Article
Neuroprotective effects of folic acid on experimental diabetic peripheral neuropathy
Toxicology and Industrial Health 1–9 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713511513 tih.sagepub.com
Mustafa Yilmaz1, Huseyin Aktug2, Fatih Oltulu3 and Oytun Erbas3 Abstract Diabetic peripheral neuropathy (DPN) is widely considered as a degenerative complication of diabetic patients. The clinical effectiveness of folic acid (FA) on DPN is uncertain. The objective of the present study was to determine the effect of FA in DPN using electromyography (EMG), histopathological examination, immunohistochemistry, inclined plane test, and malondialdehyde (MDA) levels as a marker for lipid peroxidation in experimental diabetic rats. A total of 21 Sprague Dawley rats were randomly divided into 3 groups: control group, diabetes group, and FA-treated group. In EMG, compound muscle action potential (CMAP) amplitude in the sciatic nerve was lower in the diabetes group compared with the control group. CMAP amplitude in the sciatic nerve was higher in the FA-treated group when compared with the diabetes group. Distal latency and CMAP duration in the sciatic nerve were lower in the FA-treated group when compared with the diabetes group. In histopathological examination of the sciatic nerve, peripheral fibrosis was present in the diabetic group; the fibrosis was lower in the FA-treated group. In comparison with the diabetes group, the expression of nerve growth factor (NGF) was higher in the FA-treated group. The scores for the inclined plane test were lower in the diabetes group and higher in the FA-treated group than the control group. The MDA levels were significantly lower in the FA-treated group when compared with the diabetes group. The study suggests that FA can protect diabetic rats against DPN and that the underlying mechanism for this may be related to improvement of the expression of NGF and lower MDA levels. Keywords Diabetic peripheral neuropathy, EMG, histopathology, inclined plane, NGF expression, MDA
Introduction Diabetes mellitus (DM) is a common metabolic disorder characterized by disturbed glucose metabolism (Manschot et al., 2008). DM-related complications involving vessels, kidneys, eyes, and the nervous system are a significant cause of high rates of morbidity and mortality in patients with DM (Perkins and Bril, 2005). Diabetic peripheral neuropathy (DPN) is a major neurological complication of diabetic patients and their prevalence is more than 50% (Dyck et al., 1993; Sima, 2003) DPN is characterized by a progressive loss of nerve fibers and is the leading cause of insensitive extremities, resulting in amputation (Figueroa-Romero et al., 2008). Electrophysiological studies, such as electromyography (EMG) are considered the gold standard for aiding in the diagnosis of DPN (Alp et al., 2012; Yagci et al., 2010). Changes
of sensory and motor nerve conduction velocity (NCV) and a decrease in amplitude as well as an increase in the temporal dispersion of compound muscle action potentials (CMAP) were shown in DPN. Axonal loss, demyelination, decreased NCV, decreased CMAP amplitude, and prolonged CMAP
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Department of Neurology, Faculty of Medicine, Mugla University, Mugla, Turkey 2 Department of Histology and Embryology, Faculty of Medicine, Ege University, Izmir, Turkey 3 Department of Physiology, Faculty of Medicine, Ege University, Izmir, Turkey Corresponding author: Mustafa Yilmaz, Department of Neurology, Faculty of Medicine, Mugla University, 48000 Mugla, Turkey. Email: [email protected]
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latency of the sciatic nerve were observed in diabetic rats (Cankayali et al., 2007; Malik et al., 2005). Nerve growth factor (NGF) acts as a neuroprotective agent and repairs functions. It enhances the mechanisms and treatment of DPN. In addition, inclined plane testing is important for the pathophysiologic study and development of drug therapies of neuropathy to establish convenient methods and evaluate muscle weakness. The testing is usually used in rat models with spinal cord injury. Motor and sensory impairments are a common occurrence after neuropathy in both humans and animals. Inclined plane testing is a quantitative and objective method for evaluating impairment of motor function (Rivlin and Tator, 1977; Yonemori et al., 1998). We conducted inclined plane testing after DPN development in the rats and examined the results. Folic acid (FA) is a water-soluble essential vitamin that participates in many essential cell functions (Couto et al., 2012). FA deficiency is related to increases in many neurodegenerative and cerebrovascular diseases. In addition, FA supplementation has been shown to enhance neurodevelopment and provide neuroprotection in some neurological diseases including stroke, Parkinson’s disease, Alzheimer’s disease, depression, psychosis, and spinal cord injury (Ma et al., 2009; Mastroiacovo and Leoncini, 2011). FA promotes the remethylation of homocysteine. The possible pathophysiologic mechanisms implicated in DPN are not clear, exactly. One of the possible mechanisms may have been an increase in homocysteine or oxidative stress for the development of DPN. A growing number of epidemiological studies have associated FA deficiency and resultant elevated plasma total homocysteine levels with an increased risk of vascular disease, cerebral ischemia, and neurodegenerative and neuropsychiatric diseases, including Alzheimer’s disease, depression, and schizophrenia. It has also been shown that FA supplementation can reduce the risk for these diseases. In this study, we analyzed the possible role of FA on DPN using EMG and histopathological examination, lipid peroxidation (LPO), and immunohistochemistry in diabetic rats. In addition, clinical observation such as during inclined plane testing was demonstrated to investigate the effect of FA in DPN.
were fed ad libitum and housed in pairs in steel cages having a temperature-controlled environment (22 + 2 C) with 12-h light/12-h dark cycles. The experimental procedures were approved by the Committee for Animal Research of Ege University. All animal studies strictly conformed to the animal experiment guidelines of the Committee for Human Care. DM was induced by intraperitoneal (IP) injection of single dose of streptozotocin (STZ; SigmaAldrich, Inc., St Louis, MO; 60 mg/kg in 0.9% sodium chloride, adjusted to pH 4.0 with 0.2 M sodium citrate) for 14 rats (Wayhs et al., 2013). For the control group (group 1), the level of blood glucose was below 120 mg/dl (n ¼ 7). DM was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer–Mannheim). The blood glucose levels were 250 mg/dl and higher in the diabetic rat. Then, 14 diabetic rats were randomly divided into 2 groups: group 2 (n ¼ 7; diabetes group) were given 1 ml/kg saline treatment; group 3 (n ¼ 7; FA-treated group) were given 10 mg/kg/day IP administration of FA (50 mg leucovorin; Teva Pharmaceutical Industries, Petah Tikva, Israel) for 4 weeks. At the end of the study, EMG and inclined plane testing were conducted. Then, the animals were euthanized and the sciatic nerve was removed to determine histopathological examination and LPO. EMG was obtained 3 times from the right sciatic nerve and stimulated supramaximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5–5000 Hz, 40 kHz/s sampling rate) using a bipolar subcutaneous needle stimulation electrode (BIOPAC Systems Inc., Santa Barbara, California, USA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded by unipolar needle electrodes located in the second and third interdigital muscle. Data were evaluated 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; HewlettPackard Company, Palo Alto, CA), and the temperature of each rat was kept at approximately 36–37 C by heating pad.
Materials and methods Animals
Histopathological examination of the sciatic nerve
In this study, 21 female Sprague Dawley rats aged 8 weeks and weighing 160–180 g were used. Animals
For electron microscopic evaluation, the remaining sciatic nerve tissue segments were sectioned using
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an ultramicrotome to a thickness of 4 mm. The 4 m sections came from paraffin-embedded specimens. Formalin-fixed sciatic nerve sections (4 mm) were stained with hematoxylin and eosin. The thickness of the sciatic epineurium nerve was measured, and the stained tissue sections were then examined with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Tokyo, Japan).
NGF immunoexpression For immunohistochemistry, sections were incubated with hydrogen peroxide (10%) for 30 min to eliminate endogenous standard activity and blocked with 10% normal goat serum (Invitrogen, Carlsbad, CA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (NGF, Bioss Inc., Woburn, MA; dilution 1/100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc.) against rabbit immunoglobulin G, and 3,30 diaminobenzidine was used to visualize the final product. All sections were washed in phosphatebuffered saline and photographed with an Olympus C-5050 digital camera attached to an Olympus BX51 microscope. The presence of a brown cytoplasmic staining cell was scored positive. The number of NGF positive cells was assessed systematically by scoring at least 100 cells per 10 fields of tissue sections at 100magnification by three histologists.
Figure 1. Inclined plane test.
supernatant was read at 535 nm. MDA levels were expressed in nanomoles per microgram of protein.
Inclined plane test We evaluated motor performance in rats, using a sliding apparatus described by Rivlin and Tator (1977), at 1 month after STZ was induced. The sliding apparatus had a 50 30 cm2 stainless steel plane (Figure 1). The maximum angle was then determined at the moment just when a limb of the rat slipped in order to maintain body position. The test was performed three times for each head position and averaged. Each trial was performed after a 1-min interval.
LPO level determination LPO was determined in tissue samples by measuring malondialdehyde (MDA) levels as thiobarbituric acid reactive substances (TBARS). Briefly, trichloroacetic acid and TBARS reagent were added to the tissue samples, then mixed, and incubated at 100 C for 60 min. After cooling on ice, the samples were centrifuged at 3000 r/min for 20 min, and the absorbance of the
Statistical analysis Data analyses were performed using Statistical Package for Social Sciences software, version 15.0 for Windows[SPSS, Chicago, Illinois, USA.]. The groups of parametric variables were compared by Student’s t test and analysis of variance. The groups of nonparametric variables were compared by Mann–Whitney U test. Results were given as mean + SEM. The value of p < 0.05 was accepted as statistically significant and p < 0.001 was accepted as statistically highly significant.
Results There were no deaths in the control and FA-treated groups. But there was one death in the diabetes group. The rats’ body weight was decreased in diabetic rats, especially in group 2 in comparison with the control group (p < 0.001). There was a difference in the body
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Table 1. The weights and plasma glucose levels of rats at the beginning and end of the study.
Control group Diabetes group FA-treated group
Initial weight (g)
Final weight (g)
Plasma glucose level (mg/dl)
173.4 + 10.2 175.4 + 8.4 171.6 + 6.5
185.2 + 12.4 135.8 + 7.6a 155.2 + 5.2c
94.8 + 7.6 482 + 15.4b 468 + 12.9d
FA: folic acid. a p < 0.001: comparison of diabetes group at the beginning and end of the study. b p < 0.0001 versus control group. c p < 0.05: comparison of FA-treated group at the beginning and end of the study. d p < 0.0001 versus control group.
Table 2. The maximum angle of inclined plane in rats. Maximum angle ( ) Control group Diabetes group FA-treated group
74.5 + 2.12 63.2 + 1.15a 71.6 + 2.04b
FA: folic acid. a p < 0.0001 versus control group in inclined plane test. b p < 0.05 versus control group in inclined plane test.
weight of FA-treated group compared with the diabetes and saline-treated group (p < 0.05) at the end of 1 month. The blood glucose levels of rats in diabetes and saline group were significantly increased relative to the control group at the end of first month (p < 0.0001; Table 1).
compared with the control group (p < 0.05). The latency of CMAP was significantly prolonged to 1.85 + 0.14 ms when compared with the control group (p < 0.05). However, the amplitudes of CMAP increased significantly to 10.14 + 1.27 mV in the FA-treated group when compared with the group that have been given the diabetes and saline treatment (p < 0.05). The duration of CMAP steadily shortened to 2.59 + 0.18 ms in the FA treatment group in comparison with the diabetes group (p < 0.05). The latency of CMAP was significantly shortened to 1.56 + 0.02 ms in the FAtreated group as compared to the diabetes and saline group (p < 0.05; Figure 2 and Table 3).
Inclined plane test
Epineural fibrosis and NGF immunoexpression in the sciatic nerve
We evaluated motor performance in STZ-induced diabetic rats using an inclined plane test at 1 month after DM was induced. The maximum angles at which position was maintained in the control rats were 74.5 + 2.12 , at 1 month after DM was induced (Table 2). Compared with the control group, the diabetes and saline treatment disturbed the performance of this task. The maximum angle at which position was maintained was 63.2 + 1.15 at 1 month after DM was induced, and this performance was significantly poorer than that of the control groups (p < 0.001). The performance was disturbed minimally in the FAtreated group. The maximum angle at which position could be maintained was 71.6 + 2.04 at 1 month after DM was induced (p < 0.05; Table 2).
The thickness of epineural fibrosis in the sciatic nerve tissues of the diabetic and saline groups were significantly increased when compared with the control group (p < 0.001; Figure 3 and Table 4). Similarly, the thickness of epineural fibrosis increased minimally in the FA-treated group compared with the control group (p < 0.05). It was significantly decreased in the FA treatment group as compared to the diabetes group (p < 0.01). NGF immunoexpression in the Schwann cell of the diabetes group was significantly decreased compared with the control group (p < 0.001). NGF immunoexpression in the Schwann cell was significantly increased in the FA treatment group compared with the saline group (p < 0.01).
Electromyography In the diabetes group, the amplitudes of CMAP decreased significantly to 7.69 + 0.16 mV as compared to the control group (p < 0.001). The duration of CMAP steadily prolonged to 3.18 + 0.01 ms when
LPO level Comparisons of the groups’ MDA levels are presented in Table 5. The level of MDA was increased in the diabetes group when compared with the control group
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Figure 2. A sample of CMA Precorded in the rats. (a) EMG of control group; (b) EMG of diabetes group; (c) EMG of FAtreated group. EMG: electromyography; FA: folic acid; CMAP: compound muscle action potential. Table 3. The measurements of compound second and third interdigital muscle action potentials in rats.
Control group Diabetes group FA-treated group
CMAP amplitude (mV)
Distal latans (ms)
CMAP duration
11.28 + 1.12 7.69 + 0.16a 10.14 + 1.27c
1.16 + 0.06 1.85 + 0.14b 1.56 + 0.02d
2.25 + 0.08 3.18 + 0.01b 2.59 + 0.18d
CMAP: compound muscle action potential; FA: folic acid. a p < 0.01 versus control group. b p < 0.05 versus control group. c p < 0.05 versus diabetes group. d p < 0.05 versus diabetes group.
(p < 0.0001). FA treatment significantly reduced the MDA levels compared with the diabetes group (p < 0.01).
Discussion In our study, CMAP amplitude in the sciatic nerve was higher in the FA-treated group as compared to the
diabetes group in EMG. Distal latency and CMAP duration in the sciatic nerve were lower in the FAtreated group when compared with the diabetes group. In histopathological examination of the sciatic nerve, peripheral fibrosis was present in the diabetic group; the fibrosis was lower in the FA-treated group. Comparing with the diabetes group, the expression of NGF
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Figure 3. The histological sections of sciatic nerve. (a, c, and e) H&E staining (100 magnification); (b, d, and e) NGF immunoexpression (100 magnification); (a, b): control group (p: perineurium; a: axon; m: myelin; s: Schwann cell); (c, d): diabetes group (e: epineurium; p: perineurium; a: axon; m: myelin; s: Schwann cell); and (e, f): FA-treated group. FA: folic acid; H&E: hematoxylin and eosin; NGF: nerve growth factor.
was higher in the FA-treated group. The scores for the inclined plane test were lower in the diabetes group and higher in the FA-treated group than the control group. The MDA levels were significantly lower in the FAtreated group when compared with the diabetes group. The study suggests that FA may protect DPN in diabetic rats. In a previous study, it was determined that some neurological disorders such as peripheral polyneuropathy, retrobulbar optic neuropathy, myelopathy, and leukoencephalopathy are associated with reduced FA (Lo´pez-Herna´ndez et al., 2003). In adults, FA
deficiency contributes to dementia, impaired cognition, depression, psychosis, neuropathy, and subacute combined degeneration (Ho et al., 2003; Reynolds, 2002). Preventive measures such as the early provision of appropriate nutrition can help reducing the occurrence of neuropathy. As a neuroprotectant agent, FA supplementation reduces the incidence of many diseases in adults (Van Guelpen et al., 2005). It was also showed that FA enhances repair processes following spinal injury, improving functional recovery in an adult rat model (Iskandar et al., 2004).
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Table 4. The histological sections of sciatic nerve in rats. Perineural fibrosis (percentage of control) Control group Diabetes group FA-treated group
Control group
86.4 + 4.6
100 128.5 + 13.25
NGF immunoexpression (%)
Table 5. The effects of FA treatment on MDA levels in rats.
a
107.3 + 4.32c
b
15.8 + 1.2
69.5 + 5.7d
NGF: nerve growth factor; FA: folic acid. a p < 0.01 versus control group. b p < 0.001 versus control group. c p < 0.05 versus diabetes group. d p < 0.01 versus diabetes group.
In our study, DPN was documented by EMG. Electrophysiological studies, such as EMG and biopsies of nerve cells, are considered for aiding in the diagnosis of neuropathy (Olaleye et al., 2001). Axonal loss, demyelination, decreased NCV, CMAP amplitude, and prolonged CMAP latency of the sciatic nerve was determined in diabetic patients and rat models (Cankayali et al., 2007; Confer et al., 2012; Kovrazhkina, 2012; Malik et al., 2005). We found elongation in CMAP latency and decreased CMAP amplitude in diabetic rats. It was reported in previous studies that decrease in CMAP amplitude was due to axonal loss and neurodegeneration models (Cankayali et al., 2007; Malik et al., 2005). The possible etiologies for development of DPN may be metabolic, vascular, autoimmune, oxidative stress, or hormone deficiency (Figueroa-Romero et al., 2008). The oxidative stress has been shown to be a critical factor in the development of DPN. Hyperglycemia and the resulting superoxides may have a role in increasing oxidative stress (Giacco and Brownlee, 2010). It was reported that FA supplementation ameliorates inflammation, oxidative damage, and insulin resistance in DM. In our study, MDA levels as a marker for LPO were determined. FA may be considered as an effective antioxidant in patients; this can be a result of decreased production of free radicals. We were able to verify the possibility of FA as a neuroprotector in diabetic rats. In addition, this study demonstrated that FA did not exactly prevent the increase of blood glucose. So the beneficial effects of FA without hypoglycemia are also different for some drugs such as gliclazide, b glucan, and a lipoic acid in this respect (Alp et al., 2012). FA may repair glucose tolerance and insulin resistance (Brocardo et al., 2010; Pravenec et al., 2013).
Diabetes group
FA-treated group
MDA (nmol/mg 86.7 + 5.6 314.1 + 11.8a 146.2 + 7.1b protein) MDA: malondialdehyde; FA: folic acid. a p < 0.0001 versus control group. b p < 0.01 versus diabetes group.
We analyzed the anti-apoptotic and antiinflammatory effects by measuring the NGF immunoexpression and epineural fibrosis. These are known to be key players in the processes of apoptosis and inflammation (Sousa et al., 2001). Following STZinduced diabetes, neurons and glial cells undergo apoptosis and the subsequent demyelination and axonal loss by neurodegeneration. Tyrosine kinase receptors in many different biological processes are regulated by neurotrophic factors. NGF is one of the six growth factors, and triggered tyrosine kinase receptors play a role in the survival and differentiation of neurons in the central nervous system (Dai et al., 2004). The percentage of NGF immunoexpression is related to neuronal cell loss in Schwann cells. In our study, the percentage of NGF immunoexpression was decreased in the diabetic group and was ameliorated with FA treatment. We showed that FA treatment may also decrease the epineural fibrosis and increase the NGF immunoexpression. In addition, DPN was documented by the presence of clinical signs in the study. In the study, the motor performance of the rats was tested after STZinduced diabetes. We observed that motor performance was disturbed in the diabetes group. However, the motor performance was disturbed minimally in the diabetes and FA treatment groups. In experimental studies, the inclined plane test, developed by Allmark and Bachinski (1949) and modified by Rivlin and Tator (1977) is a common method to assess neuromuscular motor function and to evaluate impairment of neurologic and motor functions in rodent models. The test recorded the maximum inclined plane degree on which the rats could stand for at least 5 s to evaluate limb motor function and coordination. The maximum angle on which the rat could stand for at least 5 s constitutes the inclined plane score (Bederson et al., 1986). Based on the inclined plane test, we may suggest using both electrophysiological and clinical observation to investigate the beneficial effect of FA on DPN.
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It was also reported that hyperhomocysteinemia was an independent risk factor for the occurrence of DPN. In the current study, it was reported that FA modulates metabolism of homocysteine, leading to mild hyperhomocysteinemia that may damage the target organs due to inducing oxidative stress in diabetes, while FA supplementation can prevent these adverse effects (Pravenec et al., 2013). FA supplementation may ameliorate the occurrence of DPN possibly related to reducing the LPO in the study. In summary, our study is not only histological and electrophysiological but is also clinical demonstrating the beneficial effects of FA on DPN. FA prevented the decrease of motor performance due to DM. This study showed the beneficial effects of FA in the prevention of DPN through improving expression of NGF and lowering MDA levels, and so FA supplementation may be considered as an adjunctive therapy in diabetes patients to improve symptoms of neuropathy. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Neuroprotective effects of folic acid on experimental diabetic peripheral neuropathy
Toxicology and Industrial Health 1–9 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713511513 tih.sagepub.com
Mustafa Yilmaz1, Huseyin Aktug2, Fatih Oltulu3 and Oytun Erbas3 Abstract Diabetic peripheral neuropathy (DPN) is widely considered as a degenerative complication of diabetic patients. The clinical effectiveness of folic acid (FA) on DPN is uncertain. The objective of the present study was to determine the effect of FA in DPN using electromyography (EMG), histopathological examination, immunohistochemistry, inclined plane test, and malondialdehyde (MDA) levels as a marker for lipid peroxidation in experimental diabetic rats. A total of 21 Sprague Dawley rats were randomly divided into 3 groups: control group, diabetes group, and FA-treated group. In EMG, compound muscle action potential (CMAP) amplitude in the sciatic nerve was lower in the diabetes group compared with the control group. CMAP amplitude in the sciatic nerve was higher in the FA-treated group when compared with the diabetes group. Distal latency and CMAP duration in the sciatic nerve were lower in the FA-treated group when compared with the diabetes group. In histopathological examination of the sciatic nerve, peripheral fibrosis was present in the diabetic group; the fibrosis was lower in the FA-treated group. In comparison with the diabetes group, the expression of nerve growth factor (NGF) was higher in the FA-treated group. The scores for the inclined plane test were lower in the diabetes group and higher in the FA-treated group than the control group. The MDA levels were significantly lower in the FA-treated group when compared with the diabetes group. The study suggests that FA can protect diabetic rats against DPN and that the underlying mechanism for this may be related to improvement of the expression of NGF and lower MDA levels. Keywords Diabetic peripheral neuropathy, EMG, histopathology, inclined plane, NGF expression, MDA
Introduction Diabetes mellitus (DM) is a common metabolic disorder characterized by disturbed glucose metabolism (Manschot et al., 2008). DM-related complications involving vessels, kidneys, eyes, and the nervous system are a significant cause of high rates of morbidity and mortality in patients with DM (Perkins and Bril, 2005). Diabetic peripheral neuropathy (DPN) is a major neurological complication of diabetic patients and their prevalence is more than 50% (Dyck et al., 1993; Sima, 2003) DPN is characterized by a progressive loss of nerve fibers and is the leading cause of insensitive extremities, resulting in amputation (Figueroa-Romero et al., 2008). Electrophysiological studies, such as electromyography (EMG) are considered the gold standard for aiding in the diagnosis of DPN (Alp et al., 2012; Yagci et al., 2010). Changes
of sensory and motor nerve conduction velocity (NCV) and a decrease in amplitude as well as an increase in the temporal dispersion of compound muscle action potentials (CMAP) were shown in DPN. Axonal loss, demyelination, decreased NCV, decreased CMAP amplitude, and prolonged CMAP
1
Department of Neurology, Faculty of Medicine, Mugla University, Mugla, Turkey 2 Department of Histology and Embryology, Faculty of Medicine, Ege University, Izmir, Turkey 3 Department of Physiology, Faculty of Medicine, Ege University, Izmir, Turkey Corresponding author: Mustafa Yilmaz, Department of Neurology, Faculty of Medicine, Mugla University, 48000 Mugla, Turkey. Email: [email protected]
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latency of the sciatic nerve were observed in diabetic rats (Cankayali et al., 2007; Malik et al., 2005). Nerve growth factor (NGF) acts as a neuroprotective agent and repairs functions. It enhances the mechanisms and treatment of DPN. In addition, inclined plane testing is important for the pathophysiologic study and development of drug therapies of neuropathy to establish convenient methods and evaluate muscle weakness. The testing is usually used in rat models with spinal cord injury. Motor and sensory impairments are a common occurrence after neuropathy in both humans and animals. Inclined plane testing is a quantitative and objective method for evaluating impairment of motor function (Rivlin and Tator, 1977; Yonemori et al., 1998). We conducted inclined plane testing after DPN development in the rats and examined the results. Folic acid (FA) is a water-soluble essential vitamin that participates in many essential cell functions (Couto et al., 2012). FA deficiency is related to increases in many neurodegenerative and cerebrovascular diseases. In addition, FA supplementation has been shown to enhance neurodevelopment and provide neuroprotection in some neurological diseases including stroke, Parkinson’s disease, Alzheimer’s disease, depression, psychosis, and spinal cord injury (Ma et al., 2009; Mastroiacovo and Leoncini, 2011). FA promotes the remethylation of homocysteine. The possible pathophysiologic mechanisms implicated in DPN are not clear, exactly. One of the possible mechanisms may have been an increase in homocysteine or oxidative stress for the development of DPN. A growing number of epidemiological studies have associated FA deficiency and resultant elevated plasma total homocysteine levels with an increased risk of vascular disease, cerebral ischemia, and neurodegenerative and neuropsychiatric diseases, including Alzheimer’s disease, depression, and schizophrenia. It has also been shown that FA supplementation can reduce the risk for these diseases. In this study, we analyzed the possible role of FA on DPN using EMG and histopathological examination, lipid peroxidation (LPO), and immunohistochemistry in diabetic rats. In addition, clinical observation such as during inclined plane testing was demonstrated to investigate the effect of FA in DPN.
were fed ad libitum and housed in pairs in steel cages having a temperature-controlled environment (22 + 2 C) with 12-h light/12-h dark cycles. The experimental procedures were approved by the Committee for Animal Research of Ege University. All animal studies strictly conformed to the animal experiment guidelines of the Committee for Human Care. DM was induced by intraperitoneal (IP) injection of single dose of streptozotocin (STZ; SigmaAldrich, Inc., St Louis, MO; 60 mg/kg in 0.9% sodium chloride, adjusted to pH 4.0 with 0.2 M sodium citrate) for 14 rats (Wayhs et al., 2013). For the control group (group 1), the level of blood glucose was below 120 mg/dl (n ¼ 7). DM was verified after 24 h by evaluating blood glucose levels with the use of glucose oxidase reagent strips (Boehringer–Mannheim). The blood glucose levels were 250 mg/dl and higher in the diabetic rat. Then, 14 diabetic rats were randomly divided into 2 groups: group 2 (n ¼ 7; diabetes group) were given 1 ml/kg saline treatment; group 3 (n ¼ 7; FA-treated group) were given 10 mg/kg/day IP administration of FA (50 mg leucovorin; Teva Pharmaceutical Industries, Petah Tikva, Israel) for 4 weeks. At the end of the study, EMG and inclined plane testing were conducted. Then, the animals were euthanized and the sciatic nerve was removed to determine histopathological examination and LPO. EMG was obtained 3 times from the right sciatic nerve and stimulated supramaximally (intensity 10 V, duration 0.05 ms, frequency 1 Hz, in the range of 0.5–5000 Hz, 40 kHz/s sampling rate) using a bipolar subcutaneous needle stimulation electrode (BIOPAC Systems Inc., Santa Barbara, California, USA) from the Achilles tendon. Compound muscle action potentials (CMAPs) were recorded by unipolar needle electrodes located in the second and third interdigital muscle. Data were evaluated 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; HewlettPackard Company, Palo Alto, CA), and the temperature of each rat was kept at approximately 36–37 C by heating pad.
Materials and methods Animals
Histopathological examination of the sciatic nerve
In this study, 21 female Sprague Dawley rats aged 8 weeks and weighing 160–180 g were used. Animals
For electron microscopic evaluation, the remaining sciatic nerve tissue segments were sectioned using
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an ultramicrotome to a thickness of 4 mm. The 4 m sections came from paraffin-embedded specimens. Formalin-fixed sciatic nerve sections (4 mm) were stained with hematoxylin and eosin. The thickness of the sciatic epineurium nerve was measured, and the stained tissue sections were then examined with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Tokyo, Japan).
NGF immunoexpression For immunohistochemistry, sections were incubated with hydrogen peroxide (10%) for 30 min to eliminate endogenous standard activity and blocked with 10% normal goat serum (Invitrogen, Carlsbad, CA) for 1 h at room temperature. Subsequently, sections were incubated with primary antibodies (NGF, Bioss Inc., Woburn, MA; dilution 1/100) for 24 h at 4 C. Antibody detection was performed with the Histostain-Plus Bulk kit (Bioss Inc.) against rabbit immunoglobulin G, and 3,30 diaminobenzidine was used to visualize the final product. All sections were washed in phosphatebuffered saline and photographed with an Olympus C-5050 digital camera attached to an Olympus BX51 microscope. The presence of a brown cytoplasmic staining cell was scored positive. The number of NGF positive cells was assessed systematically by scoring at least 100 cells per 10 fields of tissue sections at 100magnification by three histologists.
Figure 1. Inclined plane test.
supernatant was read at 535 nm. MDA levels were expressed in nanomoles per microgram of protein.
Inclined plane test We evaluated motor performance in rats, using a sliding apparatus described by Rivlin and Tator (1977), at 1 month after STZ was induced. The sliding apparatus had a 50 30 cm2 stainless steel plane (Figure 1). The maximum angle was then determined at the moment just when a limb of the rat slipped in order to maintain body position. The test was performed three times for each head position and averaged. Each trial was performed after a 1-min interval.
LPO level determination LPO was determined in tissue samples by measuring malondialdehyde (MDA) levels as thiobarbituric acid reactive substances (TBARS). Briefly, trichloroacetic acid and TBARS reagent were added to the tissue samples, then mixed, and incubated at 100 C for 60 min. After cooling on ice, the samples were centrifuged at 3000 r/min for 20 min, and the absorbance of the
Statistical analysis Data analyses were performed using Statistical Package for Social Sciences software, version 15.0 for Windows[SPSS, Chicago, Illinois, USA.]. The groups of parametric variables were compared by Student’s t test and analysis of variance. The groups of nonparametric variables were compared by Mann–Whitney U test. Results were given as mean + SEM. The value of p < 0.05 was accepted as statistically significant and p < 0.001 was accepted as statistically highly significant.
Results There were no deaths in the control and FA-treated groups. But there was one death in the diabetes group. The rats’ body weight was decreased in diabetic rats, especially in group 2 in comparison with the control group (p < 0.001). There was a difference in the body
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Table 1. The weights and plasma glucose levels of rats at the beginning and end of the study.
Control group Diabetes group FA-treated group
Initial weight (g)
Final weight (g)
Plasma glucose level (mg/dl)
173.4 + 10.2 175.4 + 8.4 171.6 + 6.5
185.2 + 12.4 135.8 + 7.6a 155.2 + 5.2c
94.8 + 7.6 482 + 15.4b 468 + 12.9d
FA: folic acid. a p < 0.001: comparison of diabetes group at the beginning and end of the study. b p < 0.0001 versus control group. c p < 0.05: comparison of FA-treated group at the beginning and end of the study. d p < 0.0001 versus control group.
Table 2. The maximum angle of inclined plane in rats. Maximum angle ( ) Control group Diabetes group FA-treated group
74.5 + 2.12 63.2 + 1.15a 71.6 + 2.04b
FA: folic acid. a p < 0.0001 versus control group in inclined plane test. b p < 0.05 versus control group in inclined plane test.
weight of FA-treated group compared with the diabetes and saline-treated group (p < 0.05) at the end of 1 month. The blood glucose levels of rats in diabetes and saline group were significantly increased relative to the control group at the end of first month (p < 0.0001; Table 1).
compared with the control group (p < 0.05). The latency of CMAP was significantly prolonged to 1.85 + 0.14 ms when compared with the control group (p < 0.05). However, the amplitudes of CMAP increased significantly to 10.14 + 1.27 mV in the FA-treated group when compared with the group that have been given the diabetes and saline treatment (p < 0.05). The duration of CMAP steadily shortened to 2.59 + 0.18 ms in the FA treatment group in comparison with the diabetes group (p < 0.05). The latency of CMAP was significantly shortened to 1.56 + 0.02 ms in the FAtreated group as compared to the diabetes and saline group (p < 0.05; Figure 2 and Table 3).
Inclined plane test
Epineural fibrosis and NGF immunoexpression in the sciatic nerve
We evaluated motor performance in STZ-induced diabetic rats using an inclined plane test at 1 month after DM was induced. The maximum angles at which position was maintained in the control rats were 74.5 + 2.12 , at 1 month after DM was induced (Table 2). Compared with the control group, the diabetes and saline treatment disturbed the performance of this task. The maximum angle at which position was maintained was 63.2 + 1.15 at 1 month after DM was induced, and this performance was significantly poorer than that of the control groups (p < 0.001). The performance was disturbed minimally in the FAtreated group. The maximum angle at which position could be maintained was 71.6 + 2.04 at 1 month after DM was induced (p < 0.05; Table 2).
The thickness of epineural fibrosis in the sciatic nerve tissues of the diabetic and saline groups were significantly increased when compared with the control group (p < 0.001; Figure 3 and Table 4). Similarly, the thickness of epineural fibrosis increased minimally in the FA-treated group compared with the control group (p < 0.05). It was significantly decreased in the FA treatment group as compared to the diabetes group (p < 0.01). NGF immunoexpression in the Schwann cell of the diabetes group was significantly decreased compared with the control group (p < 0.001). NGF immunoexpression in the Schwann cell was significantly increased in the FA treatment group compared with the saline group (p < 0.01).
Electromyography In the diabetes group, the amplitudes of CMAP decreased significantly to 7.69 + 0.16 mV as compared to the control group (p < 0.001). The duration of CMAP steadily prolonged to 3.18 + 0.01 ms when
LPO level Comparisons of the groups’ MDA levels are presented in Table 5. The level of MDA was increased in the diabetes group when compared with the control group
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Figure 2. A sample of CMA Precorded in the rats. (a) EMG of control group; (b) EMG of diabetes group; (c) EMG of FAtreated group. EMG: electromyography; FA: folic acid; CMAP: compound muscle action potential. Table 3. The measurements of compound second and third interdigital muscle action potentials in rats.
Control group Diabetes group FA-treated group
CMAP amplitude (mV)
Distal latans (ms)
CMAP duration
11.28 + 1.12 7.69 + 0.16a 10.14 + 1.27c
1.16 + 0.06 1.85 + 0.14b 1.56 + 0.02d
2.25 + 0.08 3.18 + 0.01b 2.59 + 0.18d
CMAP: compound muscle action potential; FA: folic acid. a p < 0.01 versus control group. b p < 0.05 versus control group. c p < 0.05 versus diabetes group. d p < 0.05 versus diabetes group.
(p < 0.0001). FA treatment significantly reduced the MDA levels compared with the diabetes group (p < 0.01).
Discussion In our study, CMAP amplitude in the sciatic nerve was higher in the FA-treated group as compared to the
diabetes group in EMG. Distal latency and CMAP duration in the sciatic nerve were lower in the FAtreated group when compared with the diabetes group. In histopathological examination of the sciatic nerve, peripheral fibrosis was present in the diabetic group; the fibrosis was lower in the FA-treated group. Comparing with the diabetes group, the expression of NGF
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Figure 3. The histological sections of sciatic nerve. (a, c, and e) H&E staining (100 magnification); (b, d, and e) NGF immunoexpression (100 magnification); (a, b): control group (p: perineurium; a: axon; m: myelin; s: Schwann cell); (c, d): diabetes group (e: epineurium; p: perineurium; a: axon; m: myelin; s: Schwann cell); and (e, f): FA-treated group. FA: folic acid; H&E: hematoxylin and eosin; NGF: nerve growth factor.
was higher in the FA-treated group. The scores for the inclined plane test were lower in the diabetes group and higher in the FA-treated group than the control group. The MDA levels were significantly lower in the FAtreated group when compared with the diabetes group. The study suggests that FA may protect DPN in diabetic rats. In a previous study, it was determined that some neurological disorders such as peripheral polyneuropathy, retrobulbar optic neuropathy, myelopathy, and leukoencephalopathy are associated with reduced FA (Lo´pez-Herna´ndez et al., 2003). In adults, FA
deficiency contributes to dementia, impaired cognition, depression, psychosis, neuropathy, and subacute combined degeneration (Ho et al., 2003; Reynolds, 2002). Preventive measures such as the early provision of appropriate nutrition can help reducing the occurrence of neuropathy. As a neuroprotectant agent, FA supplementation reduces the incidence of many diseases in adults (Van Guelpen et al., 2005). It was also showed that FA enhances repair processes following spinal injury, improving functional recovery in an adult rat model (Iskandar et al., 2004).
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Table 4. The histological sections of sciatic nerve in rats. Perineural fibrosis (percentage of control) Control group Diabetes group FA-treated group
Control group
86.4 + 4.6
100 128.5 + 13.25
NGF immunoexpression (%)
Table 5. The effects of FA treatment on MDA levels in rats.
a
107.3 + 4.32c
b
15.8 + 1.2
69.5 + 5.7d
NGF: nerve growth factor; FA: folic acid. a p < 0.01 versus control group. b p < 0.001 versus control group. c p < 0.05 versus diabetes group. d p < 0.01 versus diabetes group.
In our study, DPN was documented by EMG. Electrophysiological studies, such as EMG and biopsies of nerve cells, are considered for aiding in the diagnosis of neuropathy (Olaleye et al., 2001). Axonal loss, demyelination, decreased NCV, CMAP amplitude, and prolonged CMAP latency of the sciatic nerve was determined in diabetic patients and rat models (Cankayali et al., 2007; Confer et al., 2012; Kovrazhkina, 2012; Malik et al., 2005). We found elongation in CMAP latency and decreased CMAP amplitude in diabetic rats. It was reported in previous studies that decrease in CMAP amplitude was due to axonal loss and neurodegeneration models (Cankayali et al., 2007; Malik et al., 2005). The possible etiologies for development of DPN may be metabolic, vascular, autoimmune, oxidative stress, or hormone deficiency (Figueroa-Romero et al., 2008). The oxidative stress has been shown to be a critical factor in the development of DPN. Hyperglycemia and the resulting superoxides may have a role in increasing oxidative stress (Giacco and Brownlee, 2010). It was reported that FA supplementation ameliorates inflammation, oxidative damage, and insulin resistance in DM. In our study, MDA levels as a marker for LPO were determined. FA may be considered as an effective antioxidant in patients; this can be a result of decreased production of free radicals. We were able to verify the possibility of FA as a neuroprotector in diabetic rats. In addition, this study demonstrated that FA did not exactly prevent the increase of blood glucose. So the beneficial effects of FA without hypoglycemia are also different for some drugs such as gliclazide, b glucan, and a lipoic acid in this respect (Alp et al., 2012). FA may repair glucose tolerance and insulin resistance (Brocardo et al., 2010; Pravenec et al., 2013).
Diabetes group
FA-treated group
MDA (nmol/mg 86.7 + 5.6 314.1 + 11.8a 146.2 + 7.1b protein) MDA: malondialdehyde; FA: folic acid. a p < 0.0001 versus control group. b p < 0.01 versus diabetes group.
We analyzed the anti-apoptotic and antiinflammatory effects by measuring the NGF immunoexpression and epineural fibrosis. These are known to be key players in the processes of apoptosis and inflammation (Sousa et al., 2001). Following STZinduced diabetes, neurons and glial cells undergo apoptosis and the subsequent demyelination and axonal loss by neurodegeneration. Tyrosine kinase receptors in many different biological processes are regulated by neurotrophic factors. NGF is one of the six growth factors, and triggered tyrosine kinase receptors play a role in the survival and differentiation of neurons in the central nervous system (Dai et al., 2004). The percentage of NGF immunoexpression is related to neuronal cell loss in Schwann cells. In our study, the percentage of NGF immunoexpression was decreased in the diabetic group and was ameliorated with FA treatment. We showed that FA treatment may also decrease the epineural fibrosis and increase the NGF immunoexpression. In addition, DPN was documented by the presence of clinical signs in the study. In the study, the motor performance of the rats was tested after STZinduced diabetes. We observed that motor performance was disturbed in the diabetes group. However, the motor performance was disturbed minimally in the diabetes and FA treatment groups. In experimental studies, the inclined plane test, developed by Allmark and Bachinski (1949) and modified by Rivlin and Tator (1977) is a common method to assess neuromuscular motor function and to evaluate impairment of neurologic and motor functions in rodent models. The test recorded the maximum inclined plane degree on which the rats could stand for at least 5 s to evaluate limb motor function and coordination. The maximum angle on which the rat could stand for at least 5 s constitutes the inclined plane score (Bederson et al., 1986). Based on the inclined plane test, we may suggest using both electrophysiological and clinical observation to investigate the beneficial effect of FA on DPN.
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It was also reported that hyperhomocysteinemia was an independent risk factor for the occurrence of DPN. In the current study, it was reported that FA modulates metabolism of homocysteine, leading to mild hyperhomocysteinemia that may damage the target organs due to inducing oxidative stress in diabetes, while FA supplementation can prevent these adverse effects (Pravenec et al., 2013). FA supplementation may ameliorate the occurrence of DPN possibly related to reducing the LPO in the study. In summary, our study is not only histological and electrophysiological but is also clinical demonstrating the beneficial effects of FA on DPN. FA prevented the decrease of motor performance due to DM. This study showed the beneficial effects of FA in the prevention of DPN through improving expression of NGF and lowering MDA levels, and so FA supplementation may be considered as an adjunctive therapy in diabetes patients to improve symptoms of neuropathy. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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