of prolonged hemorrhagic shock on Creatinine determination (1 hour post- prolonged hemorrhagic shock) in anesthetized rats

creatinine levels in sham-operated animals (261.66 75.14 mol/L) when compared with normal rats However, a marked increase in plasma creatinine level was found in saline-

attenuated the production of creatinine during prolonged hemorrhagic shock Plasma creatinine levels were elevated in prolonged hemorrhagic shock rats receiving L-NAME- and SNAP-treated rats when compared with AG-treated rats (Figure 3.4)

Nor

mal

Sham-ope

rated

3.3.6 Effect of prolonged hemorrhagic shock Glutamic oxalacetic transaminase (1

h our post-prolonged hemorrhagic shock) in anesthetized rats

normal rat plasma No alteration of GOT activity was evidenced in sham-operated rats

hemorrhagic shock rats after receiving AG In contrast, L-NAME- and SNAP- treatments showed an increased GOT activity in prolonged hemorrhagic shock rats (Figure 3.5)

hemorrhagic shock) AG-treated rats showed significant difference (p<0.05) in creatinine levels when compared with all treatment groups (* p < 0.05: AG vs all treatment groups

of rats)

Nor

mal

Sham-ope

compared with all treatment groups (* p < 0.05: AG vs all treatment groups of rats)

3.3.7 Effect of prolonged hemorrhagic shock on brain nitrate/nitrite in conscious rats

Brain nitrate/nitrite content in sham-operated (24 h – 3.5 0.8 pmol/mg protein, 48 hours – 3.2 0.7 pmol/mg protein, 72 hours – 3.6 0.4 pmol/mg protein) was not different from that of normal rat group Brain nitrate/nitrite levels showed a marked increase at 24, 48 and 72 hours following prolonged hemorrhagic shock in saline (24 hours – 6.4 0.9 pmol/mg protein, 48 hours – 6.9 0.8 pmol/mg protein, 72 h – 5.4 0.6 pmol/mg protein) treated rats when compared with AG (24 h – 3.8 0.6 pmol/mg protein, 48 hours – 4.1 0.4 pmol/mg protein, 72 hours – 3.4 0.3 pmol/mg protein) - and L-NAME (24 hours – 2.5 0.5 pmol/mg protein, 48 h – 2.6 0.3 pmol/mg protein, 72 h – 1.9 0.8 pmol/mg protein) -treated rats (Figure 3.6)

Figure 3.6 Nitrate/nitrite content of rat brain in different groups of rats AG- and L- NAME- treated rats show significant reduction in nitrate/nitrite levels when compared with saline-treated rats at 24 and 48 and 72 hours following prolonged hemorrhagic

3.3.8 Effect of prolonged hemorrhagic shock on total brain lesion in conscious rats

Schematic diagrams of lesion brain sections are represented using the saline-treated group

at different time points (24, 48, 72 hours) after prolonged hemorrhagic shock The lesion area is enclosed within a black line (Fig 3.7)

0 1 2 3 4 5 6 7 8 9

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 saline-treated rats (A) 24 hours after prolonged hemorrhagic shock; (B) 48 hours after prolong hemorrhagic shock; (C) 72 hours after prolonged hemorrhagic shock

Lesion region bounded by a black line Sections are at 2mm intervals in descending order (anterior to posterior) beginning at the top row 3.7 mm from the bregma

A) 24 hours after prolonged hemorrhagic shock;

B) 48 hours after prolong hemorrhagic shock;

C) 72 hours after prolong hemorrhagic shock;