Article 479
Authors
T. Cavusoglu1, 2, T. Karadeniz3, E. Cagiltay4, M. Karadeniz5, G. Yigitturk1, E. Acikgoz1, Y. Uyanikgil1, 2, U. Ates6, M. İ. Tuglu7, O. Erbas8
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ medicine ● ▶ losartan ● ▶ diabetes ● ▶ electromyography ● ▶ histopathological ●
Abstract
received 28.12.2014 first decision 20.04.2015 accepted 05.05.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1550019 Published online: May 26, 2015 Exp Clin Endocrinol Diabetes 2015; 123: 479–484 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence T. Cavusoglu, MD Department of Histology and Embryology Ege University School of Medicine, Izmir, Turkey & Cord Blood, Cell and Tissue Application and Research Center Ege University Izmir Turkey Tel.: + 90/232/3905 911 Fax: + 90/232/3901 649 [email protected]
▼
Aim: Involvement of the peripheral and autonomic nervous systems is possibly the most frequent complication of diabetes. Important risk factors included hyperglycemia, dyslipidemia, hypertension, and smoking. Angiotensinconverting-enzyme inhibitor (ACE) inhibitors should be beneficial in all vascular beds, including neuropathy and retinopathy. In this study we aimed to evaluate the effect of the angiotensin receptor blocker losartan on diabetic neuropathy in a diabetic rat model. Material and Methods: 24 male, Sprague Dawley albino mature rats were divided into 3 groups; (1) control group: No drug was administered to the remainder of rats which blood glucose levels were under 120 mg/dl, (2) diabetic control: rats were given no medication, but 4 ml per day of tap water was given by oral gavage, (3) losartan groups: rats were given 10 mg/kg/ day oral of losartan for 4 weeks. Electromyography (EMG) was applied to anesthetized rats at the end of 4th weekend. Then, the animals were
Introduction
▼
Diabetes is the leading cause of peripheral neuropathy globally. Duration of diabetes, glycemic control, and preexisting cardiovascular risk factors independently correlate with the development and progression of diabetic peripheral neuropathy as well as cardiovascular autonomic neuropathy. Diabetic neuropathies are a family of nerve disorders caused by diabetes. Many trials have shown that intensive glucose control in patients with type 2 diabetes mellitus reduces the progression of microvascular disease [1, 2]. In the peripheral nervous system; sensory neurons, Schwann cells and the microvascular endothelium are vulnerable to oxidative and inflamma-
euthanized and sciatic nerve was performed for histopathological examination. Results: Compound Muscle Action Potential (CMAP) amplitude of diabetic rats receiving the Saline in the EMG was significantly reduced when compared to the control group. Distal latency value and CMAP duration of diabetic rats receiving the saline were meaningfully increased when compared to the control group. CMAP amplitude and CMAP duration of diabetic rats receiving the Losartan treatment in the EMG were meaningfully reduced when compared to diabetic rats receiving the Saline. Perineural thickness in the rats receiving the Losartan treatment was found to be significantly reduced when compared to the group receiving the Saline. Conclusions: As a result, it has been shown in this study that perineural thickness of the Losartan treatment was significantly reduced when compared to saline receiving group, significantly increased the immunoexpression of NGF, and also provided a significantly recovery in EMG when compared to Saline receiving group.
tory stress in the presence of altered metabolic substrates. Good glycemic control can decrease the incidence of diabetic neuropathy, but more than half of all patients with diabetes still develop this complication [3]. Epidemiologic publications have showed the duration and severity of hyperglycemia as important risk factors for the development of diabetic neuropathy [4]. Recent researches have extended the known risk factors beyond hyperglycemia to include dyslipidemia, hypertension, and smoking [5, 6]. More risk factors like a higher body-mass index, higher von Willebrand factor levels and urinary albumin excretion rate are all significantly associated with the cumulative incidence of neuropathy [7]. The EURODIAB Prospective Complications Study
Cavusoglu T et al. The Protective Effect of … Exp Clin Endocrinol Diabetes 2015; 123: 479–484
Downloaded by: Karolinska Institutet. Copyrighted material.
The Protective Effect of Losartan on Diabetic Neuropathy in a Diabetic Rat Model
480 Article
Materials and Methods
▼
Animals
In this study, 24 male Sprague Dawley albino mature rats at 8 weeks, weighing 220–250 gram were used. Animals were fed ad libitum and housed in pairs in steel cages having a temperaturecontrolled environment (22 ± 2 °C) with 12-h light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Celal Bayar University. All animal studies
are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
Experimental protocol
Diabetes was induced by intraperitoneal (i.p.) injection of STZ (Sigma-Aldrich, Inc.; Saint Louis, MO, USA) (60 mg/kg in 0.9 % NaCl, adjusted to a pH 4.0 with 0.2 M sodium citrate) for 16 rats. No drug was administered to the remainder of rats which blood glucose levels were under 120 mg/dl (n = 8) (control group, Group-1). Diabetes was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer- Mannheim, Indianapolis). The rats with blood glucose levels 250 mg/dl and higher were included in this study as diabetic rat group (n = 16). Then, 16 diabetic rats were randomly divided into 2 groups. Group-2 (diabetic control group, 8 rats) was given no medication, but 4 ml per day of tap water was given by oral gavage Group 3 (losartan group, 8 rats) were given 10 mg/kg/day oral of losartan for 4 weeks. Tablet containing 50 mg losartan (Merck Sharp & Dohme) was crushed and suspended in tap water to yield a concentration of 10 mg/ml. According to the weight of each rat, suspended drug solution was completed to 4 ml with tap water. The medications were given via orogastric tubes. EMG was applied to anesthetized rats at the end of 4th weekend. Then, the animals were euthanized and sciatic nerve was performed for histopathological examina▶ Table 1– 4). tion ( ●
Electrophysiological recordings
a
Electromyography (EMG) was obtained for 3 times from the right sciatic nerve stimulated supra-maximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5–5 000 Hz, 40 kHz/s with a sampling rate) by a Biopac bipolar subcutaneous needle stimulation electrode (BIOPAC Systems, Inc., Santa Barbara, CA, USA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded by unipolar needle electrodes located in the 2–3rd interdigital muscle. Data were evaluated by using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc.), with distal latency, duration and amplitude of CMAP as the parameters. During the EMG 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 °C by heating pad.
Histopathological examination of sciatic nervous
b
For histological and immunohistochemical studies, all animals were anesthetized by an i.p. of ketamin (40 mg/kg, (40 mg/kg, Alfamine®, Ege Vet, Alfasan International B.V., Holland)/xylazine (4 mg/kg, Alfazyne®, Ege Vet, Alfasan International B.V., Holland) and perfused with 200 ml of 4 % formaldehyde in 0.1 M phosphate- buffer saline (PBS). Formalin-fixed sciatic nevre sections (5 μm) were stained with hematoxylin and eosin (H&E). All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope. The thickness of the perineurium was measured.
NGF immunoexpression
c Fig. 1 a Control group EMG, b Diabetic and saline treatment EMG, c Diabetic and Losartan treatment EMG.
For immunohistochemistry, sections were incubated with H2O2 (10 %) for 30 min to eliminate endogenous standard activity and blocked with 10 % normal goat serum (Invitrogen) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (NGF, Bioss Inc.; dilution 1/100) for 24 h at
Cavusoglu T et al. The Protective Effect of … Exp Clin Endocrinol Diabetes 2015; 123: 479–484
Downloaded by: Karolinska Institutet. Copyrighted material.
baseline data and other studies claim a strong association between neuropathy and other microvascular complications, suggesting a common pathogenic mechanism [7–10]. Angiotensin receptor blockers (ARB) reverse microalbuminuria and prevent nephropathy. Extending the same analogy, ACE inhibitors should be beneficial in all vascular beds, including neuropathy and retinopathy; and reports exist in their support [11, 12]. The antihypertensive efficacy of losartan, an angiotensin II-antagonist (AT-antagonist), is similar to ACE inhibitors; however, its efficacy in neuropathy has not been extensively studied [13]. Recently, some authors found the AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting. We studied the efficacy of losartan on neuropathy in diabetic rats ▶ Fig. 1, 2). ( ●
Article 481
a
b
c
d
e
f
Table 1 The measurements of compound second and third interdigital muscle action potentials in rats. Data are expressed as mean ± SEM.
Control group Diabetes + saline treatment Diabetes + Losartan treatment
Table 2 The climbing degree and plasma glucose levels of rats. Data are expressed as mean ± SEM.
CMAP Amplitude
Distal
CMAP
Climbing degree
Plasma glucose
(mV)
Latency (ms)
duration
(degree)
(mg/dl)
12.3 ± 2.6 6.5 ± 0.8*
1.18 ± 0.04 1.87 ± 0.18 #
2.27 ± 0.05 3.21 ± 0.03
11.2 ± 1.8**
1.43 ± 0.06 ##
2.48 ± 0.24
Control group Diabetes + saline treatment Diabetes + losartan treatment
75.2 ± 3.2 62.8 ± 2.25*
95.3 ± 5.3 456.8 ± 23.5**
70.1 ± 3.12#
455.2 ± 16.8##
*, p
Authors
T. Cavusoglu1, 2, T. Karadeniz3, E. Cagiltay4, M. Karadeniz5, G. Yigitturk1, E. Acikgoz1, Y. Uyanikgil1, 2, U. Ates6, M. İ. Tuglu7, O. Erbas8
Affiliations
Affiliation addresses are listed at the end of the article
Key words ▶ medicine ● ▶ losartan ● ▶ diabetes ● ▶ electromyography ● ▶ histopathological ●
Abstract
received 28.12.2014 first decision 20.04.2015 accepted 05.05.2015 Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1550019 Published online: May 26, 2015 Exp Clin Endocrinol Diabetes 2015; 123: 479–484 © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York ISSN 0947-7349 Correspondence T. Cavusoglu, MD Department of Histology and Embryology Ege University School of Medicine, Izmir, Turkey & Cord Blood, Cell and Tissue Application and Research Center Ege University Izmir Turkey Tel.: + 90/232/3905 911 Fax: + 90/232/3901 649 [email protected]
▼
Aim: Involvement of the peripheral and autonomic nervous systems is possibly the most frequent complication of diabetes. Important risk factors included hyperglycemia, dyslipidemia, hypertension, and smoking. Angiotensinconverting-enzyme inhibitor (ACE) inhibitors should be beneficial in all vascular beds, including neuropathy and retinopathy. In this study we aimed to evaluate the effect of the angiotensin receptor blocker losartan on diabetic neuropathy in a diabetic rat model. Material and Methods: 24 male, Sprague Dawley albino mature rats were divided into 3 groups; (1) control group: No drug was administered to the remainder of rats which blood glucose levels were under 120 mg/dl, (2) diabetic control: rats were given no medication, but 4 ml per day of tap water was given by oral gavage, (3) losartan groups: rats were given 10 mg/kg/ day oral of losartan for 4 weeks. Electromyography (EMG) was applied to anesthetized rats at the end of 4th weekend. Then, the animals were
Introduction
▼
Diabetes is the leading cause of peripheral neuropathy globally. Duration of diabetes, glycemic control, and preexisting cardiovascular risk factors independently correlate with the development and progression of diabetic peripheral neuropathy as well as cardiovascular autonomic neuropathy. Diabetic neuropathies are a family of nerve disorders caused by diabetes. Many trials have shown that intensive glucose control in patients with type 2 diabetes mellitus reduces the progression of microvascular disease [1, 2]. In the peripheral nervous system; sensory neurons, Schwann cells and the microvascular endothelium are vulnerable to oxidative and inflamma-
euthanized and sciatic nerve was performed for histopathological examination. Results: Compound Muscle Action Potential (CMAP) amplitude of diabetic rats receiving the Saline in the EMG was significantly reduced when compared to the control group. Distal latency value and CMAP duration of diabetic rats receiving the saline were meaningfully increased when compared to the control group. CMAP amplitude and CMAP duration of diabetic rats receiving the Losartan treatment in the EMG were meaningfully reduced when compared to diabetic rats receiving the Saline. Perineural thickness in the rats receiving the Losartan treatment was found to be significantly reduced when compared to the group receiving the Saline. Conclusions: As a result, it has been shown in this study that perineural thickness of the Losartan treatment was significantly reduced when compared to saline receiving group, significantly increased the immunoexpression of NGF, and also provided a significantly recovery in EMG when compared to Saline receiving group.
tory stress in the presence of altered metabolic substrates. Good glycemic control can decrease the incidence of diabetic neuropathy, but more than half of all patients with diabetes still develop this complication [3]. Epidemiologic publications have showed the duration and severity of hyperglycemia as important risk factors for the development of diabetic neuropathy [4]. Recent researches have extended the known risk factors beyond hyperglycemia to include dyslipidemia, hypertension, and smoking [5, 6]. More risk factors like a higher body-mass index, higher von Willebrand factor levels and urinary albumin excretion rate are all significantly associated with the cumulative incidence of neuropathy [7]. The EURODIAB Prospective Complications Study
Cavusoglu T et al. The Protective Effect of … Exp Clin Endocrinol Diabetes 2015; 123: 479–484
Downloaded by: Karolinska Institutet. Copyrighted material.
The Protective Effect of Losartan on Diabetic Neuropathy in a Diabetic Rat Model
480 Article
Materials and Methods
▼
Animals
In this study, 24 male Sprague Dawley albino mature rats at 8 weeks, weighing 220–250 gram were used. Animals were fed ad libitum and housed in pairs in steel cages having a temperaturecontrolled environment (22 ± 2 °C) with 12-h light/dark cycles. The experimental procedures were approved by the Committee for Animal Research of Celal Bayar University. All animal studies
are strictly conformed to the animal experiment guidelines of the Committee for Human Care.
Experimental protocol
Diabetes was induced by intraperitoneal (i.p.) injection of STZ (Sigma-Aldrich, Inc.; Saint Louis, MO, USA) (60 mg/kg in 0.9 % NaCl, adjusted to a pH 4.0 with 0.2 M sodium citrate) for 16 rats. No drug was administered to the remainder of rats which blood glucose levels were under 120 mg/dl (n = 8) (control group, Group-1). Diabetes was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer- Mannheim, Indianapolis). The rats with blood glucose levels 250 mg/dl and higher were included in this study as diabetic rat group (n = 16). Then, 16 diabetic rats were randomly divided into 2 groups. Group-2 (diabetic control group, 8 rats) was given no medication, but 4 ml per day of tap water was given by oral gavage Group 3 (losartan group, 8 rats) were given 10 mg/kg/day oral of losartan for 4 weeks. Tablet containing 50 mg losartan (Merck Sharp & Dohme) was crushed and suspended in tap water to yield a concentration of 10 mg/ml. According to the weight of each rat, suspended drug solution was completed to 4 ml with tap water. The medications were given via orogastric tubes. EMG was applied to anesthetized rats at the end of 4th weekend. Then, the animals were euthanized and sciatic nerve was performed for histopathological examina▶ Table 1– 4). tion ( ●
Electrophysiological recordings
a
Electromyography (EMG) was obtained for 3 times from the right sciatic nerve stimulated supra-maximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5–5 000 Hz, 40 kHz/s with a sampling rate) by a Biopac bipolar subcutaneous needle stimulation electrode (BIOPAC Systems, Inc., Santa Barbara, CA, USA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded by unipolar needle electrodes located in the 2–3rd interdigital muscle. Data were evaluated by using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc.), with distal latency, duration and amplitude of CMAP as the parameters. During the EMG 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 °C by heating pad.
Histopathological examination of sciatic nervous
b
For histological and immunohistochemical studies, all animals were anesthetized by an i.p. of ketamin (40 mg/kg, (40 mg/kg, Alfamine®, Ege Vet, Alfasan International B.V., Holland)/xylazine (4 mg/kg, Alfazyne®, Ege Vet, Alfasan International B.V., Holland) and perfused with 200 ml of 4 % formaldehyde in 0.1 M phosphate- buffer saline (PBS). Formalin-fixed sciatic nevre sections (5 μm) were stained with hematoxylin and eosin (H&E). All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope. The thickness of the perineurium was measured.
NGF immunoexpression
c Fig. 1 a Control group EMG, b Diabetic and saline treatment EMG, c Diabetic and Losartan treatment EMG.
For immunohistochemistry, sections were incubated with H2O2 (10 %) for 30 min to eliminate endogenous standard activity and blocked with 10 % normal goat serum (Invitrogen) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (NGF, Bioss Inc.; dilution 1/100) for 24 h at
Cavusoglu T et al. The Protective Effect of … Exp Clin Endocrinol Diabetes 2015; 123: 479–484
Downloaded by: Karolinska Institutet. Copyrighted material.
baseline data and other studies claim a strong association between neuropathy and other microvascular complications, suggesting a common pathogenic mechanism [7–10]. Angiotensin receptor blockers (ARB) reverse microalbuminuria and prevent nephropathy. Extending the same analogy, ACE inhibitors should be beneficial in all vascular beds, including neuropathy and retinopathy; and reports exist in their support [11, 12]. The antihypertensive efficacy of losartan, an angiotensin II-antagonist (AT-antagonist), is similar to ACE inhibitors; however, its efficacy in neuropathy has not been extensively studied [13]. Recently, some authors found the AT2R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting. We studied the efficacy of losartan on neuropathy in diabetic rats ▶ Fig. 1, 2). ( ●
Article 481
a
b
c
d
e
f
Table 1 The measurements of compound second and third interdigital muscle action potentials in rats. Data are expressed as mean ± SEM.
Control group Diabetes + saline treatment Diabetes + Losartan treatment
Table 2 The climbing degree and plasma glucose levels of rats. Data are expressed as mean ± SEM.
CMAP Amplitude
Distal
CMAP
Climbing degree
Plasma glucose
(mV)
Latency (ms)
duration
(degree)
(mg/dl)
12.3 ± 2.6 6.5 ± 0.8*
1.18 ± 0.04 1.87 ± 0.18 #
2.27 ± 0.05 3.21 ± 0.03
11.2 ± 1.8**
1.43 ± 0.06 ##
2.48 ± 0.24
Control group Diabetes + saline treatment Diabetes + losartan treatment
75.2 ± 3.2 62.8 ± 2.25*
95.3 ± 5.3 456.8 ± 23.5**
70.1 ± 3.12#
455.2 ± 16.8##
*, p
