|Year : 2015 | Volume
| Issue : 1 | Page : 12-17
Effect of aminoguanidine on cardiovascular responses and survival time during blood loss: A study in normotensive and deoxycorticosterone acetate-salt hypertensive rats
Babak Barmaki1, Majid Khazaei2
1 Department of Physiology, Zabol University of Medical Sciences, Zabol, Iran
2 Department of Physiology, Isfahan University of Medical Sciences, Isfahan; Department of Physiology, Mashhad University of Medical Sciences, Mashhad, Iran
|Date of Submission||04-Nov-2013|
|Date of Acceptance||18-Jul-2014|
|Date of Web Publication||13-Jan-2015|
Department of Physiology, Isfahan University of Medical Sciences, Hezar Jarib Ave., Isfahan
Source of Support: The Isfahan University of Medical Sciences supported
this study (Grant No: 387413), Conflict of Interest: None
| Abstract|| |
Introduction: Hemorrhagic shock causes more circulatory disturbances and mortality in hypertensive than normotensive subjects. In the late phase of hemorrhagic shock, nitric oxide (NO) overproduction leads to vascular decompensation. In this study, we evaluated the effect of inducible NO synthase (iNOS) inhibitor, aminoguanidine (AG), on hemodynamic parameters and serum nitrite concentration in decompensated hemorrhagic shock model in normotensive and hypertensive male rats. Materials and Methods: Twenty-four male rats were divided into hypertensive and normotensive groups (n = 12 each). Hypertension was induced by subcutaneous injection of deoxycorticoesterone acetate (DOCA), 30 mg/kg in uninephrectomized rats. Decompensated hemorrhagic shock was induced by withdrawing blood until the mean arterial pressure (MAP) reached 40 mmHg. After 120 min, each group was assigned to aminguanidine (100 mg/kg) and control group. Hemodynamic parameters were monitored for next 60 min. Blood samples were taken before and after shock period and 60 min after treatment. Survival rate was monitored for 72 h. Results: Infusion of AG in normotensive animals caused a transient increase in MAP and increase of heart rate, whereas it did not affect those parameters in hypertensive animals. Hemorrhagic shock caused a significant rise in serum nitrite concentration in normotensive and hypertensive rats and infusion of AG did not significantly change it in both groups. No significant differences observed in survival rate between AG-treated and not treated groups. Conclusion: It seems that inhibition of iNOS with AG does not have beneficial effects on hemodynamatic parameters and survival rate during decompensated hemorrhagic shock in normotensive and hypertensive animals.
Keywords: Hemorrhagic shock, hypertension, nitric oxide
|How to cite this article:|
Barmaki B, Khazaei M. Effect of aminoguanidine on cardiovascular responses and survival time during blood loss: A study in normotensive and deoxycorticosterone acetate-salt hypertensive rats. Int J App Basic Med Res 2015;5:12-7
|How to cite this URL:|
Barmaki B, Khazaei M. Effect of aminoguanidine on cardiovascular responses and survival time during blood loss: A study in normotensive and deoxycorticosterone acetate-salt hypertensive rats. Int J App Basic Med Res [serial online] 2015 [cited 2020 May 31];5:12-7. Available from: http://www.ijabmr.org/text.asp?2015/5/1/12/149222
| Introduction|| |
Hypertensive patients have higher vascular resistance and sympathetic tone than normotensive ones and may demonstrate aberrant responses to bleeding.  Hemorrhagic shock induction with withdrawing 25% of total blood volume in spontaneous hypertensive rats causes more blood pressure (BP) depression and acidosis in hypertensive compared with normotensive animals. ,
Nitric oxide (NO) over production after hemorrhagic shock has been documented. ,,,, NO has several cardiovascular effects including regulation of vascular tones, neurotransmission, heart rate (HR) and contractility, inhibition of platelets aggression and leukocyte adhesion and acts as antiatherogenic factor. ,, NO is synthesized by three NO synthase (NOS) isoforms: Endothelial, neuronal and inducible NOS (iNOS). Hemorrhage leads to iNOS induction in the late phase of hemorrhagic shock with NO overproduction that lead to vascular decompensation and more mortality rate. ,
In this study, we proposed that more circulatory disturbances and mortality in hypertensive animals during hemorrhagic shock may be due to more iNOS activation compare to normotensive ones and to evaluate the effect of an iNOS inhibitor, aminoguanidine (AG), on hemodynamic parameters, serum nitrite level and survival rate in decompensated hemorrhagic shock model.
| Materials and Methods|| |
The study was conducted after getting ethical approval from the University Ethical Committee.
The male Wistar rats (age: 10-12 weeks, weight: 220 ± 20 g) were purchased from Pasteur Institute of Iran and kept in the animal room, two per cages, with 12 h light/dark cycle and temperature between 20°C and 25°C. The ethical committee of the authors' university approved the experimental procedures. Hypertension was induced by subcutaneous injection of deoxycorticoesterone acetate (DOCA) (Irandaru Co.), 30 mg/kg dissolved in almond oil, twice a week for 8 weeks in uninephrectomized rats plus NaCl 1% and KCl 0.2% solution for drinking.  In normotensive group, solvent of DOCA was injected subcutaneously and tap water was used for drinking in uninephrectomized rats. Systolic BP (SBP) was recorded by tail cuff method every week. Rats with SBP higher than 140 mmHg were considered hypertensive. 
Decompensated hemorrhagic shock induction
The animals were anesthetized by ketamine (75 mg/kg; Sigma Co. USA) and xylazine (5 mg/kg; Sigma Co. USA). The body temperature was monitored by rectal thermometer and maintained around 37°C using a heating pad. Right and left femoral arteries were cannulated by PE-50 catheters for blood withdrawal and monitoring of mean arterial pressure (MAP) and HR during the experiment. Right femoral vein was cannulated for drug administration. After 30 min (stabilizing period), decompensated hemorrhagic shock was induced by withdrawing blood using a heparinized syringe (rate: 1 ml/4-5 min) until the MAP reached 40 mmHg during the total time of 20 min  and maintained at shock state and MAP around 40 mmHg for next 120 min by withdrawing or reinfusing shed blood as necessary (shock period). Blood pressure was recorded with a physiograph (Hugo-Sachs Electronik, Germany) and data analyzed with a windows compatible software.
After shock period, AG (100 mg/kg; Sigma Co.) dissolved in normal saline (1 ml/kg) was infused during 15 min through femoral vein in hypertensive and normotensive rats and the animals were monitored for 1-h. In the control group, normal saline (1 ml/kg) was infused. After this period, the catheters were removed in survived animals. Incisions closed, and the animals returned to their cages. All the rats were returned to their cages where unlimited food and water were supplied. Mortality was recorded during the first 4 h and every 12 h for a total of 72 h.
Serum nitrite measurement
Blood samples were collected before hemorrhage, 2 h after shock induction and 1-h after AG infusion. Blood samples (0.3 ml) were centrifuged at 5000 c/s for 20 min. Serum samples were poured in eppendorf tubes and saved at - 70°C for further analysis of serum nitrite. Serum nitrite concentrations, the main metabolite of NO, were measured by griess reaction method using available reagents and kit (Promega Co, USA) with a detection limit of 2.5 μmol.
The results are expressed as mean ± standard error. One-way ANOVA test was used for comparison of data between groups. Data were compared between two groups using an independent t-test. Pre and postshock values were analyzed by paired t-test. Survival rate was evaluated by Fischer exact test. P < 0.05 was considered to be statistically significant.
| Results|| |
Mean arterial pressure and heart rate
Deoxycorticoesterone acetate-salt hypertensive animals had higher BP compare to normotensive group (MAP: 122 ± 4 vs. 81 ± 2.7 mmHg; SBP: 160 ± 5.5. vs. 106 ± 9.7 mmHg, P < 0.05). Hemorrhage decreased MAP in two groups and maintained around 40 mmHg during the shock period [Figure 1]a. Hemorrhage caused a significant decrease of HR in normotensive and hypertensive animals that continued throughout of a shock period [Figure 1]b, however, there was no significant difference between groups (P > 0.05).
|Figure 1: Changes of mean arterial pressure (a) and Heart rate (b) during the shock period in normotensive and hypertensive rats (a). *Significant difference compare to before experiment; #Significant difference compare to normotensive group.|
Click here to view
Infusion of AG during the shock period in normotensive animals caused a transient increase in MAP, however, after 1-h it returned to the base level [Figure 2]a. In hypertensive animals, AG increased MAP, although it was not statistically significant [Figure 2]b. Administration of AG caused an increase of HR in normotensive and hypertensive animals (P < 0.05) [Figure 2]c and d.
|Figure 2: Effect of aminoguanidine on mean arterial pressure and heart rate in normotensive (a and c) and hypertensive (b and d) rats|
Click here to view
Serum nitrite concentration
Basal level of serum nitrite concentration in the hypertensive group was lower than control (3.41 ± 0.17 vs. 3.97 ± 0.24 μmol/l, respectively). Hemorrhagic shock caused a significant rise in serum nitrite concentration in normotensive (5.17 ± 0.38 vs. 3.97 ± 0.24 μmol/l, P < 0.01) and hypertensive rats (4.87 ± 0.3 vs. 3.41 ± 0.17 μmol/l; P < 0.05) [Figure 3]. Infusion of AG reduced serum nitrite concentration in normotensive (4.92 ± 1.37 vs. 5.17 ± 0.38 μmol/l) and hypertensive (4.62 ± 0.3 vs. 4.87 ± 0.3. μmol/l) groups [Figure 3].
|Figure 3: Comparison of serum nitrite concentrations during the shock period and after aminoguanidine (AG) treatment in normotensive and hypertensive groups. *P < 0.05 compare to base level. #P < 0.05 compare to AG-treated group|
Click here to view
All normotensive and hypertensive rats were survived during experiment. After 4 h, two animals (33%) were died in the hypertensive group, while animals in the normotensive group were alive. After 72 h, the number of survived animals was the same in AG-treated and nontreated groups [Figure 4].
|Figure 4: Survival rate of normotensive and hypertensive animals aftershock period in aminoguanidine-treated and nontreated groups|
Click here to view
| Discussion|| |
Previous studies showed that the late phase of hemorrhagic shock is accompanied with NO overproduction and vascular decompensation. ,, In this study, we evaluate the effect of AG on hemodynamic responses, serum NO production and survival rate in decompensated hemorrhagic shock model. In this model, during the shock period, BP stabilizes at a low definite point to prevent from compensatory neural and hormonal responses. ,
Prognosis of hemorrhagic shock primarily depends on the level of hypovolemia and hypotension that restricts tissue perfusion and causes oxidative stress and tissue damages. Furthermore, endothelial cell swelling due to ischemia worsen impaired perfusion of tissues. Hemorrhagic shock causes a general ischemia in the body that usually produces systemic inflammation and degree of inflammation depends on duration of shock.  Studies demonstrated higher morbidity and mortality during hemorrhagic shock in hypertensive subjects ,, which supported the results of this study. The factors which may be involved on lower survival count are vascular decompensation, baroreflex insufficiency, systemic inflammatory response and tissue damages. ,,,
We also found that serum NO concentration in hypertensive animals was lower than normotensive, which may reflect endothelial dysfunction. Endothelial dysfunction in hypertensive subjects decreases NO bioavailability due to increased oxidative stress in the vessels. ,, Previous studies indicated increased NO production during hemorrhagic shock.  In the present study, we found increased serum NO concentration after shock period in normotensive and hypertensive groups. Hemorrhage induces inflammatory responses that lead to induction of iNOS particularly in the late phase of shock. , Increased serum NO level is accompanied with vascular decompensation.  We expected that administration of AG (iNOS inhibitor) decreases serum NO level and increases BP, however, we observed no significant effects on MAP, HR and serum NO level in normotensive and hypertensive groups. In contrast to our results, some studies demonstrated that iNOS inhibition caused a significant improvement of hemodynamic parameters and tissue injuries. ,,, Hua and Moochhala indicated that NG-nitro-L-arginine methyl ester (L-NAME) and AG (1, 10, and 100 mg/kg) increased the survival time of shocked animals.  They also showed that L-arginine reversed the beneficial effects of L-NAME and AG and suggested the involvement of NO in the pathophysiology of hemorrhagic shock. Mercaptoguanidine, an iNOS inhibitor and scavenger of peroxynitrite, prevents vascular decompensation in the late phase of shock.  In agree to our results, an iNOS inhibitor, GW274150, used in the experimental model of hemorrhagic shock did not change hemodynamic response but improved tissue injuries and renal function and decreased production of nitrotyrosine in lung and liver.  In another study, 2 h hemorrhagic shock caused a time-dependent decrease of vascular response to norepinephrine. This decrease was inhibited by L-NAME administration that indicates NO overproduction by endothelial NOS. However, in the longer period of shock, hyporeactivity of blood vessels reversed by dexamethasone that implies to NO production by iNOS.  Based on our results, it seems that the main source of higher NO production during blood loss and shock period is not the iNOS and perhaps this is the reason that AG could not alter hemodynamic response, serum NO concentration and survival rate during blood loss.
| Conclusion|| |
Administration of AG during decompensated hemorrhagic shock could not improve hemodynamic responses, serum NO concentration and survival rate in normotensive and DOCA-salt hypertensive rats.
| References|| |
Radisavljevic Z. Hypertension-induced dysfunction of circulation in hemorrhagic shock. Am J Hypertens 1995;8:761-7.
Sinert R, Guerrero P, Quintana E, Zehtabchi S, Kim CN, Agbemadzo A, et al.
The effect of hypertension on the response to blood loss in a rodent model. Acad Emerg Med 2000;7:318-26.
Sinert R, Spencer MT, Wilson R, Silverberg M, Patel M, Doty CI, et al.
The effect of hypertension on uncontrolled hemorrhage in a rodent model. Acad Emerg Med 2002;9:767-74.
Shah NS, Billiar TR. Role of nitric oxide in inflammation and tissue injury during endotoxemia and hemorrhagic shock. Environ Health Perspect 1998;106 Suppl 5:1139-43.
Hierholzer C, Billiar TR. Molecular mechanisms in the early phase of hemorrhagic shock. Langenbecks Arch Surg 2001;386:302-8.
Hierholzer C, Menezes JM, Ungeheuer A, Billiar TR, Tweardy DJ, Harbrecht BG. A nitric oxide scavenger protects against pulmonary inflammation following hemorrhagic shock. Shock 2002;17:98-103.
Hollenberg SM, Broussard M, Osman J, Parrillo JE. Increased microvascular reactivity and improved mortality in septic mice lacking inducible nitric oxide synthase. Circ Res 2000;86:774-8.
Md S, Moochhala SM, Siew-Yang KL. The role of inducible nitric oxide synthase inhibitor on the arteriolar hyporesponsiveness in hemorrhagic-shocked rats. Life Sci 2003;73:1825-34.
Förstermann U. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch 2010;459:923-39.
Hollenberg SM, Cinel I. Bench-to-bedside review: Nitric oxide in critical illness - update 2008. Crit Care 2009;13:218.
Sears CE, Ashley EA, Casadei B. Nitric oxide control of cardiac function: Is neuronal nitric oxide synthase a key component? Philos Trans R Soc Lond B Biol Sci 2004;359:1021-44.
Md S, Moochhala SM, Siew Yang KL, Lu J, Anuar F, Mok P, et al.
The role of selective nitric oxide synthase inhibitor on nitric oxide and PGE2 levels in refractory hemorrhagic-shocked rats. J Surg Res 2005;123:206-14.
Seifi B, Kadkhodaee M, Xu J, Soleimani M. Pro-inflammatory cytokines of rat vasculature in DOCA-salt treatment. Mol Biol Rep 2010;37:2111-5.
Balaszczuk AM, Arreche ND, Mc Laughlin M, Arranz C, Fellet AL. Nitric oxide synthases are involved in the modulation of cardiovascular adaptation in hemorrhaged rats. Vascul Pharmacol 2006;44:417-26.
Shirhan M, Moochhala SM, Kerwin SY, Ng KC, Lu J. Influence of selective nitric oxide synthetase inhibitor for treatment of refractory haemorrhagic shock. Resuscitation 2004;61:221-9.
Ulloa L, Messmer D. High-mobility group box1 (HMGB1) protein: Friend and foe. Cytokine Growth Factor Rev 2006;17:189-201.
Hagberg H, Haljamäe H, Johansson B, Petterson B, Wennberg E. Liver and skeletal muscle metabolism, extracellular K+concentrations, and survival in spontaneously hypertensive rats following acute blood loss. Circ Shock 1983;10:61-70.
Harbrecht BG, Wu B, Watkins SC, Billiar TR, Peitzman AB. Inhibition of nitric oxide synthesis during severe shock but not after resuscitation increases hepatic injury and neutrophil accumulation in hemorrhaged rats. Shock 1997;8:415-21.
Hierholzer C, Kalff JC, Bednarski B, Memarzadeh F, Kim YM, Billiar TR, et al.
Rapid and simultaneous activation of Stat3 and production of interleukin 6 in resuscitated hemorrhagic shock. Arch Orthop Trauma Surg 1999;119:332-6.
Hierholzer C, Harbrecht BG, Billiar TR, Tweardy DJ. Hypoxia-inducible factor-1 activation and cyclo-oxygenase-2 induction are early reperfusion-independent inflammatory events in hemorrhagic shock. Arch Orthop Trauma Surg 2001;121:219-22.
Fenning A, Harrison G, Rose'meyer R, Hoey A, Brown L. l-Arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats. Am J Physiol Heart Circ Physiol 2005;289:H1408-16.
Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, et al.
Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 2003;111:1201-9.
Neves MF, Kasal DA, Cunha AR, Medeiros F. Vascular dysfunction as target organ damage in animal models of hypertension. Int J Hypertens 2012;2012:187526.
Szabó C, Thiemermann C. Invited opinion: Role of nitric oxide in hemorrhagic, traumatic, and anaphylactic shock and thermal injury. Shock 1994;2:145-55.
Savoye G, Tamion F, Richard V, Varin R, Thuillez C. Hemorrhagic shock resuscitation affects early and selective mesenteric artery endothelial function through a free radical-dependent mechanism. Shock 2005;23:411-6.
Daughters K, Waxman K, Nguyen H. Increasing nitric oxide production improves survival in experimental hemorrhagic shock. Resuscitation 1996;31:141-4.
Hua TC, Moochhala SM. Influence of L-arginine, aminoguanidine, and NG-nitro-L-arginine methyl ester (L-name) on the survival rate in a rat model of hemorrhagic shock. Shock 1999;11:51-7.
Szabó A, Hake P, Salzman AL, Szabó C. Beneficial effects of mercaptoethylguanidine, an inhibitor of the inducible isoform of nitric oxide synthase and a scavenger of peroxynitrite, in a porcine model of delayed hemorrhagic shock. Crit Care Med 1999;27:1343-50.
McDonald MC, Izumi M, Cuzzocrea S, Thiemermann C. A novel, potent and selective inhibitor of the activity of inducible nitric oxide synthase (GW274150) reduces the organ injury in hemorrhagic shock. J Physiol Pharmacol 2002;53:555-69.
Thiemermann C, Szabó C, Mitchell JA, Vane JR. Vascular hyporeactivity to vasoconstrictor agents and hemodynamic decompensation in hemorrhagic shock is mediated by nitric oxide. Proc Natl Acad Sci U S A 1993;90:267-71.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
|This article has been cited by|
||The Cardioprotective Effects of Aminoguanidine on Lipopolysaccharide Induced Inflammation in Rats
| ||Farimah Beheshti,Mahmoud Hosseini,Milad Hashemzehi,Mohammad Reza Hadipanah,Maryam Mahmoudabady |
| ||Cardiovascular Toxicology. 2020; |
|[Pubmed] | [DOI]|
||Histological and Immunohistochemical Basis of the Effect of Aminoguanidine on Renal Changes Associated with Hemorrhagic Shock in a Rat Model
| ||Abdulmajeed Al Drees,Mahmoud Salah Khalil,Mona Soliman |
| ||Acta Histochemica et Cytochemica. 2017; 50(1): 11 |
|[Pubmed] | [DOI]|
||Aminoguanidine alleviated MMA-induced impairment of cognitive ability in rats by downregulating oxidative stress and inflammatory reaction
| ||Qiliang Li,Wenqi Song,Ze Tian,Peichang Wang |
| ||NeuroToxicology. 2017; 59: 121 |
|[Pubmed] | [DOI]|
||Co-administration of walnut (Juglans regia) prevents systemic hypertension induced by long-term use of dexamethasone: a promising strategy for steroid consumers
| ||Siyavash Joukar,Sahar Ebrahimi,Majid Khazaei,Alireza Bashiri,Mohammad Reza Shakibi,Vida Naderi,Beydolah Shahouzehi,Masoud Alasvand |
| ||Pharmaceutical Biology. 2017; 55(1): 184 |
|[Pubmed] | [DOI]|