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Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy Oytun Erbas¸ a, Fatih Oltulu b, Mustafa Yilmaz c, Altug˘ Yavas¸og˘lu b, Dilek Tas¸kiran d,* a
Istanbul Bilim University School of Medicine, Department of Physiology, Istanbul, Turkey Ege University School of Medicine, Department of Histology and Embryology, Izmir, Turkey c Mugla University School of Medicine, Department of Neurology, Mugla, Turkey d Ege University School of Medicine, Department of Physiology, Izmir, Turkey b
article info
abstract
Article history:
Objective: Diabetic neuropathy (DNP) is a frequent and serious complication of diabetes
Received 30 July 2015
mellitus (DM) that leads to progressive and length-dependent loss of peripheral nerve axons.
Received in revised form
The purpose of the present study is to assess the neuroprotective effects of levetiracetam
4 December 2015
(LEV) on DNP in a streptozotocin (STZ)-induced DM model in rats.
Accepted 28 December 2015
Methods: Adult Sprague-Dawley rats were administered with STZ (60 mg/kg) to induce
Available online xxx
diabetes. DNP was confirmed by electromyography (EMG) and motor function test on
Keywords:
21st day following STZ injection. Study groups were assigned as follows; Group 1: Naı¨ve control (n = 8), Group 2: DM + 1 mL/kg saline (n = 12), Group 3: DM + 300 mg/kg LEV (n = 10),
Diabetes mellitus
Group 4: DM + 600 mg/kg LEV (n = 10). LEV was administered i.p. for 30 consecutive days.
Diabetic neuropathy
Then, EMG, motor function test, biochemical analysis (plasma lipid peroxides and total anti-
Levetiracetam
oxidant capacity), histological and immunohistochemical analysis of sciatic nerves (TUNEL
Oxidative stress
assay, bax, caspase 3, caspase 8 and NGF) were performed to evaluate the efficacy of LEV.
Electromyography
Results: Treatment of diabetic rats with LEV significantly attenuated the inflammation and
Apoptosis
fibrosis in sciatic nerves and prevented electrophysiological alterations. Immunohistochemistry of sciatic nerves showed a considerable increase in bax, caspase 3 and caspase 8 and a decrease in NGF expression in saline-treated rats whereas LEV significantly suppressed apoptosis markers and prevented the reduction in NGF expression. Besides, LEV considerably reduced plasma lipid peroxides and increased total anti-oxidant capacity in diabetic rats. Conclusions: The results of the present study suggest that LEV may have therapeutic effects in DNP through modulation of anti-oxidant and anti-apoptotic pathways. # 2016 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Diabetic neuropathy (DNP) is the most common complication of diabetes mellitus (DM), which occurs in more than 50% of
patients and affects nerve fibers of peripheral nervous system. The patients often present with loss of feeling and numbness in their feet, hands, and legs, which may be accompanied by excessive sensitivity to nociceptive stimuli or may perceive normal stimuli as painful [1–3]. To date, numerous
* Corresponding author at: Ege University School of Medicine, Department of Physiology, Bornova, Izmir 35100, Turkey. Tel.: +90 232 3901800; fax: +90 232 3882868. ˇ E-mail address: [email protected] (D. Tas¸kˇiran). http://dx.doi.org/10.1016/j.diabres.2015.12.016 0168-8227/# 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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mechanisms have been proposed to explain the relationship between the severity of hyperglycemia and the development of DNP including increased polyol pathway activity which leads to accumulation of sorbitol and fructose, reduction in Na+K+–ATPase activity, abnormal protein kinase C (PKC) activity, formation of advanced glycation end-products and auto-oxidation of glucose leading to the generation of reactive oxygen species [1–4]. In addition, metabolic dysfunction is accompanied by vascular deficiency and nerve hypoxia, which may contribute to nerve fiber loss and injury in diabetes. Based on these studies, various therapeutic agents including aldose reductase inhibitors (ARIs), anti-oxidants, selective PKC inhibitors, and neurotrophic factors have been used to improve peripheral nerve dysfunction in diabetic animals and patients [5–8]. Levetiracetam (LEV), an analogue of the nootropic agent piracetam, is an effective antiepileptic drug. Although the molecular effects of LEV remain uncharacterized, various studies indicate that it regulates the influx of calcium into the cells, selectively blocking N-type, but not the T-type channel [9–11]. Besides, it modulates membrane depolarization and prevents irreversible cellular damage via reducing the flow of potassium within the cell [12]. It has been reported that it binds synaptic vesicle protein (SV2A) and produce neuroprotective effects via modulating SV2A function [13]. On the other hand, LEV may have a direct ability to protect cells against kainic acid-induced toxicity via inhibition of lipid peroxidation [14]. To date, numerous clinical and experimental studies have suggested the protective effects of LEV on hypoxic ischemic brain injury, traumatic brain injury, subarachnoid hemorrhage and post-stroke epilepsy [15–17]. However, there is still limited data concerning its potential neuroprotective properties against peripheral neuropathies, such as DNP. In the present study, we hypothesized that utilization of different doses of LEV might have beneficial effects on peripheral nerve damage in diabetes. To accomplish this, we tested the effects of low and high doses of LEV on the functional and architectural properties of the sciatic nerve in Type I diabetic rats using electrophysiological, histological, immunohistochemical and biochemical parameters.
2.2.
Study design
Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin (STZ; 60 mg/kg, Sigma-Aldrich Inc., St. Louis, MO) following an overnight fast. STZ was prepared in 0.9% NaCl and adjusted to a pH 4.5 with citric acid [18]. Diabetic state was verified 48 h later by determining tail vein blood glucose levels by glucose oxidase reagent strips (BoehringerMannheim, Indianapolis, IN). Animals showing blood glucose levels above 250 mg/dL were included in the study. Eight rats served as naı¨ve control group and received no treatment. Control and diabetic rats were housed in their cages for 20 days. On day 21, EMG recordings were performed from the sciatic nerve under ketamine/xylazine anesthesia to confirm DNP. Following EMG studies on day 21, rats were divided into 4 groups. Group 1: Naı¨ve control (n = 8), Group 2: DM + 1 mL/kg saline (n = 12), Group 3: DM + 300 mg/kg LEV (n = 10), Group 4: DM + 600 mg/kg LEV (n = 10). LEV (Keppra Flakon, UCB Farma) was diluted in saline and administered i.p. for 30 consecutive days. The doses were selected based on previous studies [19,20]. Following these treatments, gross motor test and EMG were performed. Blood samples were collected for biochemical analysis, and then animals were perfused for histology, quantitative immunohistochemistry (bax, caspase-3, caspase8, NGF) and TUNEL staining.
2.3.
Electrophysiological recordings (EMG)
2.
Materials and methods
Rats were anesthetized by combination of ketamine hydrochloride at a dose of 40 mg/kg (Alfamine1, Alfasan International B.V. Holland) and 4 mg/kg of xylazine hydrochloric (Alfazyne1, Alfasan International B.V. Holland). EMG was obtained for three times at the same time point from the right sciatic nerve stimulated supra-maximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5– 5000 Hz, 40 kHz/s with a sampling rate) by a Biopac bipolar subcutaneous needle stimulation electrode (BIOPAC Systems, Inc., Santa Barbara, CA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded from 2 to 3. Interosseous muscle by unipolar needle electrodes. Data were evaluated using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc., Santa Barbara, CA), 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 8C to 37 8C by heating pad [20].
2.1.
Animals
2.4.
Forty-two adult male Sprague Dawley rats weighing 210– 230 g (221.4 1.89 g) at the beginning of the experiments were used. The rats were housed in cages and maintained under standard conditions with 12-h light/dark cycles at room temperature (22 2 8C). They were fed by standard pellet diet and tap water ad libitum throughout the study. All animal care and experimental procedures were approved by the Institutional Animal Care and Ethical Committee. All chemicals were obtained from Sigma-Aldrich Inc. unless otherwise noted.
Assessment of gross motor function
The motor performances of the rats were evaluated by inclined-plate test according to the method described by Rivlin and Tator [21]. The device was an 18 18 cm2 platform, which could be adjusted to provide a slope of varying degrees. Briefly, the rats were placed with their body axis perpendicular to the inclined plane. The initial angle of the inclined plate was 50 degrees. The incline angle slowly increased and the maximum angle of the plate on which the rat preserved its position for 5 s without falling was recorded as motor score. This procedure was repeated five times per
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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rat, and the average value of the climbing angles was recorded.
2.5.
Histology and quantitative immunohistochemistry
Rats were perfused intracardially with 4% formaldehyde for histology and quantitative immunohistochemistry. Briefly, sciatic nerves were embedded in paraffin, sectioned at 5 mm thickness via microtome (Leica RM 2145) and stained with hematoxylin-eosin. All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope. The thickness of the sciatic nerve perineurium was measured using Image-Pro Express 1.4.5 (Media Cybernetics, Inc. USA). For immunohistochemical examination, sections were incubated with 10% H2O2 for 30 min to eliminate endogenous peroxidase activity and then blocked with 10% normal goat serum (Invitrogen, Life Technologies, Waltham, MA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (1/100; Santacruz Biotechnology Inc., Dallas, TX) against bax, caspase-3, caspase-8, nerve growth factor (NGF) for 24 h at 4 8C. Antibody detection was carried out with the Histostain-Plus Bulk kit (Invitrogen) against rabbit IgG and 3,30 -diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS, examined under Olympus BX51 microscope and photographed with Olympus C-5050 digital camera. Twelve animals (3 per group) and six sections from each animal were used for quantitative immunohistochemistry. Two blinded observers counted the total immune-positive Schwann cells under a light microscope at 100 magnification.
2.6.
TUNEL assay
The apoptosis in Schwann cells was detected by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) using the ApopTag Red in situ apoptosis detection kit (S7165, Chemicon, Billerica, MA) according to the manufacturer’s instructions. Schwann cells were considered positive when the nuclei stained positively and showed chromatin clumping. For quantitative assessment of apoptosis, TUNEL(+) cells were counted in each section at 100 magnification.
2.7.
Biochemical analysis
The oxidant and anti-oxidant status of diabetic and LEVtreated rats were assessed by measuring lipid peroxidation
and total antioxidant capacity (TAC) in plasma samples. Lipid peroxidation was determined by measuring malondialdehyde (MDA) levels as thiobarbituric acid reactive substances [22]. Plasma TAC was measured by ferric reducing antioxidant power (FRAP) assay according to Benzie and Strain [23].
2.8.
Statistical analysis
Data analyses were performed using SPSS for Windows, version 15.0. Results were presented as mean standard error of mean (SEM). All data were analyzed by one-way analysis of variance (ANOVA). Post-hoc testing of pairwise comparisons was performed using the Bonferroni test. The differences were considered statistically significant when p < 0.05.
3.
Results
3.1. Blood glucose levels, body weight and motor performance All animals were monitored daily for behavior and health conditions throughout the study. Two rats died in diabetic group within 48 h following STZ injection. As expected, STZreceived rats displayed typical symptoms of diabetes such as loss of body weight, polydipsia, and polyuria. The alterations in blood glucose levels, body weights and motor performance were summarized in Table 1. ANOVA results revealed significant differences between the groups ( p < 0.0005 for glycaemia, p < 0.0005 for body weight and p < 0.0005 for motor performance). All STZ-received rats showed higher blood glucose levels than those of controls ( p < 0.0005). The increase in blood glucose was observed as early as 48 h after STZ injection and was preserved during the study period. In addition, LEV treatment did not affect blood glucose levels. Baseline body weights of the animals were similar in all study groups (221.4 1.89 g). At the end of the experimental period, the saline-treated diabetic rats revealed a marked decrease in body weight when compared with naı¨ve control group (130 6.97 and 234.29 3.42, respectively; p < 0.0005). However, LEV-treated diabetic rats significantly gained weight compared to saline-treated group ( p < 0.0005). We used the inclined plate method to assess the effects of LEV treatment on gross motor performance of the rats. The diabetic rats exhibited significantly poorer scores than those of controls (59.38 1.25 vs. 69.75 0.81; p < 0.0005). However,
Table 1 – The changes in body weights, blood glucose levels and inclined plate scores in the study groups.
Naı¨ve control DM (day 2) DM (day 21) DM + saline DM + LEV (300 mg/kg) DM + LEV (600 mg/kg)
Body weight (g)
Blood glucose (mg/dL)
Inclined plate score (8)
234.29 3.42* 191.42 7.96 160.42 4.78 130 6.97 176.43 5.98# 199.29 9.75#
130.14 5.25* 499.42 17.74 501.43 22.92 508.57 23.50 516.14 18.76 522.57 28.68
69.43 0.78 70 0.67 60.43 1.33* 59.29 1.35* 66.29 0.65 67.86 0.76
One-way ANOVA and post-hoc Bonferroni test demonstrated significant differences between the groups. Data are expressed as mean SEM. p < 0.0005 different from all other groups. # p < 0.0005 different from saline group. *
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Table 2 – The alterations in compound muscle action potentials (CMAPs) parameters in the study groups.
Naı¨ve control DM (day 21) DM + saline DM + LEV (300 mg/kg) DM + LEV (600 mg/kg)
CMAP duration (ms)
CMAP amplitude (mV)
CMAP latency (ms)
2.34 0.04 3.89 0.22* 4.05 0.11* 3.73 0.10 3.36 0.09#
11.30 0.80 6.24 0.45* 5.94 0.49* 9.38 0.57# 10.23 0.69#
1.38 0.02 2.04 0.10* 2.06 0.17* 1.65 0.06y 1.66 0.04y
One-way ANOVA and post-hoc Bonferroni test demonstrated significant differences between the groups. Data are expressed as mean SEM. p < 0.0005 different from all other groups. # p < 0.005 different from DM + saline and DM (day 21) group. y p < 0.05 different from DM + saline and DM (day 21) group. *
Table 3 – The quantitative evaluation of epineurium thickness of sciatic nerves in the study groups. Naı¨ve control Epineurium thickness (mm)
2.16 0.14
DM + saline 16.54 2.80*
#
DM + LEV (300 mg/kg)
DM + LEV (600 mg/kg)
12.85 1.63
10.73 1.49
One-way ANOVA and post-hoc Bonferroni test revealed significant differences between the groups. Data are expressed as mean SEM. * p < 0.0005 different from naı¨ve control and DM + LEV (600 mg/kg) group. # p < 0.05 different from DM + LEV (300 mg/kg).
both 300 and 600 mg/kg LEV successfully improved motor performance in diabetic rats (66.25 0.61 and 67.63 0.75, respectively; p < 0.0005).
3.2.
EMG recordings
Table 2 describes the changes in EMG parameters of the study groups. ANOVA results showed significant differences between the groups ( p < 0.0005 for CMAP amplitude, CMAP duration, and distal latency). Post-hoc Bonferroni test revealed significant differences in CMAP amplitude, duration and distal latency in saline-treated diabetic rats when compared with controls ( p < 0.0005). The peak-to-peak amplitude of sciatic nerve CMAP, which essentially reflects axonal dysfunction, was reduced by 52% in diabetic rats; however, administration of both 300 and 600 mg/kg LEV significantly counteracted this reduction ( p < 0.005). Similarly, distal latency was significantly shortened ( p < 0.05) in LEV-administrated diabetic rats as compared with saline-treated rats. However, CMAP duration was reduced only in 600 mg/kg LEV group when compared with saline-treated diabetic rats ( p < 0.005), (Fig. 1).
3.3.
Morphological evaluation
Light microscopic examination of the sciatic nerves in the naı¨ve control group exhibited normal myelination and morphology while saline-treated diabetic group showed severe alterations including axonal degeneration, myelin distention, and perineurial fibrosis (Fig. 2). Quantitatively, a marked increase was observed in the thickness of perineurium of saline-treated diabetic rats (16.54 2.8 mm). Administration of 300 and 600 mg/kg LEV significantly lessened the fibrotic changes in sciatic nerve (12.85 1.63 mm and 10.73 1.49 mm; p < 0.005 and p < 0.0005, respectively), (Tables 1 and 3).
3.4.
Immunohistochemistry
To evaluate the role of apoptosis in the development of DNP, we determined the expression of bax, caspase 3, and caspase 8
immunohistochemically in the sections of sciatic nerve. Figs. 3–5 demonstrate the immunuhistochemical alterations in the study groups. ANOVA results of quantitative immunohistochemistry revealed significant differences between the study groups ( p < 0.0005 for bax, caspase 3, caspase 8, and NGF). Post-hoc test showed that while the expression of apoptotic markers including bax, caspase 3 and caspase 8 were prominently high in the saline-treated diabetic rats ( p < 0.0005), administration of 300 and 600 mg/kg LEV significantly lessened the expression of apoptotic markers compared to saline group ( p < 0.0005). In addition, no significant differences were found between the low and high doses of LEV treatments in terms of its suppressing effect on apoptotic markers. NGF expression was significantly decreased in salinetreated group ( p < 0.0005). LEV administration significantly and dose-dependently increased NGF expression in sciatic nerves of diabetic rats ( p < 0.005 and p < 0.0005), (Fig. 6).
3.5.
Tunel staining
Apoptosis was also assessed by counting the number of TUNEL (+) in the sections of sciatic nerve. ANOVA results showed significant differences between the study groups ( p < 0.0005). Diabetic rats treated with saline demonstrated significantly higher TUNEL (+) cells than those of controls ( p < 0.0005) while treatment of diabetic rats with LEV considerably lessened apoptotic cell death ( p < 0.0005), (Fig. 7).
3.6.
Evaluation of oxidative stress
Table 4 summarizes the changes in plasma lipid peroxides (MDA) and total antioxidant capacity (TAC). MDA levels were significantly elevated (0.36 0.05 mM vs. 0.13 0.01 mM; p < 0.0005), whereas plasma TAC was significantly decreased (38.09 6.33 mM vs. 72.28 5.22 mM; p < 0.05) in saline-treated diabetic rats compared to control group. On the contrary, treatment of diabetic rats with 300 mg/kg LEV significantly attenuated plasma MDA levels (0.22 0.01 mM), and improved
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 1 – Samples of CMAP recorded from (a) naı¨ve control, (b) saline-treated DM, (c) 300 mg/kg LEV-treated DM, and (d) 600 mg/kg LEV-treated DM.
diabetes research and clinical practice xxx (2016) xxx–xxx
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 2 – The histological evaluation of sciatic nerve. (a and b) naı¨ve control, (c and d) saline-treated DM, (e and f) 300 mg/kg LEVtreated DM, and (g and h) 600 mg/kg LEV-treated DM. Saline-treated group showed significant inflammation and fibrosis. p: perineurium, ax: axon, sw: Schwann cell, en: endoneurium, f: fibrosis. Hematoxylen and eosine staining (10T and 40T).
TAC (87.18 12.31 mM) when compared with those in the saline-treated diabetic rats.
4.
Discussion
In the present study, we aimed to evaluate the neuroprotective and/or neuroreparative effects of chronic treatment of LEV on
Table 4 – The biochemical evaluation of oxidative stress markers in plasma samples.
Naı¨ve control DM + saline DM + LEV (300 mg/kg)
MDA (mM)
TAC (mM)
0.13 0.01 0.36 0.05* 0.22 0.01
72.28 5.22 38.09 6.33# 87.18 12.31##
One-way ANOVA and post-hoc Bonferroni test revealed significant differences between the groups. Data are expressed as mean SEM. * p < 0.0005 different from all other groups. # p < 0.05 different from naı¨ve control. ## p < 0.005 different from DM + saline.
the development of DNP in rats. Electrophysiological, histological and biochemical findings clearly established that LEV could lessen the neurodegenerative changes in peripheral nerves due to diabetes. It has been well established that sustained elevations of blood glucose levels affect and alter the physiology of many organs, leading indirectly to the peripheral nervous system malfunctions. Hyperglycemia-induced oxidative stress and the subsequent increase in free radical production lead to cellular dysfunction and neuronal cell loss both via activation of apoptosis or necrotic cell death [3,24,25]. As indicated in previous studies, DNP is related with changes in the perineurium, including thickening of the basement membrane of the perineurial cells. Electrophysiological measurements reflect the underlying pathology and alterations in nerve conduction velocity (NCV) or amplitude correlate with myelinated nerve fiber density. There have been several studies in diabetic patients and animal models that indicate reduced NCV, CMAP amplitude, and prolonged CMAP latency in sciatic nerve [26–28]. Streptozotocin induced diabetes in rats is the most extensively studied animal model of DNP. It leads a significant lessening in nerve blood flow and
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 3 – Bax immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEVtreated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate bax (+) cells. Saline-treated group showed increased number of bax (+) cells. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M.
Fig. 4 – Caspase 3 immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEV-treated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate caspase 3 (+) cells. Salinetreated group displayed significantly higher caspase 3 expression compared to naı¨ve controls. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M. Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 5 – Immunoexpression of caspase 8 in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEV-treated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate caspase 8 (+) cells. Salinetreated group showed increased expression of caspase 8 compared to naı¨ve controls. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M.
Fig. 6 – NGF immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEVtreated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Saline-treated group displays reduced NGF immunoreactivity. # p < 0.0005 different from control, * p < 0.005 different from saline-treated group, ** p < 0.0005 different from saline-treated group. Data represent mean W S.E.M. Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 7 – Effect of LEV treatment on the number of apoptotic cells. (a and b) naı¨ve control, (c and d) saline-treated DM, (e and f) 300 mg/kg LEV-treated DM, and (g and h) 600 mg/kg LEV-treated DM. Black arrows indicate TUNEL (+) cells. The number of TUNEL (+) (apoptotic) cells in the sciatic nerve sections was significantly increased in saline-treated DM group (* p < 0.0005). The number of TUNEL (+) cells decreased with LEV-treatment. ** p < 0.0005 different from saline-treated group. Data represent mean W S.E.M.
slowing of NCV, followed by axonal loss of both the motor and sensory nerve fibers [29]. In line with previous reports, we observed significantly reduced CMAP amplitude and elongation in CMAP latency in saline-treated diabetic rats compared to naı¨ve rats. Furthermore, histological findings of neuropathy, such as axonal degeneration, myelin distention, and perineurial fibrosis, are evident in the sciatic nerve of diabetic rats. In the present study, in order to elucidate the mechanisms underlying neuroaxonal changes in DNP we evaluated particular apoptotic indicators such as bax, caspase 3, caspase 8 and Tunel assay. The qualitative and quantitative immunohistochemical assessment of sciatic nerves clearly confirmed the apoptotic death of Schwann cells in saline-treated diabetic rats. Besides, NGF immunoexpression, which is crucial for survival of neurons, was significantly reduced in diabetic group. On the other hand, the measurement of plasma lipid peroxides (MDA) and total anti-oxidant capacity (TAC) demonstrated a significant imbalance between oxidative and anti-oxidative status in saline- treated diabetic rats. There is growing support for the neuroprotective effects of conventional, as well as recently introduced, antiepileptic agents in experimental models of hypoxic/ischemic and traumatic brain injury [16,17,30,31]. It has been suggested that these drugs exert their effects by modulating of GABAergic and glutamatergic transmission (via inhibition of voltage-dependent ion-channels), receptors and secondary messengers [31–33]. LEV, a second-generation new antiepileptic drug, is used for both focal and generalized epilepsy. To date, several in vitro and in vivo studies have indicated the
neuroprotective effects of LEV due to its anti-apoptotic, antioxidative and anti-inflammatory properties [30,34–36]. For instance, in a pilocarpine-induced injury model, it has been demonstrated that LEV could counteract oxidative stress by lessening lipid peroxidation and nitric oxide generation, and also preserving catalase activity in the hippocampus [36]. More recently, Ueda et al. have revealed that LEV could modify the expressions of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS) in connection with lipid peroxidation and also enhance basal endogenous antioxidant ability in the hippocampus [35]. Evidence suggests that LEV has therapeutic effects on neuropathic pain models in mice [19,37]. LEV has been found to generate a dose- (20–200 mg/kg) and time-dependent (0– 75 min) inhibition of the diabetes-induced hyperalgesia [19]. Also, it has been showed that LEV (10–100 mg/kg) could exert synergistic interactions with analgesics in producing antinociception in diabetic mice that developed thermal/mechanical hypersensitivity [37]. In our model, we used two different doses (300 mg/kg and 600 mg/kg) of LEV to evaluate its protective effects against diabetes-induced alterations in peripheral nerves. These doses were chosen based on the results of our previous study, which indicated a beneficial effect of LEV in a model of sepsisinduced polyneuropathy in rats. Our results confirmed that both doses of LEV caused a significant reduction in the number of apoptotic cells and immunoexpression of bax, caspase-3 and capase-8 in sciatic nerve. Another important finding of our study was that NGF immunoexpression in the Schwann cells
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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was significantly preserved in LEV treated diabetic rats compared to saline-treated group. Besides, histomorphometric examination of sciatic nerve showed a significant reduction in fibrosis in LEV treated diabetic rats. The assessment of oxidative and anti-oxidative status of study groups revealed that LEV was able to decrease plasma lipid peroxides and enhance total antioxidant capacity in diabetic rats. In addition to biochemical and histological findings, our results showed that LEV could successfully improve the electrophysiological parameters including CMAP duration, amplitude and latency in diabetic rats. Overall, these results suggest a direct evidence of cytoprotective potential of LEV against oxidative stress and apoptotic cell death in diabetes related neuropathy in rats. Previously, several experimental models have established that inhibitors of ion channels could lessen inflammation and apoptotic cell death [30,34]. However, the probable mechanisms of the anti-apoptotic effects of LEV in neuronal damages are not completely understood. In an in vitro study, Stettner et al. have demonstrated the potential of LEV to protect Schwann cells from oxidative stress and inflammation through reducing tumor necrosis factor alpha (TNF-a) and matrix metalloproteinase 9 (MMP-9) expression and caspase-6 activity [34]. In a neonatal rat model of hypoxic ischemic brain injury (HIBI), it has been reported that LEV treatment following hypoxic ischemia could lead to a significant reduction in the number of apoptotic cells [30]. Similar to these findings, Komur et. al., showed that LEV can act as a neuroprotective agent by lessening the number of apoptotic neurons in rat model of HIBI in the early period. They also found that administration of rats with LEV significantly reduced the plasma lipid peroxides and improved anti-oxidant enzyme activities such as glutathione peroxidase and superoxide dismutase [17]. We also consider the importance of LEV receptor, SV2A, in mediation of neuroprotection and neurorepair, as had been suggested by previous studies [13,38,39]. SV2A receptor protein has been implied as a presynaptic calcium sensor and a transmitter vesicular uptake and release moderator like synaptotagmin. Without SV2A, presynaptic calcium accumulates in neurons and leads to abnormal increases in the neurotransmitter release. Therefore, the binding of the SV2A receptor with LEV may provide the ability of a neuron to lessen excessive glutamate release [13,37]. However, further studies are required to define the precise mechanism underlying the neuroprotective effects of LEV through SV2A modulation. Taken together, our results provide in vivo evidence that LEV can effectively reduces neuronal damage in diabetic rats through its anti-oxidant and anti-apoptotic properties. On the basis of our findings, we can propose that lower doses LEV may be as efficacious as higher doses in preventing of DNP. Future experimental and clinical studies that evaluate its efficacy and long-term safety may encourage the application of LEV as a treatment modality for diabetic neuropathy.
Conflict of interest statement The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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Diabetes Research and Clinical Practice journ al h ome pa ge : www .elsevier.co m/lo cate/diabres
Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy Oytun Erbas¸ a, Fatih Oltulu b, Mustafa Yilmaz c, Altug˘ Yavas¸og˘lu b, Dilek Tas¸kiran d,* a
Istanbul Bilim University School of Medicine, Department of Physiology, Istanbul, Turkey Ege University School of Medicine, Department of Histology and Embryology, Izmir, Turkey c Mugla University School of Medicine, Department of Neurology, Mugla, Turkey d Ege University School of Medicine, Department of Physiology, Izmir, Turkey b
article info
abstract
Article history:
Objective: Diabetic neuropathy (DNP) is a frequent and serious complication of diabetes
Received 30 July 2015
mellitus (DM) that leads to progressive and length-dependent loss of peripheral nerve axons.
Received in revised form
The purpose of the present study is to assess the neuroprotective effects of levetiracetam
4 December 2015
(LEV) on DNP in a streptozotocin (STZ)-induced DM model in rats.
Accepted 28 December 2015
Methods: Adult Sprague-Dawley rats were administered with STZ (60 mg/kg) to induce
Available online xxx
diabetes. DNP was confirmed by electromyography (EMG) and motor function test on
Keywords:
21st day following STZ injection. Study groups were assigned as follows; Group 1: Naı¨ve control (n = 8), Group 2: DM + 1 mL/kg saline (n = 12), Group 3: DM + 300 mg/kg LEV (n = 10),
Diabetes mellitus
Group 4: DM + 600 mg/kg LEV (n = 10). LEV was administered i.p. for 30 consecutive days.
Diabetic neuropathy
Then, EMG, motor function test, biochemical analysis (plasma lipid peroxides and total anti-
Levetiracetam
oxidant capacity), histological and immunohistochemical analysis of sciatic nerves (TUNEL
Oxidative stress
assay, bax, caspase 3, caspase 8 and NGF) were performed to evaluate the efficacy of LEV.
Electromyography
Results: Treatment of diabetic rats with LEV significantly attenuated the inflammation and
Apoptosis
fibrosis in sciatic nerves and prevented electrophysiological alterations. Immunohistochemistry of sciatic nerves showed a considerable increase in bax, caspase 3 and caspase 8 and a decrease in NGF expression in saline-treated rats whereas LEV significantly suppressed apoptosis markers and prevented the reduction in NGF expression. Besides, LEV considerably reduced plasma lipid peroxides and increased total anti-oxidant capacity in diabetic rats. Conclusions: The results of the present study suggest that LEV may have therapeutic effects in DNP through modulation of anti-oxidant and anti-apoptotic pathways. # 2016 Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
Diabetic neuropathy (DNP) is the most common complication of diabetes mellitus (DM), which occurs in more than 50% of
patients and affects nerve fibers of peripheral nervous system. The patients often present with loss of feeling and numbness in their feet, hands, and legs, which may be accompanied by excessive sensitivity to nociceptive stimuli or may perceive normal stimuli as painful [1–3]. To date, numerous
* Corresponding author at: Ege University School of Medicine, Department of Physiology, Bornova, Izmir 35100, Turkey. Tel.: +90 232 3901800; fax: +90 232 3882868. ˇ E-mail address: [email protected] (D. Tas¸kˇiran). http://dx.doi.org/10.1016/j.diabres.2015.12.016 0168-8227/# 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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mechanisms have been proposed to explain the relationship between the severity of hyperglycemia and the development of DNP including increased polyol pathway activity which leads to accumulation of sorbitol and fructose, reduction in Na+K+–ATPase activity, abnormal protein kinase C (PKC) activity, formation of advanced glycation end-products and auto-oxidation of glucose leading to the generation of reactive oxygen species [1–4]. In addition, metabolic dysfunction is accompanied by vascular deficiency and nerve hypoxia, which may contribute to nerve fiber loss and injury in diabetes. Based on these studies, various therapeutic agents including aldose reductase inhibitors (ARIs), anti-oxidants, selective PKC inhibitors, and neurotrophic factors have been used to improve peripheral nerve dysfunction in diabetic animals and patients [5–8]. Levetiracetam (LEV), an analogue of the nootropic agent piracetam, is an effective antiepileptic drug. Although the molecular effects of LEV remain uncharacterized, various studies indicate that it regulates the influx of calcium into the cells, selectively blocking N-type, but not the T-type channel [9–11]. Besides, it modulates membrane depolarization and prevents irreversible cellular damage via reducing the flow of potassium within the cell [12]. It has been reported that it binds synaptic vesicle protein (SV2A) and produce neuroprotective effects via modulating SV2A function [13]. On the other hand, LEV may have a direct ability to protect cells against kainic acid-induced toxicity via inhibition of lipid peroxidation [14]. To date, numerous clinical and experimental studies have suggested the protective effects of LEV on hypoxic ischemic brain injury, traumatic brain injury, subarachnoid hemorrhage and post-stroke epilepsy [15–17]. However, there is still limited data concerning its potential neuroprotective properties against peripheral neuropathies, such as DNP. In the present study, we hypothesized that utilization of different doses of LEV might have beneficial effects on peripheral nerve damage in diabetes. To accomplish this, we tested the effects of low and high doses of LEV on the functional and architectural properties of the sciatic nerve in Type I diabetic rats using electrophysiological, histological, immunohistochemical and biochemical parameters.
2.2.
Study design
Diabetes was induced by a single intraperitoneal (i.p.) injection of streptozotocin (STZ; 60 mg/kg, Sigma-Aldrich Inc., St. Louis, MO) following an overnight fast. STZ was prepared in 0.9% NaCl and adjusted to a pH 4.5 with citric acid [18]. Diabetic state was verified 48 h later by determining tail vein blood glucose levels by glucose oxidase reagent strips (BoehringerMannheim, Indianapolis, IN). Animals showing blood glucose levels above 250 mg/dL were included in the study. Eight rats served as naı¨ve control group and received no treatment. Control and diabetic rats were housed in their cages for 20 days. On day 21, EMG recordings were performed from the sciatic nerve under ketamine/xylazine anesthesia to confirm DNP. Following EMG studies on day 21, rats were divided into 4 groups. Group 1: Naı¨ve control (n = 8), Group 2: DM + 1 mL/kg saline (n = 12), Group 3: DM + 300 mg/kg LEV (n = 10), Group 4: DM + 600 mg/kg LEV (n = 10). LEV (Keppra Flakon, UCB Farma) was diluted in saline and administered i.p. for 30 consecutive days. The doses were selected based on previous studies [19,20]. Following these treatments, gross motor test and EMG were performed. Blood samples were collected for biochemical analysis, and then animals were perfused for histology, quantitative immunohistochemistry (bax, caspase-3, caspase8, NGF) and TUNEL staining.
2.3.
Electrophysiological recordings (EMG)
2.
Materials and methods
Rats were anesthetized by combination of ketamine hydrochloride at a dose of 40 mg/kg (Alfamine1, Alfasan International B.V. Holland) and 4 mg/kg of xylazine hydrochloric (Alfazyne1, Alfasan International B.V. Holland). EMG was obtained for three times at the same time point from the right sciatic nerve stimulated supra-maximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5– 5000 Hz, 40 kHz/s with a sampling rate) by a Biopac bipolar subcutaneous needle stimulation electrode (BIOPAC Systems, Inc., Santa Barbara, CA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded from 2 to 3. Interosseous muscle by unipolar needle electrodes. Data were evaluated using Biopac Student Lab Pro version 3.6.7 software (BIOPAC Systems, Inc., Santa Barbara, CA), 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 8C to 37 8C by heating pad [20].
2.1.
Animals
2.4.
Forty-two adult male Sprague Dawley rats weighing 210– 230 g (221.4 1.89 g) at the beginning of the experiments were used. The rats were housed in cages and maintained under standard conditions with 12-h light/dark cycles at room temperature (22 2 8C). They were fed by standard pellet diet and tap water ad libitum throughout the study. All animal care and experimental procedures were approved by the Institutional Animal Care and Ethical Committee. All chemicals were obtained from Sigma-Aldrich Inc. unless otherwise noted.
Assessment of gross motor function
The motor performances of the rats were evaluated by inclined-plate test according to the method described by Rivlin and Tator [21]. The device was an 18 18 cm2 platform, which could be adjusted to provide a slope of varying degrees. Briefly, the rats were placed with their body axis perpendicular to the inclined plane. The initial angle of the inclined plate was 50 degrees. The incline angle slowly increased and the maximum angle of the plate on which the rat preserved its position for 5 s without falling was recorded as motor score. This procedure was repeated five times per
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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rat, and the average value of the climbing angles was recorded.
2.5.
Histology and quantitative immunohistochemistry
Rats were perfused intracardially with 4% formaldehyde for histology and quantitative immunohistochemistry. Briefly, sciatic nerves were embedded in paraffin, sectioned at 5 mm thickness via microtome (Leica RM 2145) and stained with hematoxylin-eosin. All sections were photographed with Olympus C-5050 digital camera mounted on Olympus BX51 microscope. The thickness of the sciatic nerve perineurium was measured using Image-Pro Express 1.4.5 (Media Cybernetics, Inc. USA). For immunohistochemical examination, sections were incubated with 10% H2O2 for 30 min to eliminate endogenous peroxidase activity and then blocked with 10% normal goat serum (Invitrogen, Life Technologies, Waltham, MA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (1/100; Santacruz Biotechnology Inc., Dallas, TX) against bax, caspase-3, caspase-8, nerve growth factor (NGF) for 24 h at 4 8C. Antibody detection was carried out with the Histostain-Plus Bulk kit (Invitrogen) against rabbit IgG and 3,30 -diaminobenzidine (DAB) was used to visualize the final product. All sections were washed in PBS, examined under Olympus BX51 microscope and photographed with Olympus C-5050 digital camera. Twelve animals (3 per group) and six sections from each animal were used for quantitative immunohistochemistry. Two blinded observers counted the total immune-positive Schwann cells under a light microscope at 100 magnification.
2.6.
TUNEL assay
The apoptosis in Schwann cells was detected by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) using the ApopTag Red in situ apoptosis detection kit (S7165, Chemicon, Billerica, MA) according to the manufacturer’s instructions. Schwann cells were considered positive when the nuclei stained positively and showed chromatin clumping. For quantitative assessment of apoptosis, TUNEL(+) cells were counted in each section at 100 magnification.
2.7.
Biochemical analysis
The oxidant and anti-oxidant status of diabetic and LEVtreated rats were assessed by measuring lipid peroxidation
and total antioxidant capacity (TAC) in plasma samples. Lipid peroxidation was determined by measuring malondialdehyde (MDA) levels as thiobarbituric acid reactive substances [22]. Plasma TAC was measured by ferric reducing antioxidant power (FRAP) assay according to Benzie and Strain [23].
2.8.
Statistical analysis
Data analyses were performed using SPSS for Windows, version 15.0. Results were presented as mean standard error of mean (SEM). All data were analyzed by one-way analysis of variance (ANOVA). Post-hoc testing of pairwise comparisons was performed using the Bonferroni test. The differences were considered statistically significant when p < 0.05.
3.
Results
3.1. Blood glucose levels, body weight and motor performance All animals were monitored daily for behavior and health conditions throughout the study. Two rats died in diabetic group within 48 h following STZ injection. As expected, STZreceived rats displayed typical symptoms of diabetes such as loss of body weight, polydipsia, and polyuria. The alterations in blood glucose levels, body weights and motor performance were summarized in Table 1. ANOVA results revealed significant differences between the groups ( p < 0.0005 for glycaemia, p < 0.0005 for body weight and p < 0.0005 for motor performance). All STZ-received rats showed higher blood glucose levels than those of controls ( p < 0.0005). The increase in blood glucose was observed as early as 48 h after STZ injection and was preserved during the study period. In addition, LEV treatment did not affect blood glucose levels. Baseline body weights of the animals were similar in all study groups (221.4 1.89 g). At the end of the experimental period, the saline-treated diabetic rats revealed a marked decrease in body weight when compared with naı¨ve control group (130 6.97 and 234.29 3.42, respectively; p < 0.0005). However, LEV-treated diabetic rats significantly gained weight compared to saline-treated group ( p < 0.0005). We used the inclined plate method to assess the effects of LEV treatment on gross motor performance of the rats. The diabetic rats exhibited significantly poorer scores than those of controls (59.38 1.25 vs. 69.75 0.81; p < 0.0005). However,
Table 1 – The changes in body weights, blood glucose levels and inclined plate scores in the study groups.
Naı¨ve control DM (day 2) DM (day 21) DM + saline DM + LEV (300 mg/kg) DM + LEV (600 mg/kg)
Body weight (g)
Blood glucose (mg/dL)
Inclined plate score (8)
234.29 3.42* 191.42 7.96 160.42 4.78 130 6.97 176.43 5.98# 199.29 9.75#
130.14 5.25* 499.42 17.74 501.43 22.92 508.57 23.50 516.14 18.76 522.57 28.68
69.43 0.78 70 0.67 60.43 1.33* 59.29 1.35* 66.29 0.65 67.86 0.76
One-way ANOVA and post-hoc Bonferroni test demonstrated significant differences between the groups. Data are expressed as mean SEM. p < 0.0005 different from all other groups. # p < 0.0005 different from saline group. *
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Table 2 – The alterations in compound muscle action potentials (CMAPs) parameters in the study groups.
Naı¨ve control DM (day 21) DM + saline DM + LEV (300 mg/kg) DM + LEV (600 mg/kg)
CMAP duration (ms)
CMAP amplitude (mV)
CMAP latency (ms)
2.34 0.04 3.89 0.22* 4.05 0.11* 3.73 0.10 3.36 0.09#
11.30 0.80 6.24 0.45* 5.94 0.49* 9.38 0.57# 10.23 0.69#
1.38 0.02 2.04 0.10* 2.06 0.17* 1.65 0.06y 1.66 0.04y
One-way ANOVA and post-hoc Bonferroni test demonstrated significant differences between the groups. Data are expressed as mean SEM. p < 0.0005 different from all other groups. # p < 0.005 different from DM + saline and DM (day 21) group. y p < 0.05 different from DM + saline and DM (day 21) group. *
Table 3 – The quantitative evaluation of epineurium thickness of sciatic nerves in the study groups. Naı¨ve control Epineurium thickness (mm)
2.16 0.14
DM + saline 16.54 2.80*
#
DM + LEV (300 mg/kg)
DM + LEV (600 mg/kg)
12.85 1.63
10.73 1.49
One-way ANOVA and post-hoc Bonferroni test revealed significant differences between the groups. Data are expressed as mean SEM. * p < 0.0005 different from naı¨ve control and DM + LEV (600 mg/kg) group. # p < 0.05 different from DM + LEV (300 mg/kg).
both 300 and 600 mg/kg LEV successfully improved motor performance in diabetic rats (66.25 0.61 and 67.63 0.75, respectively; p < 0.0005).
3.2.
EMG recordings
Table 2 describes the changes in EMG parameters of the study groups. ANOVA results showed significant differences between the groups ( p < 0.0005 for CMAP amplitude, CMAP duration, and distal latency). Post-hoc Bonferroni test revealed significant differences in CMAP amplitude, duration and distal latency in saline-treated diabetic rats when compared with controls ( p < 0.0005). The peak-to-peak amplitude of sciatic nerve CMAP, which essentially reflects axonal dysfunction, was reduced by 52% in diabetic rats; however, administration of both 300 and 600 mg/kg LEV significantly counteracted this reduction ( p < 0.005). Similarly, distal latency was significantly shortened ( p < 0.05) in LEV-administrated diabetic rats as compared with saline-treated rats. However, CMAP duration was reduced only in 600 mg/kg LEV group when compared with saline-treated diabetic rats ( p < 0.005), (Fig. 1).
3.3.
Morphological evaluation
Light microscopic examination of the sciatic nerves in the naı¨ve control group exhibited normal myelination and morphology while saline-treated diabetic group showed severe alterations including axonal degeneration, myelin distention, and perineurial fibrosis (Fig. 2). Quantitatively, a marked increase was observed in the thickness of perineurium of saline-treated diabetic rats (16.54 2.8 mm). Administration of 300 and 600 mg/kg LEV significantly lessened the fibrotic changes in sciatic nerve (12.85 1.63 mm and 10.73 1.49 mm; p < 0.005 and p < 0.0005, respectively), (Tables 1 and 3).
3.4.
Immunohistochemistry
To evaluate the role of apoptosis in the development of DNP, we determined the expression of bax, caspase 3, and caspase 8
immunohistochemically in the sections of sciatic nerve. Figs. 3–5 demonstrate the immunuhistochemical alterations in the study groups. ANOVA results of quantitative immunohistochemistry revealed significant differences between the study groups ( p < 0.0005 for bax, caspase 3, caspase 8, and NGF). Post-hoc test showed that while the expression of apoptotic markers including bax, caspase 3 and caspase 8 were prominently high in the saline-treated diabetic rats ( p < 0.0005), administration of 300 and 600 mg/kg LEV significantly lessened the expression of apoptotic markers compared to saline group ( p < 0.0005). In addition, no significant differences were found between the low and high doses of LEV treatments in terms of its suppressing effect on apoptotic markers. NGF expression was significantly decreased in salinetreated group ( p < 0.0005). LEV administration significantly and dose-dependently increased NGF expression in sciatic nerves of diabetic rats ( p < 0.005 and p < 0.0005), (Fig. 6).
3.5.
Tunel staining
Apoptosis was also assessed by counting the number of TUNEL (+) in the sections of sciatic nerve. ANOVA results showed significant differences between the study groups ( p < 0.0005). Diabetic rats treated with saline demonstrated significantly higher TUNEL (+) cells than those of controls ( p < 0.0005) while treatment of diabetic rats with LEV considerably lessened apoptotic cell death ( p < 0.0005), (Fig. 7).
3.6.
Evaluation of oxidative stress
Table 4 summarizes the changes in plasma lipid peroxides (MDA) and total antioxidant capacity (TAC). MDA levels were significantly elevated (0.36 0.05 mM vs. 0.13 0.01 mM; p < 0.0005), whereas plasma TAC was significantly decreased (38.09 6.33 mM vs. 72.28 5.22 mM; p < 0.05) in saline-treated diabetic rats compared to control group. On the contrary, treatment of diabetic rats with 300 mg/kg LEV significantly attenuated plasma MDA levels (0.22 0.01 mM), and improved
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Fig. 1 – Samples of CMAP recorded from (a) naı¨ve control, (b) saline-treated DM, (c) 300 mg/kg LEV-treated DM, and (d) 600 mg/kg LEV-treated DM.
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Fig. 2 – The histological evaluation of sciatic nerve. (a and b) naı¨ve control, (c and d) saline-treated DM, (e and f) 300 mg/kg LEVtreated DM, and (g and h) 600 mg/kg LEV-treated DM. Saline-treated group showed significant inflammation and fibrosis. p: perineurium, ax: axon, sw: Schwann cell, en: endoneurium, f: fibrosis. Hematoxylen and eosine staining (10T and 40T).
TAC (87.18 12.31 mM) when compared with those in the saline-treated diabetic rats.
4.
Discussion
In the present study, we aimed to evaluate the neuroprotective and/or neuroreparative effects of chronic treatment of LEV on
Table 4 – The biochemical evaluation of oxidative stress markers in plasma samples.
Naı¨ve control DM + saline DM + LEV (300 mg/kg)
MDA (mM)
TAC (mM)
0.13 0.01 0.36 0.05* 0.22 0.01
72.28 5.22 38.09 6.33# 87.18 12.31##
One-way ANOVA and post-hoc Bonferroni test revealed significant differences between the groups. Data are expressed as mean SEM. * p < 0.0005 different from all other groups. # p < 0.05 different from naı¨ve control. ## p < 0.005 different from DM + saline.
the development of DNP in rats. Electrophysiological, histological and biochemical findings clearly established that LEV could lessen the neurodegenerative changes in peripheral nerves due to diabetes. It has been well established that sustained elevations of blood glucose levels affect and alter the physiology of many organs, leading indirectly to the peripheral nervous system malfunctions. Hyperglycemia-induced oxidative stress and the subsequent increase in free radical production lead to cellular dysfunction and neuronal cell loss both via activation of apoptosis or necrotic cell death [3,24,25]. As indicated in previous studies, DNP is related with changes in the perineurium, including thickening of the basement membrane of the perineurial cells. Electrophysiological measurements reflect the underlying pathology and alterations in nerve conduction velocity (NCV) or amplitude correlate with myelinated nerve fiber density. There have been several studies in diabetic patients and animal models that indicate reduced NCV, CMAP amplitude, and prolonged CMAP latency in sciatic nerve [26–28]. Streptozotocin induced diabetes in rats is the most extensively studied animal model of DNP. It leads a significant lessening in nerve blood flow and
Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 3 – Bax immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEVtreated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate bax (+) cells. Saline-treated group showed increased number of bax (+) cells. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M.
Fig. 4 – Caspase 3 immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEV-treated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate caspase 3 (+) cells. Salinetreated group displayed significantly higher caspase 3 expression compared to naı¨ve controls. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M. Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 5 – Immunoexpression of caspase 8 in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEV-treated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Black arrows indicate caspase 8 (+) cells. Salinetreated group showed increased expression of caspase 8 compared to naı¨ve controls. * p < 0.0005 different from other groups, # p < 0.0005 different from saline group. Data represent mean W S.E.M.
Fig. 6 – NGF immunoexpression in the sciatic nerve of naı¨ve control (a and b), saline-treated DM (c and d), 300 mg/kg LEVtreated DM (e and f) and 600 mg/kg LEV-treated DM. (20T and 40T). Saline-treated group displays reduced NGF immunoreactivity. # p < 0.0005 different from control, * p < 0.005 different from saline-treated group, ** p < 0.0005 different from saline-treated group. Data represent mean W S.E.M. Please cite this article in press as: Erbas¸ O, et al. Neuroprotective effects of chronic administration of levetiracetam in a rat model of diabetic neuropathy. Diabetes Res Clin Pract (2016), http://dx.doi.org/10.1016/j.diabres.2015.12.016
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Fig. 7 – Effect of LEV treatment on the number of apoptotic cells. (a and b) naı¨ve control, (c and d) saline-treated DM, (e and f) 300 mg/kg LEV-treated DM, and (g and h) 600 mg/kg LEV-treated DM. Black arrows indicate TUNEL (+) cells. The number of TUNEL (+) (apoptotic) cells in the sciatic nerve sections was significantly increased in saline-treated DM group (* p < 0.0005). The number of TUNEL (+) cells decreased with LEV-treatment. ** p < 0.0005 different from saline-treated group. Data represent mean W S.E.M.
slowing of NCV, followed by axonal loss of both the motor and sensory nerve fibers [29]. In line with previous reports, we observed significantly reduced CMAP amplitude and elongation in CMAP latency in saline-treated diabetic rats compared to naı¨ve rats. Furthermore, histological findings of neuropathy, such as axonal degeneration, myelin distention, and perineurial fibrosis, are evident in the sciatic nerve of diabetic rats. In the present study, in order to elucidate the mechanisms underlying neuroaxonal changes in DNP we evaluated particular apoptotic indicators such as bax, caspase 3, caspase 8 and Tunel assay. The qualitative and quantitative immunohistochemical assessment of sciatic nerves clearly confirmed the apoptotic death of Schwann cells in saline-treated diabetic rats. Besides, NGF immunoexpression, which is crucial for survival of neurons, was significantly reduced in diabetic group. On the other hand, the measurement of plasma lipid peroxides (MDA) and total anti-oxidant capacity (TAC) demonstrated a significant imbalance between oxidative and anti-oxidative status in saline- treated diabetic rats. There is growing support for the neuroprotective effects of conventional, as well as recently introduced, antiepileptic agents in experimental models of hypoxic/ischemic and traumatic brain injury [16,17,30,31]. It has been suggested that these drugs exert their effects by modulating of GABAergic and glutamatergic transmission (via inhibition of voltage-dependent ion-channels), receptors and secondary messengers [31–33]. LEV, a second-generation new antiepileptic drug, is used for both focal and generalized epilepsy. To date, several in vitro and in vivo studies have indicated the
neuroprotective effects of LEV due to its anti-apoptotic, antioxidative and anti-inflammatory properties [30,34–36]. For instance, in a pilocarpine-induced injury model, it has been demonstrated that LEV could counteract oxidative stress by lessening lipid peroxidation and nitric oxide generation, and also preserving catalase activity in the hippocampus [36]. More recently, Ueda et al. have revealed that LEV could modify the expressions of the cystine/glutamate exchanger (xCT) and the inducible nitric oxide synthase (iNOS) in connection with lipid peroxidation and also enhance basal endogenous antioxidant ability in the hippocampus [35]. Evidence suggests that LEV has therapeutic effects on neuropathic pain models in mice [19,37]. LEV has been found to generate a dose- (20–200 mg/kg) and time-dependent (0– 75 min) inhibition of the diabetes-induced hyperalgesia [19]. Also, it has been showed that LEV (10–100 mg/kg) could exert synergistic interactions with analgesics in producing antinociception in diabetic mice that developed thermal/mechanical hypersensitivity [37]. In our model, we used two different doses (300 mg/kg and 600 mg/kg) of LEV to evaluate its protective effects against diabetes-induced alterations in peripheral nerves. These doses were chosen based on the results of our previous study, which indicated a beneficial effect of LEV in a model of sepsisinduced polyneuropathy in rats. Our results confirmed that both doses of LEV caused a significant reduction in the number of apoptotic cells and immunoexpression of bax, caspase-3 and capase-8 in sciatic nerve. Another important finding of our study was that NGF immunoexpression in the Schwann cells
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was significantly preserved in LEV treated diabetic rats compared to saline-treated group. Besides, histomorphometric examination of sciatic nerve showed a significant reduction in fibrosis in LEV treated diabetic rats. The assessment of oxidative and anti-oxidative status of study groups revealed that LEV was able to decrease plasma lipid peroxides and enhance total antioxidant capacity in diabetic rats. In addition to biochemical and histological findings, our results showed that LEV could successfully improve the electrophysiological parameters including CMAP duration, amplitude and latency in diabetic rats. Overall, these results suggest a direct evidence of cytoprotective potential of LEV against oxidative stress and apoptotic cell death in diabetes related neuropathy in rats. Previously, several experimental models have established that inhibitors of ion channels could lessen inflammation and apoptotic cell death [30,34]. However, the probable mechanisms of the anti-apoptotic effects of LEV in neuronal damages are not completely understood. In an in vitro study, Stettner et al. have demonstrated the potential of LEV to protect Schwann cells from oxidative stress and inflammation through reducing tumor necrosis factor alpha (TNF-a) and matrix metalloproteinase 9 (MMP-9) expression and caspase-6 activity [34]. In a neonatal rat model of hypoxic ischemic brain injury (HIBI), it has been reported that LEV treatment following hypoxic ischemia could lead to a significant reduction in the number of apoptotic cells [30]. Similar to these findings, Komur et. al., showed that LEV can act as a neuroprotective agent by lessening the number of apoptotic neurons in rat model of HIBI in the early period. They also found that administration of rats with LEV significantly reduced the plasma lipid peroxides and improved anti-oxidant enzyme activities such as glutathione peroxidase and superoxide dismutase [17]. We also consider the importance of LEV receptor, SV2A, in mediation of neuroprotection and neurorepair, as had been suggested by previous studies [13,38,39]. SV2A receptor protein has been implied as a presynaptic calcium sensor and a transmitter vesicular uptake and release moderator like synaptotagmin. Without SV2A, presynaptic calcium accumulates in neurons and leads to abnormal increases in the neurotransmitter release. Therefore, the binding of the SV2A receptor with LEV may provide the ability of a neuron to lessen excessive glutamate release [13,37]. However, further studies are required to define the precise mechanism underlying the neuroprotective effects of LEV through SV2A modulation. Taken together, our results provide in vivo evidence that LEV can effectively reduces neuronal damage in diabetic rats through its anti-oxidant and anti-apoptotic properties. On the basis of our findings, we can propose that lower doses LEV may be as efficacious as higher doses in preventing of DNP. Future experimental and clinical studies that evaluate its efficacy and long-term safety may encourage the application of LEV as a treatment modality for diabetic neuropathy.
Conflict of interest statement The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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