diabetes research and clinical practice 107 (2015) 377–383
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Granulocyte colony-stimulating factor provides protection against cardiovascular autonomic neuropathy in streptozotocin-induced diabetes in rats Oytun Erbas¸ a, Volkan Solmaz b, Dilek Tas¸kiran c,* a
Istanbul Bilim University School of Medicine, Department of Physiology, Istanbul, Turkey Gaziosmanpas¸a University School of Medicine, Department of Neurology, Tokat, Turkey c Ege University School of Medicine, Department of Physiology, Izmir, Turkey b
article info
abstract
Article history:
Aims: Cardiovascular autonomic neuropathy (CAN) is a relatively common and detrimental
Received 9 June 2014
complication of diabetes mellitus (DM). Dysregulation of neuropeptides, such as calcitonin
Received in revised form
gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), are thought to play
23 October 2014
significant roles in diabetes-related cardiovascular disease. Accumulating evidence indi-
Accepted 29 December 2014
cates the neuroprotective effects of granulocyte-colony stimulating factor (G-CSF) in differ-
Available online 21 January 2015
ent neurological disorders. The purpose of the study is to investigate the role of CGRP and
Keywords:
Methods: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) for 14
VIP and possible effects of G-CSF on CAN in type I DM model in rats. Diabetes mellitus
rats. Seven rats served as controls and 6 rats were administered G-CSF alone. DM group was
Cardiovascular autonomic
randomly divided into 2 groups and received either 1 mL/kg saline (DM + saline group) or
neuropathy
100 mg/kg/day G-CSF (DM + G-CSF group) for 4 weeks. Following electrocardiography (ECG),
Granulocyte-colony stimulating
GCRP and VIP levels were measured in plasma samples.
factor
Results: Diabetes promoted a significant prolongation in the corrected QT interval (cQT)
QT interval
(P < 0.001) whereas G-CSF administration significantly shortened cQT interval (P < 0.05).
Calcitonin gene-related peptide
Plasma VIP and CGRP levels of saline treated DM group were significantly lower than those of
Vasoactive intestinal peptide
control group (P < 0.05). G-CSF treatment significantly prevented the reduction in plasma VIP and CGRP levels (P < 0.01 and P < 0.05, respectively). Also, correlation analysis showed a significant negative correlation between the cQT and neuropeptide levels. Conclusions: This study suggests that G-CSF can be effective in CAN by means of neuroprotection, and plasma VIP and CGRP levels can be used for the assessment of autonomic and sensory functions in diabetes. # 2015 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Ege University School of Medicine, Department of Physiology, 35100 Izmir, Turkey. Tel.: +90 232 390 1800; fax: +90 232 388 2868. ˇ E-mail address: [email protected] (D. Tas¸kˇiran). http://dx.doi.org/10.1016/j.diabres.2014.12.018 0168-8227/# 2015 Elsevier Ireland Ltd. All rights reserved.
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1.
diabetes research and clinical practice 107 (2015) 377–383
Introduction
Diabetic neuropathy is a frequent and severe complication that involves the several clinical syndromes affecting motor, sensory and autonomic nerves [1]. In case of diabetes-related autonomic neuropathy, dysfunction is observed in many systems including cardiovascular, gastrointestinal, genitourinary and neurovascular systems [2]. Cardiovascular autonomic neuropathy (CAN) is a condition, developed in relation to diabetes mellitus (DM) and frequently associated with high mortality. It has been reported that the mortality rate associated with CAN is five times as high as other autonomic involvement [3]. The condition has been estimated to develop more frequently in people with type 2 DM than those with type 1 [4]. In CAN, fatal complications such as cardiac failure and sudden cardiac arrhythmia develop in patients in association with damage to both parasympathetic and sympathetic parts of autonomic nervous system [5]. The QT interval on the electrocardiography (ECG) reflects the total duration of ventricular depolarization and repolarization, and its measurement has been proposed as a simple and noninvasive method for the cardiovascular mortality in various conditions, including diabetes [6–9]. Since the QT interval differs inversely with heart rate, heart rate-corrected QT (cQT) interval is preferably used. In patients with diabetes, cQT prolongation and autonomic dysfunction are closely correlated, and cQT prolongation is considered to be a specific sign of autonomic cardiac dysfunction and high mortality risk [8,10]. Cardioactive neuropeptides, such as calcitonin gene-related peptide (CGRP), atrial natriuretic peptide (ANP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and substance P (SP), maintain cardiovascular homeostasis through their vasomodulatory properties [11]. In diabetes, the levels of both autonomic and sensory neuropeptides are dysregulated in the myocardium. Accumulating evidence suggests that dysregulated neurohormonal activation, an outcome of neuropathy, has a major pathophysiological role in diabetes-related cardiovascular disease. Many previous studies have revealed that plasma levels of these neuropeptides are lower in those with autonomic neuropathy compared to healthy controls [12– 15]. Among these neuropeptides, VIP, an indicator of parasympathetic function, is stored with acetylcholine in parasympathetic nerves innervating blood vessels. It is also produced and released by intrinsic neurons in the heart, and improves cardiac perfusion and function [16]. CGRP is the major neuropeptide released from sensory nerve terminals in the heart. CGRP has both positive inotropic and potent vasodilatory effects in the heart. Besides, CGRP exerts modulatory effect on peripheral insulin sensitivity and trophic effects on the pancreas, motor neurons and peripheral nerves [11,17]. Granulocyte colony-stimulating factor (G-CSF), which is in widespread clinical use for the treatment of chemotherapyassociated neutropenia, plays a critical role in the regulation of the proliferation, differentiation, and survival of myeloid progenitor cells. Although there are several publications stating the beneficial effects of G-CSF in the treatment of some neurological diseases [18–20], there is a lack of data with regard to the use of this agent in CAN. Considering this, we
examined the role of CGRP and VIP and possible effects of GCSF on CAN in an experimental type I diabetes model in rats.
2.
Materials and methods
2.1.
Animals
In this study, 27 Sprague Dawley albino mature rats, weighing 200–220 g, were used. Animals were fed ad libitum and housed in pairs in plastic cages having a temperature-controlled environment (22 2 8C) with 12-h light/dark cycles. All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Committee for Animal Research of Ege University.
2.2.
Experimental protocol
Diabetes was induced with a single intraperitoneal (IP) injection of streptozotocin (STZ) (Sigma–Aldrich, Inc.; Saint Louis, MO) for 14 rats. STZ solution (60 mg/kg of body weight) was prepared fresh in citrate buffer (pH = 4.0). Seven animals served as control group and received the buffer alone. Six animals were treated with 100 mg/kg/day G-CSF (Neupogen, 48 mIU/0.5 mL, F. Hoffmann-La Roche Ltd., Switzerland) only to test the effects of G-CSF on the study parameters. Diabetes was verified after 24 h by evaluating tail blood glucose levels with glucose oxidase reagent strips (Boehringer Mannheim, Indianapolis, IN). The rats with 250 mg/dL and higher blood glucose levels were accepted diabetic and included in the study. Then, rats with diabetes were randomly divided into 2 groups; one group was treated with 1 mL/kg saline (n = 7, DM + saline group), and the other group (n = 7, DM + G-CSF group) was treated with 100 mg/kg/day G-CSF. The injections were performed intraperitoneally for 4 weeks. Following ECG, the animals were euthanized and blood samples were collected by cardiac puncture for biochemical analysis. The plasma was separated from cells by centrifugation at 3000 rpm for 10 min, and aliquots were frozen at 30 8C for further use.
2.3.
ECG recording
Rats were anesthetized by a combination of ketamine hydrochloride at a dose of 40 mg/kg (Alfamine1, Ege Vet, Alfasan International B.V. Woerden, Holland) and 4 mg/kg of xylazine hydrochloride (Alfazyne1, Ege Vet, Alfasan International B.V. Woerden, Holland), IP. The electrodes constructed of 26 gauge hypodermic needles were placed subcutaneously in the gently extended limbs of the animal. After the subcutaneous insertion of the electrodes, rats were observed for about 20 min until stabilization of the respiration and ECG patterns. Then, a 5 min ECG was taken in derivation I (DI) using BIOPAC MP 150 system (BIOPAC Systems, Inc., Goleta, CA, USA). During the ECG recordings, rectal temperatures of the rats were monitored by a rectal probe (HP Viridia 24-C; Hewlett-Packard Company, Palo Alto, CA, USA), and the temperature of each rat was kept at approximately 36–37 8C by heating pad. QT interval (duration of ventricular myocardial depolarization and repolarization), T duration and heart rate (BPM-beat per
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diabetes research and clinical practice 107 (2015) 377–383
were significantly higher than those of control group [88 mg/dL (min = 76–max = 106), P < 0.01]. There was no significant difference between the plasma glucose levels of saline and G-CSF group (P > 0.05) (Table 1). Table 1 summarizes the significant changes in ECG recordings of the study groups. The Kruskal–Wallis test showed significant differences among the study groups with regard to heart rate [x2(3) = 11.44, P < 0.01], T duration P < 0.0005], QT duration [x2(3) = 18.23, [x2(3) = 19.94, P < 0.001] and cQT [x2(3) = 21.09, P < 0.005]. Saline treated diabetes group had significantly lower heart rate compared with the control rats (P < 0.05). Furthermore, T duration, QT duration and cQT of saline treated diabetes group were significantly prolonged compared with the control group (P < 0.005, P < 0.005 and P < 0.001, respectively). G-CSF treatment significantly shortened T duration, QT duration and cQT in rats compared to saline treated diabetes group (P < 0.05, P < 0.005 and P < 0.05, respectively). No significant changes were observed in G-CSF administered group (Fig. 1). Fig. 2 represents the alterations in plasma neuropeptide levels of study groups. The analysis of neuropeptide levels with Kruskal–Wallis test revealed significant differences among the groups with regard to CGRP [x2(3) = 9.2, P < 0.05] and VIP [x2(3) = 11.57, P < 0.01] levels. Plasma VIP and CGRP levels of saline treated group were significantly lower than those of control group (P < 0.05 and P < 0.01, respectively). However, treatment of rats with G-CSF for 4 weeks significantly prevented the reduction in plasma VIP and CGRP levels compared to saline treated diabetes group (P < 0.05 and P < 0.01, respectively). No significant changes were found in G-CSF group (Fig. 2). We also examined the association between electrocardiography and biochemical parameters. Pearson’s correlation analysis revealed a significant negative correlation between the cQT and VIP level (r = 0.533, P < 0.05), and CGRP level (r = 0.594, P < 0.01). Fig. 3 demonstrates the correlation analysis between the cQT and plasma neuropeptide levels.
minute) were calculated from the ECG data using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc., Goleta, CA, USA) [21]. The heart rate was determined from the 5-min average of all R–R intervals in the ECG. QT interval was measured from the onset of QRS to end of the T wave. QT interval was further processed to incorporate beat-to-beat changes in the heart rate using Bazett’s formula [22].
2.4.
Measurement of plasma VIP and CGRP levels
Measurements of plasma VIP and CGRP levels were carried out using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Cusabio, Biotech Co., Ltd., Wuhan) specific for rats. All samples from each animal were measured in duplicate according to the manufacturer’s guidelines. The detection limit for each ELISA kit was 2.31 pg/mL, and 7.71 pg/ mL, respectively. Intraassay and interassay coefficients of variation were less than 10% in each determination.
2.5.
Statistical analysis
Data analyses were performed using SPSS software (version 15.0, SPSS Inc., Chicago, IL). Non-parametric tests were used for statistical analysis. Statistical differences in ECG parameters and neuropeptide levels among the groups were tested with the Kruskal–Wallis test. If the difference significant, Mann–Whitney U test with Bonferroni correction was used. Data were presented as median and interquartile range (IQR), which is between the 25th and 75th percentiles. Pearson’s correlation analysis was conducted to examine the association between electrocardiography and biochemical parameters. The value of P < 0.05 was accepted as statistically significant.
3.
Results
In the present study, STZ administration to animals caused typical characteristics of diabetes mellitus, including hyperglycemia, polyuria, and weight loss. The Kruskal–Wallis test showed significant differences among the study groups with regard to glycemia [x2(3) = 15.82, P < 0.001]. Plasma glucose levels of saline treated DM group [398 mg/dl (min = 365–max = 465)] and G-CSF treated DM group [392 mg/dL (min = 325–max = 468)]
4.
Discussion
In the present study, our results revealed that plasma CGRP and VIP levels significantly decreased in rats during the first 4 weeks of experimental type 1 diabetes. Furthermore, we found
Table 1 – Alterations in plasma glucose levels (mg/dL), BMP (beat per minute), cQT (ms), QT duration (ms) and T duration (ms) in study groups. Data are expressed as median (min–max). Control (n = 7) Glucose level (mg/dL) BPM (beat per min) QT duration (ms) cQT (ms) T duration (ms) #
88 256 62 124 29
(76–106) (234–285) (60–69) (112–132) (26–38)
P < 0.05 different from control and G-CSF. P < 0.005 different from control and G-CSF. * P < 0.001 different from control and G-CSF. ** P < 0.05 different from DM + saline. *** P < 0.005 different from DM + saline. ##
G-CSF (n = 6) 99 258 63 130 31
(83–113) (235–289) (57–68) (125–135) (28–30)
DM + saline (n = 7) 398 218 112 187 74
(365–465)## (190–245)# (100–125)## (150–232)* (58–88)##
DM + G-CSF (n = 7) 392 214 83 152 57
(325–468)## (186–235)# (76–91)*** (140–180)** (48–64)**
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diabetes research and clinical practice 107 (2015) 377–383
Fig. 1 – Examples of ECG recordings from the left ventricle of study groups. (a) Control, (b) G-CSF, (c) DM + saline group, and (d) DM + G-CSF group.
Fig. 2 – Measurement of plasma CGRP and VIP levels in control, G-CSF, DM + saline and DM + G-CSF groups. Data are expressed as median with 25th and 75th percentile. Boxes enclose the interquartile range and the median, while whiskers enclose the range (min–max values). # P < 0.01 and *P < 0.05 different from control and G-CSF. ## P < 0.01 and **P < 0.05 different from DM + saline.
a significant negative correlation between the neuropeptide levels and ECG data. The other important finding of our study is that G-CSF, recognized originally as a main regulator of hematopoietic stem cells, can also provide a significant protection against diabetic CAN. It has been well established that sustained elevations of blood glucose levels affect and alter the physiology of many organs, leading to the peripheral nervous system malfunctions. Diabetes related neuropathy could be explained by several mechanisms, such as generation of reactive oxygen species, activation of the polyol pathway, and formation of advanced glycation end products and depletion of the neurotrophic factors [2,23]. Clinically, the electrocardiogram of patients with diabetes may exhibit a number of abnormalities including prolonged QT interval and altered T-waves, which may lead to cardiac arrhythmias and sudden cardiac death [10,24,25]. Numerous abnormalities including autonomic dysfunction, alterations in carbohydrate and lipid metabolism and in electrolyte concentrations may have significant role in the pathogenesis of electrical instability in diabetic heart [2,26]. Experimentally, in a rat heart with diabetes, the most significant electrophysiological change is an increase in action potential duration with a subsequent prolongation of the QT interval. Additionally, prolongation of ventricular action potential duration may produce ST segment and T-wave
changes on the ECG by altering the endocardial–epicardial action potential gradient [27]. In the present study, our results revealed significant reduction in heart rate whereas QT, cQT and T durations were all prolonged in STZ-induced diabetes group compared to controls. In accordance with our present results, reductions in heart rate and/or prolonged QT interval have been previously demonstrated in STZ-induced diabetes model in rodents [22,28–31]. Reviewing the literature, there is much evidence indicating the relationship between neuropeptides and diabetic neuropathy. Also, dysregulation of the expression of neuropeptides or activation of the neuropeptide signaling pathways can harmfully affect cardiac functions. Noda et al. [32] have revealed considerably reduced VIP content in sciatic nerve of diabetic rats that were also negatively correlated with blood glucose levels. Furthermore, significantly reduced VIP levels have been reported in CAN patients with rheumatic diseases [15]. More recently, Dvora´kova´ et al. [16] have provided evidence for progressive decrease of endogenous VIP production in the hearts of rats with diabetes during the first months after onset of diabetes, with concomitant alterations in the expression of VIP receptors. In the heart, CGRP is released from sensory nerve terminals and improves cardiac perfusion and function. Clinically, decreased levels of CGRP and substance B have been reported in patients with diabetes and coronary
diabetes research and clinical practice 107 (2015) 377–383
Fig. 3 – Relationship between corrected QT interval (cQT) and neuropeptide levels. Pearson correlation analysis revealed a significant negative correlation between cQT and CGRP level (P < 0.01) and VIP level (P < 0.05).
artery disease [33]. Experimental studies have demonstrated decreased CGRP levels in diabetes model created by using STZ [17,34,35], and also significantly lower CGRP immunoexpression in perivascular nerves in heart tissues of rats with diabetes [36]. However, in female rats, Dvora´kova´ et al. [37] have found elevated content of CGRP in the heart tissue during the first 4 months of STZ-induced diabetes, as a result of impaired neuronal release and intra-axonal accumulation of the peptide rather than structural degeneration of sensory terminals. In accordance with these studies, the present data reveal significant alterations in plasma VIP and CGRP levels in rats with diabetes, which is probably associated with the combination of lessened expression and impaired release of peptides from nerve terminals. The other important finding of the present study is that GCSF can provide a significant protection against diabetes related neuropathy. This finding is consistent with previous studies that have demonstrated the beneficial effects of G-CSF in some neurological diseases such as ischemic cerebrovascular disease, spinal cord damage and Parkinson’s disease due to its neuroprotective properties. The neuroprotective effects of G-CSF have been mainly attributed to its anti-inflammatory and anti-apoptotic effects [38,39]. Besides, G-CSF increases both stem cell production in bone marrow and their transmission to central nervous system (CNS) [18–20]. In a rat model with ischemic cerebral infarct, it has been mentioned that G-CSF fastened the healing of neurological
381
symptoms by decreasing infarct volume. It has been suggested that G-CSF shows this effect by means of increasing the production of stem cells in bone marrow, facilitating the transmission of increased bone marrow-originated cells to CNS, and increasing angiogenesis and neurogenesis [18]. On the other hand, in a spinal cord injury model, it has been shown that G-CSF repairs neurological functions by inhibiting apoptosis [40]. Similarly, in a rat model with spinal cord injury, Hayashi et al. [19] have reported that G-CSF treatment both induces neurogenesis and oligodendrogliogenesis, and repairs locomotor functions at early stages by increasing the production of brain-derived neurotrophic factor (BDNF). In a clinical study, G-CSF treatment of the patients with acute ischemic stroke has given better results in terms of neurological functions improvement compared to control group after the 12 months of follow-up period [41]. It has been demonstrated that G-CSF inhibits apoptosis by increasing the activation of extracellular-regulated kinase (ERK) signaling pathway [42], which was previously proven to be effective in neuronal cell survival [43]. Also, it has been reported that G-CSF reduces inflammatory activity by inhibiting the production or activity of the main inflammatory mediators interleukin-1, tumor necrosis factor-alpha, and interferon gamma [44]. At this point, its anti-inflammatory effect can be considered as another hypothesis to explain the neuroprotective effect of this agent in diabetic autonomic neuropathy. As can be seen in previous studies, assessment of plasma neuropeptide levels suggests impaired neuropeptide synthesis or release mechanism in diabetes. These findings have led us to think that the related peptides can be used as markers for the diagnosis of diabetes related CAN. Furthermore, in our study, G-CSF treatment was observed to prevent the reduction in plasma levels of these peptides, and hence it was considered as a proof of beneficial effects of G-CSF on this disease. Although this is the first report to assess the effects of G-CSF on cardiovascular neuropathy in a rat model of diabetes, the absence of dose-response studies with varying concentrations of G-CSF and the absence of myocardial analysis of neuropeptides are the main limitations of our study.
5.
Conclusions
This study reports that G-CSF may have beneficial effects on diabetic cardiovascular autonomic neuropathy in rats by means of neuroprotection. In addition, plasma VIP and CGRP levels can be used as valuable laboratory markers of autonomic and sensory innervation in diabetes related CAN. Although the underlying mechanisms of action remain to be elucidated, we can propose that G-CSF may be a novel therapeutic drug in diabetic autonomic neuropathy. However, more comprehensive and detailed further studies are needed to explore the use of G-CSF in the clinical setting of diabetes related CAN.
Conflict of interest statement The authors declare that they have no conflict of interest.
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references
[1] Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4–14. [2] Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Semin Neurol 2003;23:365–72. [3] Ziegler D, Zentai C, Perz S, Rathmann W, Haastert B, Meisinger C, et al. Selective contribution of diabetes and other cardiovascular risk factors to cardiac autonomic dysfunction in the general population. Exp Clin Endocrinol Diabetes 2006;114:153–9. [4] Vinik AI, Erbas T. Cardiovascular autonomic neuropathy: diagnosis and management. Curr Diab Rep 2006;6:424–30. [5] Weimer LH. Autonomic testing: common techniques and clinical applications. Neurologist 2010;16:215–22. [6] Ewing DJ, Boland O, Neilson JM, Cho CG, Clarke BF. Autonomic neuropathy, QT interval lengthening, and unexpected deaths in male diabetic patients. Diabetologia 1991;34:182–5. [7] Tentolouris N, Katsilambros N, Papazachos G, Papadogiannis D, Linos A, Stamboulis E, et al. Corrected QT interval in relation to the severity of diabetic autonomic neuropathy. Eur J Clin Invest 1997;27:1049–54. [8] Rossing P, Breum L, Major-Pedersen A, Sato A, Winding H, Pietersen A, et al. Prolonged QTc interval predicts mortality in patients with Type 1 diabetes mellitus. Diabet Med 2001;18:199–205. [9] Maser RE, Mitchell BD, Vinik AI, Freeman R. The association between cardiovascular autonomic neuropathy and mortality in individuals with diabetes: a meta-analysis. Diabetes Care 2003;26:1895–901. [10] Khoharo HK, Halepoto AW. QTc-interval, heart rate variability and postural hypotension as an indicator of cardiac autonomic neuropathy in type 2 diabetic patients. J Pak Med Assoc 2012;62:328–31. [11] Ejaz A, LoGerfo FW, Pradhan L. Diabetic neuropathy and heart failure: role of neuropeptides. Expert Rev Mol Med 2011;13:e26. http://dx.doi.org/10.1017/S1462399411001979. [12] Ralevic V, Aberdeen JA, Burnstock G. Acrylamide-induced autonomic neuropathy of rat mesenteric vessels: histological and pharmacological studies. J Auton Nerv Syst 1991;34:77–87. [13] Sundkvist G, Bramnert M, Bergstro¨m B, Manhem P, Lilja B, Ahre´n B. Plasma neuropeptide Y (NPY) and galanin before and during exercise in type 1 diabetic patients with autonomic dysfunction. Diabetes Res Clin Pract 1992;15:219–26. [14] Bolinder J, Sjo¨berg S, Persson A, Ahre´n B, Sundkvist G. Autonomic neuropathy is associated with impaired pancreatic polypeptide and neuropeptide Y responses to insulin-induced hypoglycaemia in Type I diabetic patients. Diabetologia 2002;45:1043–4. [15] El-Sayed ZA, Mostafa GA, Aly GS, El-Shahed GS, El-Aziz MM, El-Emam SM. Cardiovascular autonomic function assessed by autonomic function tests and serum autonomic neuropeptides in Egyptian children and adolescents with rheumatic diseases. Rheumatology (Oxford) 2009;48:843–8. [16] Dvora´kova´ MC, Pfeil U, Kuncova´ J, Svı´glerova´ J, Galvis G, Krasteva G, et al. Down-regulation of vasoactive intestinal peptide and altered expression of its receptors in rat diabetic cardiomyopathy. Cell Tissue Res 2006;323:383–93. [17] Zheng LR, Han J, Yao L, Sun YL, Jiang DM, Hu SJ, et al. Up-regulation of calcitonin gene-related peptide protects streptozotocin-induced diabetic hearts from ischemia/ reperfusion injury. Int J Cardiol 2012;156:192–8.
[18] Shyu WC, Lin SZ, Yang HI, Tzeng YS, Pang CY, Yen PS, et al. Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation 2004;110:1847–54. [19] Hayashi K, Ohta S, Kawakami Y, Toda M. Activation of dendritic-like cells and neural stem/progenitor cells in injured spinal cord by GM-CSF. Neurosci Res 2009;64:96–103. [20] Song S, Sava V, Rowe A, Li K, Cao C, Mori T, et al. Granulocyte-colony stimulating factor (G-CSF) enhances recovery in mouse model of Parkinson’s disease. Neurosci Lett 2011;487:153–7. [21] Erbas O, Yilmaz M. Metoprolol and diltiazem ameliorate ziprasidone-induced prolonged corrected QT interval in rats. Toxicol Ind Health 2013. http://dx.doi.org/10.1177/ 0748233713487249. [22] Howarth FC, Adeghate E, Jacobson M. Heart rate and QT interval in streptozotocin-induced diabetic rat. J Med Sci 2009;2:108–18. [23] Singh R, Kishore L, Kaur N. Diabetic peripheral neuropathy: current perspective and future directions. Pharmacol Res 2014;80C:21–35. [24] Veglio M, Chinaglia A, Cavallo-Perin P. QT interval, cardiovascular risk factors and risk of death in diabetes. J Endocrinol Invest 2004;27:175–81. [25] Pappachan JM, Sebastian J, Bino BC, Jayaprakash K, Vijayakumar K, Sujathan P, et al. Cardiac autonomic neuropathy in diabetes mellitus: prevalence, risk factors and utility of corrected QT interval in the ECG for its diagnosis. Postgrad Med J 2008;84:205–10. [26] Edwards JL, Vincent AM, Cheng HT, Feldman EL. Diabetic neuropathy: mechanisms to management. Pharmacol Ther 2008;120:1–34. [27] Okin PM, Devereux RB, Lee ET, Galloway JM, Howard BV. Electrocardiographic repolarization complexity and abnormality predict all-cause and cardiovascular mortality in diabetes. Diabetes 2004;53:434–40. [28] Howarth FC, Jacobson M, Naseer O, Adeghate E. Short-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats. Exp Physiol 2005;90:237–45. [29] Costa EC, Gonc¸alves AA, Areas MA, Morgabel RG. Effects of metformin on QT and QTc interval dispersion of diabetic rats. Arq Bras Cardiol 2008;90:232–8. [30] Vaykshnorayte MA, Ovechkin AO, Azarov JE. The effect of diabetes mellitus on the ventricular epicardial activation and repolarization in mice. Physiol Res 2012;61:363–70. [31] Huang W, Wang Y, Cao YG, Qi HP, Li L, Bai B, et al. Antiarrhythmic effects and ionic mechanisms of allicin on myocardial injury of diabetic rats induced by streptozotocin. Naunyn Schmiedebergs Arch Pharmacol 2013;386:697–704. [32] Noda K, Umeda F, Ono H, Hisatomi A, Chijiiwa Y, Nawata H, et al. Decreased VIP content in peripheral nerve from streptozocin-induced diabetic rats. Diabetes 1990;39: 608–12. [33] Wang LH, Zhou SX, Li RC, Zheng LR, Zhu JH, Hu SJ, et al. Serum levels of calcitonin gene-related peptide and substance P are decreased in patients with diabetes mellitus and coronary artery disease. J Int Med Res 2012;40:134–40. [34] Song JX, Wang LH, Yao L, Xu C, Wei ZH, Zheng LR. Impaired transient receptor potential vanilloid 1 in streptozotocininduced diabetic hearts. Int J Cardiol 2009;134:290–2. [35] Boer PA, Rossi Cde L, Mesquita FF, Gontijo JA. Early potential impairment of renal sensory nerves in streptozotocin-induced diabetic rats: role of neurokinin receptors. Nephrol Dial Transplant 2011;26:823–32. [36] Wharton J, Gulbenkian S, Mulderry PK, Ghatei MA, McGregor GP, Bloom SR, et al. Capsaicin induces a depletion
diabetes research and clinical practice 107 (2015) 377–383
[37]
[38]
[39]
[40]
of calcitonin gene-related peptide (CGRP)-immunoreactive nerves in the cardiovascular system of the guinea pig and rat. J Auton Nerv Syst 1986;16:289–309. Dvora´kova´ MC, Kuncova´ J, Pfeil U, McGregor GP, Svı´glerova´ J, Slavı´kova´ J, et al. Cardiomyopathy in streptozotocininduced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats. Neuroscience 2005;134:51–8. Solaroglu I, Cahill J, Jadhav V, Zhang JH. A novel neuroprotectant granulocyte-colony stimulating factor. Stroke 2006;37:1123–8. Solaroglu I, Tsubokawa T, Cahill J, Zhang JH. Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat. Neuroscience 2006;143(4):965–74. Ha Y, Park HS, Park CW, Yoon SH, Park SR, Hyun DK, et al. Granulocyte macrophage colony stimulating factor
[41]
[42]
[43]
[44]
383
(GM-CSF) prevents apoptosis and improves functional outcome in experimental spinal cord contusion injury. Clin Neurosurg 2005;52:341–7. Shyu WC, Lin SZ, Lee CC, Liu DD, Li H. Granulocyte colony-stimulating factor for acute ischemic stroke: a randomized controlled trial. CMAJ 2006;174:927–33. Huang HY, Lin SZ, Kuo JS, Chen WF, Wang MJ. G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway. Neurobiol Aging 2007;28:1258–69. Nicholson SE, Novak U, Ziegler SF, Layton JE. Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. Blood 1995;86:3698–704. Hartung T. Anti-inflammatory effects of granulocyte colony-stimulating factor. Curr Opin Hematol 1998;5:221–5.
Contents available at ScienceDirect
Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Granulocyte colony-stimulating factor provides protection against cardiovascular autonomic neuropathy in streptozotocin-induced diabetes in rats Oytun Erbas¸ a, Volkan Solmaz b, Dilek Tas¸kiran c,* a
Istanbul Bilim University School of Medicine, Department of Physiology, Istanbul, Turkey Gaziosmanpas¸a University School of Medicine, Department of Neurology, Tokat, Turkey c Ege University School of Medicine, Department of Physiology, Izmir, Turkey b
article info
abstract
Article history:
Aims: Cardiovascular autonomic neuropathy (CAN) is a relatively common and detrimental
Received 9 June 2014
complication of diabetes mellitus (DM). Dysregulation of neuropeptides, such as calcitonin
Received in revised form
gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP), are thought to play
23 October 2014
significant roles in diabetes-related cardiovascular disease. Accumulating evidence indi-
Accepted 29 December 2014
cates the neuroprotective effects of granulocyte-colony stimulating factor (G-CSF) in differ-
Available online 21 January 2015
ent neurological disorders. The purpose of the study is to investigate the role of CGRP and
Keywords:
Methods: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) for 14
VIP and possible effects of G-CSF on CAN in type I DM model in rats. Diabetes mellitus
rats. Seven rats served as controls and 6 rats were administered G-CSF alone. DM group was
Cardiovascular autonomic
randomly divided into 2 groups and received either 1 mL/kg saline (DM + saline group) or
neuropathy
100 mg/kg/day G-CSF (DM + G-CSF group) for 4 weeks. Following electrocardiography (ECG),
Granulocyte-colony stimulating
GCRP and VIP levels were measured in plasma samples.
factor
Results: Diabetes promoted a significant prolongation in the corrected QT interval (cQT)
QT interval
(P < 0.001) whereas G-CSF administration significantly shortened cQT interval (P < 0.05).
Calcitonin gene-related peptide
Plasma VIP and CGRP levels of saline treated DM group were significantly lower than those of
Vasoactive intestinal peptide
control group (P < 0.05). G-CSF treatment significantly prevented the reduction in plasma VIP and CGRP levels (P < 0.01 and P < 0.05, respectively). Also, correlation analysis showed a significant negative correlation between the cQT and neuropeptide levels. Conclusions: This study suggests that G-CSF can be effective in CAN by means of neuroprotection, and plasma VIP and CGRP levels can be used for the assessment of autonomic and sensory functions in diabetes. # 2015 Elsevier Ireland Ltd. All rights reserved.
* Corresponding author at: Ege University School of Medicine, Department of Physiology, 35100 Izmir, Turkey. Tel.: +90 232 390 1800; fax: +90 232 388 2868. ˇ E-mail address: [email protected] (D. Tas¸kˇiran). http://dx.doi.org/10.1016/j.diabres.2014.12.018 0168-8227/# 2015 Elsevier Ireland Ltd. All rights reserved.
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1.
diabetes research and clinical practice 107 (2015) 377–383
Introduction
Diabetic neuropathy is a frequent and severe complication that involves the several clinical syndromes affecting motor, sensory and autonomic nerves [1]. In case of diabetes-related autonomic neuropathy, dysfunction is observed in many systems including cardiovascular, gastrointestinal, genitourinary and neurovascular systems [2]. Cardiovascular autonomic neuropathy (CAN) is a condition, developed in relation to diabetes mellitus (DM) and frequently associated with high mortality. It has been reported that the mortality rate associated with CAN is five times as high as other autonomic involvement [3]. The condition has been estimated to develop more frequently in people with type 2 DM than those with type 1 [4]. In CAN, fatal complications such as cardiac failure and sudden cardiac arrhythmia develop in patients in association with damage to both parasympathetic and sympathetic parts of autonomic nervous system [5]. The QT interval on the electrocardiography (ECG) reflects the total duration of ventricular depolarization and repolarization, and its measurement has been proposed as a simple and noninvasive method for the cardiovascular mortality in various conditions, including diabetes [6–9]. Since the QT interval differs inversely with heart rate, heart rate-corrected QT (cQT) interval is preferably used. In patients with diabetes, cQT prolongation and autonomic dysfunction are closely correlated, and cQT prolongation is considered to be a specific sign of autonomic cardiac dysfunction and high mortality risk [8,10]. Cardioactive neuropeptides, such as calcitonin gene-related peptide (CGRP), atrial natriuretic peptide (ANP), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and substance P (SP), maintain cardiovascular homeostasis through their vasomodulatory properties [11]. In diabetes, the levels of both autonomic and sensory neuropeptides are dysregulated in the myocardium. Accumulating evidence suggests that dysregulated neurohormonal activation, an outcome of neuropathy, has a major pathophysiological role in diabetes-related cardiovascular disease. Many previous studies have revealed that plasma levels of these neuropeptides are lower in those with autonomic neuropathy compared to healthy controls [12– 15]. Among these neuropeptides, VIP, an indicator of parasympathetic function, is stored with acetylcholine in parasympathetic nerves innervating blood vessels. It is also produced and released by intrinsic neurons in the heart, and improves cardiac perfusion and function [16]. CGRP is the major neuropeptide released from sensory nerve terminals in the heart. CGRP has both positive inotropic and potent vasodilatory effects in the heart. Besides, CGRP exerts modulatory effect on peripheral insulin sensitivity and trophic effects on the pancreas, motor neurons and peripheral nerves [11,17]. Granulocyte colony-stimulating factor (G-CSF), which is in widespread clinical use for the treatment of chemotherapyassociated neutropenia, plays a critical role in the regulation of the proliferation, differentiation, and survival of myeloid progenitor cells. Although there are several publications stating the beneficial effects of G-CSF in the treatment of some neurological diseases [18–20], there is a lack of data with regard to the use of this agent in CAN. Considering this, we
examined the role of CGRP and VIP and possible effects of GCSF on CAN in an experimental type I diabetes model in rats.
2.
Materials and methods
2.1.
Animals
In this study, 27 Sprague Dawley albino mature rats, weighing 200–220 g, were used. Animals were fed ad libitum and housed in pairs in plastic cages having a temperature-controlled environment (22 2 8C) with 12-h light/dark cycles. All experimental procedures were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Committee for Animal Research of Ege University.
2.2.
Experimental protocol
Diabetes was induced with a single intraperitoneal (IP) injection of streptozotocin (STZ) (Sigma–Aldrich, Inc.; Saint Louis, MO) for 14 rats. STZ solution (60 mg/kg of body weight) was prepared fresh in citrate buffer (pH = 4.0). Seven animals served as control group and received the buffer alone. Six animals were treated with 100 mg/kg/day G-CSF (Neupogen, 48 mIU/0.5 mL, F. Hoffmann-La Roche Ltd., Switzerland) only to test the effects of G-CSF on the study parameters. Diabetes was verified after 24 h by evaluating tail blood glucose levels with glucose oxidase reagent strips (Boehringer Mannheim, Indianapolis, IN). The rats with 250 mg/dL and higher blood glucose levels were accepted diabetic and included in the study. Then, rats with diabetes were randomly divided into 2 groups; one group was treated with 1 mL/kg saline (n = 7, DM + saline group), and the other group (n = 7, DM + G-CSF group) was treated with 100 mg/kg/day G-CSF. The injections were performed intraperitoneally for 4 weeks. Following ECG, the animals were euthanized and blood samples were collected by cardiac puncture for biochemical analysis. The plasma was separated from cells by centrifugation at 3000 rpm for 10 min, and aliquots were frozen at 30 8C for further use.
2.3.
ECG recording
Rats were anesthetized by a combination of ketamine hydrochloride at a dose of 40 mg/kg (Alfamine1, Ege Vet, Alfasan International B.V. Woerden, Holland) and 4 mg/kg of xylazine hydrochloride (Alfazyne1, Ege Vet, Alfasan International B.V. Woerden, Holland), IP. The electrodes constructed of 26 gauge hypodermic needles were placed subcutaneously in the gently extended limbs of the animal. After the subcutaneous insertion of the electrodes, rats were observed for about 20 min until stabilization of the respiration and ECG patterns. Then, a 5 min ECG was taken in derivation I (DI) using BIOPAC MP 150 system (BIOPAC Systems, Inc., Goleta, CA, USA). During the ECG recordings, rectal temperatures of the rats were monitored by a rectal probe (HP Viridia 24-C; Hewlett-Packard Company, Palo Alto, CA, USA), and the temperature of each rat was kept at approximately 36–37 8C by heating pad. QT interval (duration of ventricular myocardial depolarization and repolarization), T duration and heart rate (BPM-beat per
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diabetes research and clinical practice 107 (2015) 377–383
were significantly higher than those of control group [88 mg/dL (min = 76–max = 106), P < 0.01]. There was no significant difference between the plasma glucose levels of saline and G-CSF group (P > 0.05) (Table 1). Table 1 summarizes the significant changes in ECG recordings of the study groups. The Kruskal–Wallis test showed significant differences among the study groups with regard to heart rate [x2(3) = 11.44, P < 0.01], T duration P < 0.0005], QT duration [x2(3) = 18.23, [x2(3) = 19.94, P < 0.001] and cQT [x2(3) = 21.09, P < 0.005]. Saline treated diabetes group had significantly lower heart rate compared with the control rats (P < 0.05). Furthermore, T duration, QT duration and cQT of saline treated diabetes group were significantly prolonged compared with the control group (P < 0.005, P < 0.005 and P < 0.001, respectively). G-CSF treatment significantly shortened T duration, QT duration and cQT in rats compared to saline treated diabetes group (P < 0.05, P < 0.005 and P < 0.05, respectively). No significant changes were observed in G-CSF administered group (Fig. 1). Fig. 2 represents the alterations in plasma neuropeptide levels of study groups. The analysis of neuropeptide levels with Kruskal–Wallis test revealed significant differences among the groups with regard to CGRP [x2(3) = 9.2, P < 0.05] and VIP [x2(3) = 11.57, P < 0.01] levels. Plasma VIP and CGRP levels of saline treated group were significantly lower than those of control group (P < 0.05 and P < 0.01, respectively). However, treatment of rats with G-CSF for 4 weeks significantly prevented the reduction in plasma VIP and CGRP levels compared to saline treated diabetes group (P < 0.05 and P < 0.01, respectively). No significant changes were found in G-CSF group (Fig. 2). We also examined the association between electrocardiography and biochemical parameters. Pearson’s correlation analysis revealed a significant negative correlation between the cQT and VIP level (r = 0.533, P < 0.05), and CGRP level (r = 0.594, P < 0.01). Fig. 3 demonstrates the correlation analysis between the cQT and plasma neuropeptide levels.
minute) were calculated from the ECG data using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc., Goleta, CA, USA) [21]. The heart rate was determined from the 5-min average of all R–R intervals in the ECG. QT interval was measured from the onset of QRS to end of the T wave. QT interval was further processed to incorporate beat-to-beat changes in the heart rate using Bazett’s formula [22].
2.4.
Measurement of plasma VIP and CGRP levels
Measurements of plasma VIP and CGRP levels were carried out using commercially available enzyme-linked immunosorbent assay (ELISA) kits (Cusabio, Biotech Co., Ltd., Wuhan) specific for rats. All samples from each animal were measured in duplicate according to the manufacturer’s guidelines. The detection limit for each ELISA kit was 2.31 pg/mL, and 7.71 pg/ mL, respectively. Intraassay and interassay coefficients of variation were less than 10% in each determination.
2.5.
Statistical analysis
Data analyses were performed using SPSS software (version 15.0, SPSS Inc., Chicago, IL). Non-parametric tests were used for statistical analysis. Statistical differences in ECG parameters and neuropeptide levels among the groups were tested with the Kruskal–Wallis test. If the difference significant, Mann–Whitney U test with Bonferroni correction was used. Data were presented as median and interquartile range (IQR), which is between the 25th and 75th percentiles. Pearson’s correlation analysis was conducted to examine the association between electrocardiography and biochemical parameters. The value of P < 0.05 was accepted as statistically significant.
3.
Results
In the present study, STZ administration to animals caused typical characteristics of diabetes mellitus, including hyperglycemia, polyuria, and weight loss. The Kruskal–Wallis test showed significant differences among the study groups with regard to glycemia [x2(3) = 15.82, P < 0.001]. Plasma glucose levels of saline treated DM group [398 mg/dl (min = 365–max = 465)] and G-CSF treated DM group [392 mg/dL (min = 325–max = 468)]
4.
Discussion
In the present study, our results revealed that plasma CGRP and VIP levels significantly decreased in rats during the first 4 weeks of experimental type 1 diabetes. Furthermore, we found
Table 1 – Alterations in plasma glucose levels (mg/dL), BMP (beat per minute), cQT (ms), QT duration (ms) and T duration (ms) in study groups. Data are expressed as median (min–max). Control (n = 7) Glucose level (mg/dL) BPM (beat per min) QT duration (ms) cQT (ms) T duration (ms) #
88 256 62 124 29
(76–106) (234–285) (60–69) (112–132) (26–38)
P < 0.05 different from control and G-CSF. P < 0.005 different from control and G-CSF. * P < 0.001 different from control and G-CSF. ** P < 0.05 different from DM + saline. *** P < 0.005 different from DM + saline. ##
G-CSF (n = 6) 99 258 63 130 31
(83–113) (235–289) (57–68) (125–135) (28–30)
DM + saline (n = 7) 398 218 112 187 74
(365–465)## (190–245)# (100–125)## (150–232)* (58–88)##
DM + G-CSF (n = 7) 392 214 83 152 57
(325–468)## (186–235)# (76–91)*** (140–180)** (48–64)**
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diabetes research and clinical practice 107 (2015) 377–383
Fig. 1 – Examples of ECG recordings from the left ventricle of study groups. (a) Control, (b) G-CSF, (c) DM + saline group, and (d) DM + G-CSF group.
Fig. 2 – Measurement of plasma CGRP and VIP levels in control, G-CSF, DM + saline and DM + G-CSF groups. Data are expressed as median with 25th and 75th percentile. Boxes enclose the interquartile range and the median, while whiskers enclose the range (min–max values). # P < 0.01 and *P < 0.05 different from control and G-CSF. ## P < 0.01 and **P < 0.05 different from DM + saline.
a significant negative correlation between the neuropeptide levels and ECG data. The other important finding of our study is that G-CSF, recognized originally as a main regulator of hematopoietic stem cells, can also provide a significant protection against diabetic CAN. It has been well established that sustained elevations of blood glucose levels affect and alter the physiology of many organs, leading to the peripheral nervous system malfunctions. Diabetes related neuropathy could be explained by several mechanisms, such as generation of reactive oxygen species, activation of the polyol pathway, and formation of advanced glycation end products and depletion of the neurotrophic factors [2,23]. Clinically, the electrocardiogram of patients with diabetes may exhibit a number of abnormalities including prolonged QT interval and altered T-waves, which may lead to cardiac arrhythmias and sudden cardiac death [10,24,25]. Numerous abnormalities including autonomic dysfunction, alterations in carbohydrate and lipid metabolism and in electrolyte concentrations may have significant role in the pathogenesis of electrical instability in diabetic heart [2,26]. Experimentally, in a rat heart with diabetes, the most significant electrophysiological change is an increase in action potential duration with a subsequent prolongation of the QT interval. Additionally, prolongation of ventricular action potential duration may produce ST segment and T-wave
changes on the ECG by altering the endocardial–epicardial action potential gradient [27]. In the present study, our results revealed significant reduction in heart rate whereas QT, cQT and T durations were all prolonged in STZ-induced diabetes group compared to controls. In accordance with our present results, reductions in heart rate and/or prolonged QT interval have been previously demonstrated in STZ-induced diabetes model in rodents [22,28–31]. Reviewing the literature, there is much evidence indicating the relationship between neuropeptides and diabetic neuropathy. Also, dysregulation of the expression of neuropeptides or activation of the neuropeptide signaling pathways can harmfully affect cardiac functions. Noda et al. [32] have revealed considerably reduced VIP content in sciatic nerve of diabetic rats that were also negatively correlated with blood glucose levels. Furthermore, significantly reduced VIP levels have been reported in CAN patients with rheumatic diseases [15]. More recently, Dvora´kova´ et al. [16] have provided evidence for progressive decrease of endogenous VIP production in the hearts of rats with diabetes during the first months after onset of diabetes, with concomitant alterations in the expression of VIP receptors. In the heart, CGRP is released from sensory nerve terminals and improves cardiac perfusion and function. Clinically, decreased levels of CGRP and substance B have been reported in patients with diabetes and coronary
diabetes research and clinical practice 107 (2015) 377–383
Fig. 3 – Relationship between corrected QT interval (cQT) and neuropeptide levels. Pearson correlation analysis revealed a significant negative correlation between cQT and CGRP level (P < 0.01) and VIP level (P < 0.05).
artery disease [33]. Experimental studies have demonstrated decreased CGRP levels in diabetes model created by using STZ [17,34,35], and also significantly lower CGRP immunoexpression in perivascular nerves in heart tissues of rats with diabetes [36]. However, in female rats, Dvora´kova´ et al. [37] have found elevated content of CGRP in the heart tissue during the first 4 months of STZ-induced diabetes, as a result of impaired neuronal release and intra-axonal accumulation of the peptide rather than structural degeneration of sensory terminals. In accordance with these studies, the present data reveal significant alterations in plasma VIP and CGRP levels in rats with diabetes, which is probably associated with the combination of lessened expression and impaired release of peptides from nerve terminals. The other important finding of the present study is that GCSF can provide a significant protection against diabetes related neuropathy. This finding is consistent with previous studies that have demonstrated the beneficial effects of G-CSF in some neurological diseases such as ischemic cerebrovascular disease, spinal cord damage and Parkinson’s disease due to its neuroprotective properties. The neuroprotective effects of G-CSF have been mainly attributed to its anti-inflammatory and anti-apoptotic effects [38,39]. Besides, G-CSF increases both stem cell production in bone marrow and their transmission to central nervous system (CNS) [18–20]. In a rat model with ischemic cerebral infarct, it has been mentioned that G-CSF fastened the healing of neurological
381
symptoms by decreasing infarct volume. It has been suggested that G-CSF shows this effect by means of increasing the production of stem cells in bone marrow, facilitating the transmission of increased bone marrow-originated cells to CNS, and increasing angiogenesis and neurogenesis [18]. On the other hand, in a spinal cord injury model, it has been shown that G-CSF repairs neurological functions by inhibiting apoptosis [40]. Similarly, in a rat model with spinal cord injury, Hayashi et al. [19] have reported that G-CSF treatment both induces neurogenesis and oligodendrogliogenesis, and repairs locomotor functions at early stages by increasing the production of brain-derived neurotrophic factor (BDNF). In a clinical study, G-CSF treatment of the patients with acute ischemic stroke has given better results in terms of neurological functions improvement compared to control group after the 12 months of follow-up period [41]. It has been demonstrated that G-CSF inhibits apoptosis by increasing the activation of extracellular-regulated kinase (ERK) signaling pathway [42], which was previously proven to be effective in neuronal cell survival [43]. Also, it has been reported that G-CSF reduces inflammatory activity by inhibiting the production or activity of the main inflammatory mediators interleukin-1, tumor necrosis factor-alpha, and interferon gamma [44]. At this point, its anti-inflammatory effect can be considered as another hypothesis to explain the neuroprotective effect of this agent in diabetic autonomic neuropathy. As can be seen in previous studies, assessment of plasma neuropeptide levels suggests impaired neuropeptide synthesis or release mechanism in diabetes. These findings have led us to think that the related peptides can be used as markers for the diagnosis of diabetes related CAN. Furthermore, in our study, G-CSF treatment was observed to prevent the reduction in plasma levels of these peptides, and hence it was considered as a proof of beneficial effects of G-CSF on this disease. Although this is the first report to assess the effects of G-CSF on cardiovascular neuropathy in a rat model of diabetes, the absence of dose-response studies with varying concentrations of G-CSF and the absence of myocardial analysis of neuropeptides are the main limitations of our study.
5.
Conclusions
This study reports that G-CSF may have beneficial effects on diabetic cardiovascular autonomic neuropathy in rats by means of neuroprotection. In addition, plasma VIP and CGRP levels can be used as valuable laboratory markers of autonomic and sensory innervation in diabetes related CAN. Although the underlying mechanisms of action remain to be elucidated, we can propose that G-CSF may be a novel therapeutic drug in diabetic autonomic neuropathy. However, more comprehensive and detailed further studies are needed to explore the use of G-CSF in the clinical setting of diabetes related CAN.
Conflict of interest statement The authors declare that they have no conflict of interest.
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references
[1] Shaw JE, Sicree RA, Zimmet PZ. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 2010;87:4–14. [2] Vinik AI, Maser RE, Mitchell BD, Freeman R. Diabetic autonomic neuropathy. Semin Neurol 2003;23:365–72. [3] Ziegler D, Zentai C, Perz S, Rathmann W, Haastert B, Meisinger C, et al. Selective contribution of diabetes and other cardiovascular risk factors to cardiac autonomic dysfunction in the general population. Exp Clin Endocrinol Diabetes 2006;114:153–9. [4] Vinik AI, Erbas T. Cardiovascular autonomic neuropathy: diagnosis and management. Curr Diab Rep 2006;6:424–30. [5] Weimer LH. Autonomic testing: common techniques and clinical applications. Neurologist 2010;16:215–22. [6] Ewing DJ, Boland O, Neilson JM, Cho CG, Clarke BF. Autonomic neuropathy, QT interval lengthening, and unexpected deaths in male diabetic patients. Diabetologia 1991;34:182–5. [7] Tentolouris N, Katsilambros N, Papazachos G, Papadogiannis D, Linos A, Stamboulis E, et al. Corrected QT interval in relation to the severity of diabetic autonomic neuropathy. Eur J Clin Invest 1997;27:1049–54. [8] Rossing P, Breum L, Major-Pedersen A, Sato A, Winding H, Pietersen A, et al. Prolonged QTc interval predicts mortality in patients with Type 1 diabetes mellitus. Diabet Med 2001;18:199–205. [9] Maser RE, Mitchell BD, Vinik AI, Freeman R. The association between cardiovascular autonomic neuropathy and mortality in individuals with diabetes: a meta-analysis. Diabetes Care 2003;26:1895–901. [10] Khoharo HK, Halepoto AW. QTc-interval, heart rate variability and postural hypotension as an indicator of cardiac autonomic neuropathy in type 2 diabetic patients. J Pak Med Assoc 2012;62:328–31. [11] Ejaz A, LoGerfo FW, Pradhan L. Diabetic neuropathy and heart failure: role of neuropeptides. Expert Rev Mol Med 2011;13:e26. http://dx.doi.org/10.1017/S1462399411001979. [12] Ralevic V, Aberdeen JA, Burnstock G. Acrylamide-induced autonomic neuropathy of rat mesenteric vessels: histological and pharmacological studies. J Auton Nerv Syst 1991;34:77–87. [13] Sundkvist G, Bramnert M, Bergstro¨m B, Manhem P, Lilja B, Ahre´n B. Plasma neuropeptide Y (NPY) and galanin before and during exercise in type 1 diabetic patients with autonomic dysfunction. Diabetes Res Clin Pract 1992;15:219–26. [14] Bolinder J, Sjo¨berg S, Persson A, Ahre´n B, Sundkvist G. Autonomic neuropathy is associated with impaired pancreatic polypeptide and neuropeptide Y responses to insulin-induced hypoglycaemia in Type I diabetic patients. Diabetologia 2002;45:1043–4. [15] El-Sayed ZA, Mostafa GA, Aly GS, El-Shahed GS, El-Aziz MM, El-Emam SM. Cardiovascular autonomic function assessed by autonomic function tests and serum autonomic neuropeptides in Egyptian children and adolescents with rheumatic diseases. Rheumatology (Oxford) 2009;48:843–8. [16] Dvora´kova´ MC, Pfeil U, Kuncova´ J, Svı´glerova´ J, Galvis G, Krasteva G, et al. Down-regulation of vasoactive intestinal peptide and altered expression of its receptors in rat diabetic cardiomyopathy. Cell Tissue Res 2006;323:383–93. [17] Zheng LR, Han J, Yao L, Sun YL, Jiang DM, Hu SJ, et al. Up-regulation of calcitonin gene-related peptide protects streptozotocin-induced diabetic hearts from ischemia/ reperfusion injury. Int J Cardiol 2012;156:192–8.
[18] Shyu WC, Lin SZ, Yang HI, Tzeng YS, Pang CY, Yen PS, et al. Functional recovery of stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells. Circulation 2004;110:1847–54. [19] Hayashi K, Ohta S, Kawakami Y, Toda M. Activation of dendritic-like cells and neural stem/progenitor cells in injured spinal cord by GM-CSF. Neurosci Res 2009;64:96–103. [20] Song S, Sava V, Rowe A, Li K, Cao C, Mori T, et al. Granulocyte-colony stimulating factor (G-CSF) enhances recovery in mouse model of Parkinson’s disease. Neurosci Lett 2011;487:153–7. [21] Erbas O, Yilmaz M. Metoprolol and diltiazem ameliorate ziprasidone-induced prolonged corrected QT interval in rats. Toxicol Ind Health 2013. http://dx.doi.org/10.1177/ 0748233713487249. [22] Howarth FC, Adeghate E, Jacobson M. Heart rate and QT interval in streptozotocin-induced diabetic rat. J Med Sci 2009;2:108–18. [23] Singh R, Kishore L, Kaur N. Diabetic peripheral neuropathy: current perspective and future directions. Pharmacol Res 2014;80C:21–35. [24] Veglio M, Chinaglia A, Cavallo-Perin P. QT interval, cardiovascular risk factors and risk of death in diabetes. J Endocrinol Invest 2004;27:175–81. [25] Pappachan JM, Sebastian J, Bino BC, Jayaprakash K, Vijayakumar K, Sujathan P, et al. Cardiac autonomic neuropathy in diabetes mellitus: prevalence, risk factors and utility of corrected QT interval in the ECG for its diagnosis. Postgrad Med J 2008;84:205–10. [26] Edwards JL, Vincent AM, Cheng HT, Feldman EL. Diabetic neuropathy: mechanisms to management. Pharmacol Ther 2008;120:1–34. [27] Okin PM, Devereux RB, Lee ET, Galloway JM, Howard BV. Electrocardiographic repolarization complexity and abnormality predict all-cause and cardiovascular mortality in diabetes. Diabetes 2004;53:434–40. [28] Howarth FC, Jacobson M, Naseer O, Adeghate E. Short-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats. Exp Physiol 2005;90:237–45. [29] Costa EC, Gonc¸alves AA, Areas MA, Morgabel RG. Effects of metformin on QT and QTc interval dispersion of diabetic rats. Arq Bras Cardiol 2008;90:232–8. [30] Vaykshnorayte MA, Ovechkin AO, Azarov JE. The effect of diabetes mellitus on the ventricular epicardial activation and repolarization in mice. Physiol Res 2012;61:363–70. [31] Huang W, Wang Y, Cao YG, Qi HP, Li L, Bai B, et al. Antiarrhythmic effects and ionic mechanisms of allicin on myocardial injury of diabetic rats induced by streptozotocin. Naunyn Schmiedebergs Arch Pharmacol 2013;386:697–704. [32] Noda K, Umeda F, Ono H, Hisatomi A, Chijiiwa Y, Nawata H, et al. Decreased VIP content in peripheral nerve from streptozocin-induced diabetic rats. Diabetes 1990;39: 608–12. [33] Wang LH, Zhou SX, Li RC, Zheng LR, Zhu JH, Hu SJ, et al. Serum levels of calcitonin gene-related peptide and substance P are decreased in patients with diabetes mellitus and coronary artery disease. J Int Med Res 2012;40:134–40. [34] Song JX, Wang LH, Yao L, Xu C, Wei ZH, Zheng LR. Impaired transient receptor potential vanilloid 1 in streptozotocininduced diabetic hearts. Int J Cardiol 2009;134:290–2. [35] Boer PA, Rossi Cde L, Mesquita FF, Gontijo JA. Early potential impairment of renal sensory nerves in streptozotocin-induced diabetic rats: role of neurokinin receptors. Nephrol Dial Transplant 2011;26:823–32. [36] Wharton J, Gulbenkian S, Mulderry PK, Ghatei MA, McGregor GP, Bloom SR, et al. Capsaicin induces a depletion
diabetes research and clinical practice 107 (2015) 377–383
[37]
[38]
[39]
[40]
of calcitonin gene-related peptide (CGRP)-immunoreactive nerves in the cardiovascular system of the guinea pig and rat. J Auton Nerv Syst 1986;16:289–309. Dvora´kova´ MC, Kuncova´ J, Pfeil U, McGregor GP, Svı´glerova´ J, Slavı´kova´ J, et al. Cardiomyopathy in streptozotocininduced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats. Neuroscience 2005;134:51–8. Solaroglu I, Cahill J, Jadhav V, Zhang JH. A novel neuroprotectant granulocyte-colony stimulating factor. Stroke 2006;37:1123–8. Solaroglu I, Tsubokawa T, Cahill J, Zhang JH. Anti-apoptotic effect of granulocyte-colony stimulating factor after focal cerebral ischemia in the rat. Neuroscience 2006;143(4):965–74. Ha Y, Park HS, Park CW, Yoon SH, Park SR, Hyun DK, et al. Granulocyte macrophage colony stimulating factor
[41]
[42]
[43]
[44]
383
(GM-CSF) prevents apoptosis and improves functional outcome in experimental spinal cord contusion injury. Clin Neurosurg 2005;52:341–7. Shyu WC, Lin SZ, Lee CC, Liu DD, Li H. Granulocyte colony-stimulating factor for acute ischemic stroke: a randomized controlled trial. CMAJ 2006;174:927–33. Huang HY, Lin SZ, Kuo JS, Chen WF, Wang MJ. G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway. Neurobiol Aging 2007;28:1258–69. Nicholson SE, Novak U, Ziegler SF, Layton JE. Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK. Blood 1995;86:3698–704. Hartung T. Anti-inflammatory effects of granulocyte colony-stimulating factor. Curr Opin Hematol 1998;5:221–5.
