| Abstract|| |
Background: Insulin has been shown to exert positive inotropic effects in several in vivo ex vivo models and in human hearts. Resuscitation following hemorrhagic shock results in myocardial contractile dysfunction. However, the optimal timing for treatment with insulin for the cardioprotection effects is unclear. Objectives: The objective of this study was to test the hypothesis that treatment with insulin before resuscitation provides better cardioprotection than treatment with insulin after resuscitation. Materials and Methods: Rats were assigned to 3 experimental groups (n = 6 per group): (1) Hemorrhagic shock and resuscitation, (2) hemorrhagic shock resuscitated then treated with insulin and (3) hemorrhagic shock treated with insulin before resuscitation. Rats were hemorrhaged for 60 min to rach mean arterial blood pressure of 40 mmHg. Rats were resuscitated in vivo by reinfusion of the shedded blood to restore normotension and monitored for 60 min. Rats were treated or not with insulin 200 μU/g body weight intramuscularly either before or after resuscitation. The maximum of the left ventricular developed pressure (+dP/dt) was measured for 60 min in the isolated perfused hearts using the Langendorff method. Blood samples were obtained for measurements of tumor necrosis factor-alpha (TNF-α). Results: Treatment with insulin before resuscitation following hemorrhagic shock significantly elevated max dP/dt compared with insulin treatment after resuscitation and the untreated group. TNF-α levels were lower in the insulin treatment before resuscitation compared to the treatment after resuscitation and the untreated group. Conclusion: Insulin treatment before resuscitation following hemorrhagic shock provides better cardiac protection than treatment with insulin after resuscitation, as evidenced by the improved myocardial contractility, preservation of myocardial structure. The mechanism of cardiac protection involves decrease in the inflammatory response to shock by lowering the levels of TNF.
Keywords: Contractility, hemorrhage, insulin, resuscitation, shock
|How to cite this article:|
Soliman M. Insulin treatment before resuscitation following hemorrhagic shock improves cardiac contractility and protects the myocardium in the isolated rat heart. J Emerg Trauma Shock 2015;8:144-8
|How to cite this URL:|
Soliman M. Insulin treatment before resuscitation following hemorrhagic shock improves cardiac contractility and protects the myocardium in the isolated rat heart. J Emerg Trauma Shock [serial online] 2015 [cited 2019 Dec 9];8:144-8. Available from: http://www.onlinejets.org/text.asp?2015/8/3/144/160714
| Introduction|| |
The effects of insulin on the heart has been of great interest for researches with the recognition of the side-effects of hyperglycemia on heart resulting in decrease myocardial contractility, organ dysfunction, and mortality. , Studies have shown the beneficial effects of insulin therapy in cardiac surgery patients. , Several studies have shown that insulin therapy reduced the mortality in patients with acute myocardial infarction. ,, Previous studies have demonstrated that insulin may play an important role in attenuating both myocardial ischemia and reperfusion injury. ,,
The exact mechanism of cardioprotection by insulin is not fully understood. Studies have shown that the cardioprotective effects of insulin are mediated in part metabolically by altering the hyperglycemic effects on the heart. , In addition, insulin exerts its cardioprotective effects by promoting cardiomyocyte survival pathway by activation of the phosphatidylinositol 3 kinase and its antiapoptotic effect.  Another important mechanism of insulin cardioprotection is via reducing the oxidative stress by reducing the nitric oxide synthase (NOS) production.  Data showed that insulin has anti-inflammatory effects and that glucose proinflammatory and pro-oxidant actions suppress the anti-inflammatory effects of insulin. 
Hemorrhagic shock and resuscitation (HS-R) has been shown to result in myocardial contractile dysfunction, failure, and injury. ,,,, The exact mechanism is not known. Activation of the inflammatory pathways by hemorrhage and resuscitation and the production of inflammatory mediators have been shown to result in myocardial injury. , Another mechanism of cardiac injury following hemorrhagic shock is by the activation of NOS and the production of NO. ,
Timing of insulin therapy in relation to ischemia was found to be a crucial factor for its cardioprotective effects.  It has been shown that insulin administration prior to ischemia provides better cardioprotection than insulin administered only at reperfusion in the isolated ischemic rat heart. 
The aim of the present study was to test the hypothesis that treatment with insulin before resuscitation following hemorrhagic shock provides better cardioprotection than treatment with insulin after resuscitation in hemorrhagic shock rat models. The study measured the myocardial contractility and examined myocardial structure as well as the levels of tumor necrosis factor-alpha (TNF-α) for the possible involvement of inflammatory pathways.
| Materials and Methods|| |
The National Plan for Sciences and Technologies, King Saud University approved this study. Male Sprague-Dawley weighing 300-350 g were anesthetized using intraperitoneal injection of urethane (125 mg/kg). The rats were injected with heparin sodium intraperitoneally (2000 IU) 15 min prior to anesthesia to prevent coagulation of the blood in the isolated hearts and vasculature.
The animals were assigned randomly to the three experimental groups (n = 6 per group):
- Hemorrhagic shock treated with insulin before resuscitation (HS-I-R) and
- Hemorrhagic shock treated with insulin after resuscitation (HS-R-I) [Figure 1].
Hemorrhagic shock and resuscitation rat model
The left carotid artery was cannulated, and the mean arterial blood pressure (MABP) was monitored using a blood pressure transducer. The animals were allowed to stabilize for a period of 30 min. After stabilization, hemorrhagic shock was induced by aspiration of blood at a rate of 1 ml/min to reach MABP of approximately 35-40 mmHg using a reservoir (a 10 ml syringe). The rats were resuscitated in vivo by reinfusion of the shed blood to restore normotension, and the MABP was monitored for 60 min.
Measurement of myocardial contractile function in the isolated hearts
After 60 min hemorrhagic shock and resuscitated, with or without treatment, hearts were excised quickly and mounted on a cannula of the Langendorff apparatus. Retrograde perfusion was performed at a flow rate of 10 ml/min with Krebs Hanseleit bicarbonate buffer (KHB, in mM: NaCl 118, CaCl 2 1.25, KCl 4.7, NaHCO 3 21, MgSO 4 1.2, glucose, 11, KH 2 PO 4 1.2, and ethylenediaminetetraacetic acid 0.5). Perfusate temperature was maintained at 37°C and was gassed with a mixture of 95% O 2 and 5% CO 2 at a pH of 7.4 as described previously.  Hearts were stimulated electrically at 5 Hz using an electrical stimulator (6020 stimulator from harvard apparatus). Left ventricle pressure was measured by the use of a saline-filled cellophane balloon-tipped catheter which was placed into the left ventricle via the mitral valve and inflated to maintain an end diastolic pressure at 5 mmHg. Left ventricular maximum developed pressure (+dP/dt) was calculated by the acknowledge software as the first derivative of pressure over time. Data were collected and analyzed using the BIOPAC system and the acknowledge software.
In the preresuscitation insulin treated group, insulin 200 μU/g body weight in 1 ml saline was injected intramuscularly before resuscitation. The postresuscitation insulin group (HS-R-I), insulin was injected after resuscitation period of 60 min [Figure 1].
Measurement of tumor necrosis factor-alpha
Blood (0.5 ml) was collected from the left carotid artery cannula at the end of the experimental period harvesting the heart, and centrifuged at 2500 g for 10 min and plasma was stored at −80°C until analysis for TNF-a measurement. Serum samples were analyzed by ELISA (R&D systems).
Data were presented as means ± standard deviation Data were analyzed with one-way ANOVA. The values of P < 0.05 were considered significant. The Student's t-test was used to compare mean values between the two experimental groups.
| Results|| |
Baseline measurements are shown in [Table 1]. There was no significant difference in baseline measurements. Coronary perfusion pressure was comparable among all experimental groups [Table 1].
Left ventricular max dP/dt was significantly increased in the experimental group treated with insulin before resuscitation compared to the group treated after resuscitation. After resuscitation, left ventricular dP/dt was significantly lower compared to the resuscitated groups treated with insulin [Figure 2].
|Figure 2: Left ventricular max dP/dt in the three experimental groups (n = 6 per group). The data are presented as means ± standard deviation *P < 0.05 compared to hemorrhagic shock resuscitated then treated with insulin (HS-R-I) group and to the untreated group. •P < 0.05 compared to the hemorrhagic shock treated with insulin before resuscitation and HS-R-I|
Click here to view
The TNF-α levels were significantly elevated in the HS-R untreated group compared to the HS-I-R and HS-R-I groups [Figure 3]. Treatment with insulin before resuscitation significantly lowered the levels of TNF-α compared to the group treated after resuscitation.
|Figure 3: Tumor necrosis factor alpha levels measured at the end of the experimental period after resuscitation and treatment in the three experimental groups (n = 6 per group). The data are presented as means ± standard deviation *P < 0.05 compared to hemorrhagic shock resuscitated then treated with insulin (HS-R-I) group and to the untreated group. •P < 0.05 compared to the hemorrhagic shock treated with insulin before resuscitation and HS-R-I|
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| Discussion|| |
The present study showed that treatment with insulin before resuscitation following hemorrhagic shock improved cardiac contractility compared to treatment after resuscitation in HS-R rat model. Insulin has been shown to induce cardioprotection effects.  The exact mechanism of the cardioprotection is not known. Insulin has been shown to have positive inotropic effects in animals and in human hearts. , Several cardiopretection effects of insulin have been identified. Insulin has been shown to have antioxidant effects by preventing the formation of free radicals after myocardial ischemi-reperfusion in rats.  Another extensively studied mechanism of insulin cardioprotection is by inhibiting apoptosis in the context of ischemia-reperfusion.  The present study showed that the positive inotropic effect of insulin was mediated, in part, via the anti-inflammatory effect.
The present study demonstrated that the levels of TNF-α was elevated in the HS-R without treatment. Hemorrhagic shock has been shown to result in myocardial contractile dysfunction and injury. , One of the possible mechanisms of cardiac injury following HS-R is by activation of the inflammatory pathways,  and increasing the levels of inflammatory mediators, including TNF-α. , Treatment with insulin before resuscitation significantly lowered the levels of TNF-α compared to the untreated group and even compared to treatment before resuscitation. Insulin is known to exert anti-inflammatory effects.  Hyperglycemia is known to activate inflammatory pathways.  In addition, insulin has been shown to have direct anti-inflammatory effects by inhibiting interleukin-6.  Insulin has been also shown to inhibit the pro-inflammatory effects of TNF-α.  Studies have shown that insulin lowered the levels of TNF-α and reduced apoptosis in cardiomyocyte preparation in ischemia-reperfusion. In human studies, insulin has been shown to inhibit TNF-α in human aortic endothelium. 
The optimal timing for insulin administration for heart cardioprotection remains unclear. Timing for insulin therapy is important in ischemia for its cardioprotective effects. Studies have shown that insulin treatment after ischemia was effective in reducing the infarct size in isolated rat heart of acute myocardial infarction.  In 2014, Sato et al. demonstrated that treatment with insulin prior to ischemia provides better cardioprotection than insulin administration only at reperfusion.  However, studies have demonstrated that insulin treatment before cardiopulmonary bypass (coronary bypass graft) is cardioprotective in human.  Several studies emphasize the importance of timing of insulin treatment in relation to myocardial ischemia.  However, the optimal timing for treatment with insulin in resuscitation of hemorrhagic shock is not known. The present study showed that treatment with insulin before resuscitation was significantly effective in cardioprotection than after resuscitation as demonstrated by the significant increase in myocardial contractility.
| Limitations|| |
One limitation of the present study is that blood glucose levels were not measured.
Future studies are needed to examine the effects of treatment with insulin on cardiac function and monitor the blood glucose changes.
| Conclusion|| |
Treatment with insulin before resuscitation following hemorrhagic shock provided better cardioprotection than insulin treatment after resuscitation. The present study suggested that one of the possible mechanisms of cardioprotection by insulin is by inhibiting the inflammatory pathways.
| Acknowledgments|| |
This work was supported by a grant from the National Plan for Science, Technology and Innovation (08-MED560-02) at King Saud University, Riyadh, Saudi Arabia. Technical help from Mr. Sabirine is acknowledged.
| References|| |
Sato H, Sakaeda M, Ishii J, Kashiwagi K, Shimoyamada H, Okudela K, et al.
Insulin-like growth factor binding protein-4 gene silencing in lung adenocarcinomas. Pathol Int 2011;61:19-27.
Carvalho G, Pelletier P, Albacker T, Lachapelle K, Joanisse DR, Hatzakorzian R, et al
. Cardioprotective effects of glucose and insulin administration while maintaining normoglycemia (GIN therapy) in patients undergoing coronary artery bypass grafting. J Clin Endocrinol Metab 2011;96:1469-77.
Haga KK, McClymont KL, Clarke S, Grounds RS, Ng KY, Glyde DW, et al.
The effect of tight glycaemic control, during and after cardiac surgery, on patient mortality and morbidity: A systematic review and meta-analysis. J Cardiothorac Surg 2011;6:3.
Keng NT, Lin HH, Lin HR, Hsieh WK, Lai CC. Dual regulation by ethanol of the inhibitory effects of ketamine on spinal NMDA-induced pressor responses in rats. J Biomed Sci 2012 2;19:11.
Fath-Ordoubadi F, Beatt KJ. Glucose-insulin-potassium therapy for treatment of acute myocardial infarction: An overview of randomized placebo-controlled trials. Circulation 1997;96:1152-6.
Díaz R, Paolasso EA, Piegas LS, Tajer CD, Moreno MG, Corvalán R, et al.
Metabolic modulation of acute myocardial infarction. The ECLA (Estudios Cardiológicos Latinoamérica) Collaborative Group. Circulation 1998;98:2227-34.
Malmberg K, Rydén L, Hamsten A, Herlitz J, Waldenström A, Wedel H. Mortality prediction in diabetic patients with myocardial infarction: Experiences from the DIGAMI study. Cardiovasc Res 1997;34:248-53.
Gao F, Gao E, Yue TL, Ohlstein EH, Lopez BL, Christopher TA, et al.
Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: The roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation. Circulation 2002;105:1497-502.
Malmberg K. Prospective randomised study of intensive insulin treatment on long term survival after acute myocardial infarction in patients with diabetes mellitus. DIGAMI (Diabetes Mellitus, Insulin Glucose Infusion in Acute Myocardial Infarction) Study Group. BMJ 1997;314:1512-5.
Ma H, Zhang HF, Yu L, Zhang QJ, Li J, Huo JH, et al.
Vasculoprotective effect of insulin in the ischemic/reperfused canine heart: Role of Akt-stimulated NO production. Cardiovasc Res 2006;69:57-65.
Jonassen AK, Sack MN, Mjøs OD, Yellon DM. Myocardial protection by insulin at reperfusion requires early administration and is mediated via Akt and p70s6 kinase cell-survival signaling. Circ Res 2001;89:1191-8.
Jonassen AK, Mjøs OD, Sack MN. p70s6 kinase is a functional target of insulin activated Akt cell-survival signaling. Biochem Biophys Res Commun 2004;315:160-5.
Koo JR, Vaziri ND. Effects of diabetes, insulin and antioxidants on NO synthase abundance and NO interaction with reactive oxygen species. Kidney Int 2003;63:195-201.
Dandona P, Chaudhuri A, Ghanim H, Mohanty P. Insulin as an anti-inflammatory and antiatherogenic modulator. J Am Coll Cardiol 2009;53 5 Suppl:S14-20.
Soliman M. Dimethyl amiloride, a Na+-H+ exchange inhibitor, and its cardioprotective effects in hemorrhagic shock in in vivo
resuscitated rats. J Physiol Sci 2009;59:175-80.
Soliman MM, Arafah MM. Treatment with dipyridamole improves cardiac function and prevent injury in a rat model of hemorrhage. Eur J Pharmacol 2012;678:26-31.
Moore FA, Sauaia A, Moore EE, Haenel JB, Burch JM, Lezotte DC. Postinjury multiple organ failure: A bimodal phenomenon. J Trauma 1996;40:501-10.
Kirkman E, Watts S. Haemodynamic changes in trauma. Br J Anaesth 2014;113:266-75.
Soliman M. Preservation of myocardial contractile function by aminoguanidine, a nitric oxide synthase inhibitors, in a rat model of hemorrhagic shock. Pak J Med Sci 2013;29:1415-9.
Junger WG, Rhind SG, Rizoli SB, Cuschieri J, Shiu MY, Baker AJ, et al.
Resuscitation of traumatic hemorrhagic shock patients with hypertonic saline-without dextran-inhibits neutrophil and endothelial cell activation. Shock 2012;38:341-50.
Hazeldine J, Hampson P, Lord JM. The impact of trauma on neutrophil function. Injury 2014;45:1824-833.
Collins JL, Vodovotz Y, Hierholzer C, Villavicencio RT, Liu S, Alber S, et al.
Characterization of the expression of inducible nitric oxide synthase in rat and human liver during hemorrhagic shock. Shock 2003;19:117-22.
Sato T, Sato H, Oguchi T, Fukushima H, Carvalho G, Lattermann R, et al.
Insulin preconditioning elevates p-Akt and cardiac contractility after reperfusion in the isolated ischemic rat heart. Biomed Res Int 2014;2014:536510.
Ng KW, Allen ML, Desai A, Macrae D, Pathan N. Cardioprotective effects of insulin: How intensive insulin therapy may benefit cardiac surgery patients. Circulation 2012;125:721-8.
Hsu CH, Wei J, Chen YC, Yang SP, Tsai CS, Lin CI. Cellular mechanisms responsible for the inotropic action of insulin on failing human myocardium. J Heart Lung Transplant 2006;25:1126-34.
Lucchesi BR, Medina M, Kniffen FJ. The positive inotropic action of insulin in the canine heart. Eur J Pharmacol 1972;18:107-15.
Ji L, Fu F, Zhang L, Liu W, Cai X, Zhang L, et al.
Insulin attenuates myocardial ischemia/reperfusion injury via reducing oxidative/nitrative stress. Am J Physiol Endocrinol Metab 2010;298:E871-80.
Wong VW, Mardini M, Cheung NW, Mihailidou AS. High-dose insulin in experimental myocardial infarction in rabbits: Protection against effects of hyperglycaemia. J Diabetes Complications 2011;25:122-8.
Soliman M. Inhibition of Na(+)-H(+) exchange before resuscitation following hemorrhagic shock is cardioprotective in rats. J Saudi Heart Assoc 2009;21:159-63.
Cotton BA, Guy JS, Morris JA Jr, Abumrad NN. The cellular, metabolic, and systemic consequences of aggressive fluid resuscitation strategies. Shock 2006;26:115-21.
Lomas-Neira J, Perl M, Venet F, Chung CS, Ayala A. The role and source of tumor necrosis factor-α in hemorrhage-induced priming for septic lung injury. Shock 2012;37:611-20.
Lomas-Neira JL, Ayala A. What's new in Shock, March 2012? Shock 2012;37:239-41.
Esposito K, Nappo F, Marfella R, Giugliano G, Giugliano F, Ciotola M, et al.
Inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: Role of oxidative stress. Circulation 2002;106:2067-72.
Andersson CX, Sopasakis VR, Wallerstedt E, Smith U. Insulin antagonizes interleukin-6 signaling and is anti-inflammatory in 3T3-L1 adipocytes. J Biol Chem 2007 30;282:9430-5.
Bortoff KD, Keeton AB, Franklin JL, Messina JL. Anti-Inflammatory Action of Insulin via Induction of Gadd45-ß Transcription by the mTOR Signaling Pathway. Hepat Med 2010 1;2001:79-85.
Aljada A, Ghanim H, Saadeh R, Dandona P. Insulin inhibits NFkappaB and MCP-1 expression in human aortic endothelial cells. J Clin Endocrinol Metab 2001;86:450-3.
Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, et al.
Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo
. Circulation 2001;104:330-5.
Sato H, Hatzakorzian R, Carvalho G, Sato T, Lattermann R, Matsukawa T, et al.
High-dose insulin administration improves left ventricular function after coronary artery bypass graft surgery. J Cardiothorac Vasc Anesth 2011;25:1086-91.
Department of Physiology, College of Medicine, King Saud University, Riyadh
Source of Support: The National Plan for Science, Technology and Innovation at King Saud University, Riyadh, Saudi Arabia., Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]