LFS-14339; No of Pages 7 Life Sciences xxx (2015) xxx–xxx
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Life Sciences journal homepage: www.elsevier.com/locate/lifescie
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Sung Tae Kim a,1, Yoon Hee Chung b,1, Ho Sung Lee c, Su Jin Chung c, Jong Hyuk Lee c, Uy Dong Sohn a, Yong Kyoo Shin c, Eon Sub Park d, Hyoung-Chun Kim e, Ji Hoon Jeong c,⁎
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Article history: Received 27 November 2014 Received in revised form 9 February 2015 Accepted 13 March 2015 Available online xxxx
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Keywords: Oxaliplatin Phosphatidylcholine (PC) Peripheral neuropathy Oxidative stress Microglial activation
Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 156-756, Republic of Korea Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea d Department of Pathology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea e Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea
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Aims: The present study was designed to investigate the therapeutic potential of phosphatidylcholine (PC) on oxaliplatin-induced peripheral neuropathy. Main methods: Male Sprague–Dawley rats were randomly divided into three groups: the control group, the oxaliplatin group (4 mg/kg, twice per week for 4 weeks) and the oxaliplatin + PC (300 mg/kg) group. To evaluate the effect of PC, we examined the thermal nociceptive threshold changes in oxaliplatin-induced peripheral neuropathy by conducting paw pressure, hot-plate and tail-flick tests. Additional experiments on the degree of oxidative stress in the sciatic nerves were performed by measuring the level of MDA, total glutathione (GSH), glutathione peroxidase (GPx) activity and superoxide dismutase (SOD) activity. We also used histopathological and immunohistochemical methods to observe neuronal damage and glial activation. Key findings: PC attenuated oxidative stress by increasing antioxidant levels. In histopathological evaluation, the PC administrated group maintained normal morphologic appearance of sciatic nerves, similar to the control group. In spinal cords, however, no significant difference between the oxaliplatin-alone group and the oxaliplatin + PC group was observed. In the immunohistochemical evaluation, PC administration ameliorated oxaliplatin-induced microglial activation. Significance: It is suggested that PC has a therapeutic potential against oxaliplatin-induced peripheral neuropathy due to its antioxidant property and modulation of microglial activities. © 2015 Published by Elsevier Inc.
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Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats
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1. Introduction
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Oxaliplatin is a third-generation platinum-based antineoplastic agent, which is commonly used in treating advanced colorectal cancer, and as adjuvant therapy in several types of cancer [12]. Although oxaliplatin has less ototoxicity and nephrotoxicity than other platinum-based chemotherapeutic agents, it causes acute and chronic peripheral neurotoxicity [1,13]. Acute neuropathy can be observed in almost all patients. This neuropathy occurs within hours of injection, and can be resolved within
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Abbreviations:PC, phosphatidylcholine; GSH, total glutathione; GPx, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; PMSF, phenylmethylsulfonyl fluoride; TBA, thiobarbituric acid; GSSG, glutathione disulfide; GFAP, glial fibrillary acidic protein. ⁎ Corresponding author at: Department of Pharmacology, College of Medicine, ChungAng University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea. Tel.: +82 2 820 5688; fax: +82 2 826 8752. E-mail address: [email protected] (J.H. Jeong). 1 These authors contributed equally to this work and share the first authorship.
days [4,18]. On the other hand, chronic neuropathy is observed in 10–15% of patients after cumulative injection of oxaliplatin, which cannot be resolved easily [10,16]. It is one of the main reasons that patients do not continue their cancer treatments; therefore, it is important to protect cancer patients from chemotherapy-induced neuropathic pain. Developing effective treatments to attenuate peripheral neuropathy is difficult because knowledge about the mechanism of oxaliplatininduced neuropathy is still insufficient [13]. Many studies suggest that oxidative stress associated with oxaliplatin is a direct cause of neuropathy. It is generally known that chemotherapeutic agents generate reactive oxygen species (ROS) to induce apoptosis in cancer cells [11]. However, ROS also affects normal cells and tissues and may be associated with neurotoxicity. In particular, peripheral nerves can be physically damaged by demyelination, mitochondrial dysfunction, inflammation, and apoptosis [26]. Thus, levels of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and activities of mitochondrial enzymes are good biomarkers for determining neuropathy. A recent study suggests that the spinal cord and its subpopulation are directly
http://dx.doi.org/10.1016/j.lfs.2015.03.013 0024-3205/© 2015 Published by Elsevier Inc.
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Oxaliplatin (5 mg/ml) was purchased from the Sanofi-Aventis pharmaceutical company. Phosphatidylcholine was purchased from Lipoid GmbH (Phospholipon90G). MDA, SOD activity, total GSH and GPx activity assay kits were purchased from Biovision Inc. (San Francisco, CA, USA). All other essential chemicals were purchased from SigmaAldrich chemical Co. (St. Louis, MO, USA).
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2.2. Animals
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Male Sprague–Dawley rats, 5 weeks old and weighing about 180 g, were purchased from Samtako Biotechnology (Osan, Republic of Korea). Animals were housed in standard laboratory conditions (22 ± 2 °C, light:dark cycle of 12:12 h), with food (Purina, Republic of Korea) and tap water ad libitum for 1 week before starting the experiment. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chung-Ang University, in accordance with the guidelines for the Care and Use of Laboratory Animals in Seoul, Republic of Korea.
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2.3. Treatments
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To determine the proper dosage of PC, we conducted preliminary experiments in which rats were orally administered several dosages of PC (200 mg, 300 mg or 500 mg/kg) and injected intraperitoneally with 4 mg/kg of oxaliplatin [1,31]. As a result, the PC dosage 300 mg/kg was chosen because it showed the most desired effect against oxaliplatin-induced neuronal damage and thermal & mechanical hypersensitivity. Rats were divided into three groups (n = 6 for each group): the control group was injected intraperitoneally with 0.9% saline twice a week for 4 weeks and orally administered distilled water five times a week for
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All behavioral tests were performed on days 0, 7, 14, 21, and 28. Each 135 test was conducted by the same researcher to minimize variation. 136 2.5. Paw pressure test
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Mechanical nociceptive threshold was assessed by using an analgesiometer (Ugo Basile, Varese, Italy). Briefly, rats were lightly restrained and mechanical pressure was applied to a hind paw of the rat. Pressure was constantly increased until withdrawal reflex was observed, then operators read the scale and recorded the force. The cutoff pressure was 180 g.
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4 weeks. The oxaliplatin group was injected intraperitoneally with oxaliplatin (4 mg/kg) twice a week for 4 weeks and orally administered distilled water. The oxaliplatin (4 mg/kg) + PC group was injected intraperitoneally with oxaliplatin twice a week for 4 weeks and orally administered PC (300 mg/kg) five times a week for 4 weeks. PC was suspended in distilled water (100 mg/ml).
The thermal nociceptive threshold was assessed using the hot-plate method. Rats were placed inside an acryl pipe with a hot-plate floor. The hot plate's temperature was kept constant at 49–50 °C. Once the painreflex behavior was observed, operators recorded the time (seconds). The cut-off time of the latency to pain-reflex behavior was 60 s.
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2.7. Tail-flick test
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The thermal nociceptive threshold was measured in rats using a tailflick unit (Ugo Basile, Varese, Italy). Briefly, rats were lightly restrained and infrared heat stimulus was applied to the tail. When the animal flicked its tail due to thermal stimulus, a sensor detected it and automatically recorded the reaction time. The cut-off time of the latency to tailflick was 10 s.
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After the final behavioral tests (28 days after starting the experiment), the animals were anesthetized with ethyl ether. The right sciatic nerve was isolated and washed with 0.9% saline, then homogenized in lysis buffer containing a protease inhibitor and PMSF. After homogenization, the homogenate was incubated on ice for 1 h then sonicated. The obtained material was centrifuged (13,000 g for 15 min at 4 °C) and stored at − 70 °C, pending biochemical analysis. The left sciatic nerve was excised and fixed in 10% neutral formalin for histopathological examination.
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2.9. Biochemical measurements
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2.9.1. Lipid peroxidation Lipid peroxidation was determined by measuring MDA production using the lipid peroxidation assay kit (K739-100, Biovision). Briefly, sciatic nerve tissue homogenate was diluted with MDA lysis buffer and the total sample volume was 200 μl. A 600 μl of thiobarbituric acid (TBA) reagent was added to each sample and incubated at 95 °C for 60 min. The tube was cooled to room temperature in an ice bath for 10 min. After cooling, 300 μl of n-butanol was added and centrifuged (3 min at 16,000 g). n-Butanol was removed and the MDA–TBA adduct was placed into a 96-well plate and absorbance was measured at 532 nm. MDA content was calculated with MDA standards.
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damaged by oxaliplatin, and oxidative stress is the major reason for this neuronal damage [7]. To ameliorate oxaliplatin-induced neuropathy, various treatments have been suggested including acetyl-L-carnitine, vitamin E, vitamin C, glutathione, and amifostine [2,3,15]. Among these treatments, antioxidants (glutathione, N-acetylcysteine, and vitamin E) are known for their neuron-protecting actions, alleviating functional impairments of neurons [6,8,9,22]. However, their abilities to relieve pain have proven to be insufficient. There are several reasons that can explain this weakness: 1) irreversibility of established oxidative damage; 2) radical specificity of antioxidants; and 3) interference with oxidation-reduction signaling pathways [17]. Phosphatidylcholine (PC, 1,2-diacyl-sn-glycero-3 phosphocholine) is a major component of biological membranes and several studies suggest that PC has antioxidant effects and prevents lipid peroxidation [23,27]. In our previous study, the treatment with PC resulted in a significant attenuation on the increase in serum levels of TNF-α and IL-6, proinflammatory cytokines in lipopolysaccharide-induced acute inflammation in multiple organ injury, suggesting that PC may be a functional material for its use as an anti-inflammatory agent [20]. In addition, decreases in choline level are associated with oxidative damage, resulting in cellular injury and necrosis [30]. Therefore, we evaluated the protective effect of PC in rats by recording the thermal nociceptive threshold changes in oxaliplatin-induced peripheral neuropathy by conducting paw pressure, hot-plate and tail-flick tests. Then, we examined quantity of MDA, total GSH, glutathione peroxidase (GPx) activity and superoxide dismutase activity to determine the oxidative stress level. We used histopathological and immunohistochemical methods to observe neuronal damage and glial activation in sciatic nerves and lumbar spinal cords.
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Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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GPx activity was determined using a glutathione peroxidase activity colorimetric assay kit (K762-100, Biovision). In this assay, GPx reduces hydroperoxide while oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG). The generated GSSH consumes NADPH (by glutathione reductase), which is reduced to GSH. The decrease of NADPH was measured at 340 nm, and the result was proportional to GPx activity.
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SOD activity was determined using a SOD activity assay kit (K335100, Biovision). In this assay, superoxide anion reacts with WST− 1 (water-soluble tetrazolium dye) and water-soluble formazan dye was produced. The rate of the reaction with a superoxide anion was proportional to xanthine oxidase (the enzyme that converts xanthine to uric
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2.14. Immunohistochemical evaluations
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Sections were made after fixing and incubating at room temperature for 1 h in a blocking solution. Tissues were incubated overnight at 4 °C in primary antisera against nitrotyrosine (05-233, Millipore, MA), myelin protein zero (MPZ, ab31851, Abcam, Cambridge, MA), GFAP (ab7260, Abcam, Cambridge) and Iba1 (ab15690, Abcam, Cambridge). Finally, the sections were washed in PBS and visualized with Dako REAL™ EnVision™ Detection System, Peroxidase/DAB, Rabbit/Mouse. Digital images of stained sections were obtained with an Olympus microscope (BX-50). Quantifications were conducted using the graphic editor software Paint.NET.
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The left sciatic nerve and lumbar spinal cord of rats from all groups were excised and fixed in 10% neutral formalin, then dehydrated in 70% ethanol. Each nerve was then embedded in paraffin. 5 μm sections were prepared from the paraffin block and stained by hematoxylin– eosin. Demyelination and degeneration of myelinated fibers in sciatic nerves were evaluated.
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The total GSH was determined using a glutathione detection kit (K251-100, Biovision). Each sample was added to a 96-well plate and assay buffer was added to each well to bring the total volume to 100 μl. Next, 2 μl of glutathione S-transferase reagent and 2 μl of monochlorobimane was added and the plate was incubated for 1 h at 37 °C. Total glutathione was measured by reading absorbance (Ex/Em = 380/461 nm).
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acid, forming a superoxide as a by-product) activity, and this activity was inhibited by SOD; thus, the formazan dye's color was reduced. SOD's inhibition activity was determined by reading the absorbance at 450 nm.
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Fig. 1. Effect of PC on body weight (A), mechanical nociceptive threshold (B) and thermal nociceptive threshold (C, D) in oxaliplatin-treated rats. Body weights (A), paw pressure (B), hotplate test (C) and tail-flick test (D) were measured on days 0, 7, 14, 21 and 28 (n = 6 in each group). Data are expressed as mean ± S.E.M. (#p b 0.05, ##p b 0.01 or ###p b 0.001 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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3. Results
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3.1. Effect of PC on body weights in oxaliplatin-treated rats
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Body weight changes of all groups are shown in Fig. 1A. On day 0, there were no significant differences in body weights between groups. Body weights of the oxaliplatin and oxaliplatin + PC groups were significantly lower than those of the control group on days 7, 14, 21 and 28 (p b 0.01 on days 7 and 14; p b 0.001 on days 21 and 28). However, there were no significant differences in body weights between the oxaliplatin group and the oxaliplatin + PC group.
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MDA levels were measured in each group to evaluate the level of lipid peroxidation. MDA levels of sciatic nerve tissue are shown in Fig. 2A. Oxaliplatin administration caused a significant increase in the MDA levels of sciatic nerve tissue compared with the control group (p b 0.001). The oxaliplatin + PC group showed significantly lower
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Mechanical nociceptive threshold changes in the paw pressure test are shown in Fig. 1B. No significant differences were observed on days 0, 7 and 14. Oxaliplatin significantly lowered paw withdrawal thresholds compared to the control group on days 21 and 28 (p b 0.01 on days 21 and 28). The oxaliplatin + PC group showed no significant difference compared to the oxaliplatin group on days 0, 7, 14 and 21;
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3.4. Effect of PC on lipid peroxidation of sciatic nerve tissue in oxaliplatin- 261 treated rats 262
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The thermal nociceptive threshold was evaluated by the hot-plate test and tail-flick test, and the results are shown in Fig. 1C and D, respectively. In the hot-plate test (Fig. 1C), paw withdrawal thresholds in the oxaliplatin group were significantly lower than those in the control group on days 21 and 28 (p b 0.01 on day 21; p b 0.001 on day 28). The oxaliplatin + PC group showed slightly higher thresholds than the oxaliplatin group, but no statistically significant differences were observed. In the tail-flick test (Fig. 1D), oxaliplatin administration significantly lowered the tail withdrawal thresholds compared with the control group on days 21 and 28 (p b 0.05 on day 21; p b 0.001 on day 28). The oxaliplatin + PC group showed a statistically significant increase of tail withdrawal thresholds compared with the oxaliplatin group on day 28 (p b 0.05).
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The data are expressed as mean ± S.E.M. Statistical differences between the means were analyzed by Turkey's multiple comparison after analysis of variance (ANOVA). Differences were considered significant at error probabilities b 0.05.
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however, on day 28, paw withdrawal thresholds were significantly 245 higher than the oxaliplatin group (p b 0.05). 246
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Fig. 2. Effect of PC on levels of MDA (A), total GSH (B), GPx activity (C) and SOD activity (D) of sciatic nerve tissue in oxaliplatin-treated rats. Levels of MDA (A), total GSH (B), GPx activity (C) and SOD activity (D) were measured in rat sciatic nerves after final behavioral tests (n = 5 in each group). Data are expressed as mean ± S.E.M. (#p b 0.05, ##p b 0.01 or ###p b 0.001 vs. control group; *p b 0.05 or **p b 0.01 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Oxaliplatin administration significantly decreased total GSH level and GPx activity in sciatic nerve tissue compared with the control group (Fig. 2B–D, p b 0.05 for tGSH; p b 0.01 for GPx; p b 0.001 for SOD). Significant increases in total GSH level, GPx and SOD activities were observed in the oxaliplatin + PC group compared to the oxaliplatin group (p b 0.05 for tGSH and GPx; p b 0.01 for SOD).
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Photomicrographs of sciatic nerves from each group after 28 days of oxaliplatin or PC treatment are shown in Fig. 3. In H&E staining, sciatic nerves of the control group showed no histological abnormalities, while oxaliplatin injection induced decreases in the density of nerve fibers. PC administration attenuated these oxaliplatin-induced decreases, resulting in a morphological appearance similar to the control group. To assess the extent of cellular injury in the peripheral nervous system of oxaliplatin-treated rats, we examined the expression of nitrotyrosine in the sciatic nerve by immunohistochemistry. Nitrotyrosine immunoreactivity was increased in the oxaliplatin-treated group (7.57 ± 0.43 vs. 15.67 ± 0.92% area positively stained, p b 0.01). These oxaliplatininduced increases were attenuated by PC administration (15.67 ±
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Representative micrographs of lumbar spinal cords from each group are shown in Fig. 4. In the H&E staining of spinal cords, the control group showed a normal appearance of the dorsal horn of the lumbar spinal cord. In the oxaliplatin group, neuronal cell bodies were increased and the number of cells was significantly decreased compared with the control group (10.67 ± 2.94 vs. 4.66 ± 1.37, p b 0.01). The PC-administered group showed normal morphologic appearance, while the number of cells was not different from the oxaliplatin group. To determine whether neurochemical reorganization in the spinal cord occurs following intraperitoneal oxaliplatin administration, we examined lumbar spinal cord sections using immunohistochemistry with antibodies against GFAP and Iba1 to label astrocytes and microglia, respectively. Astrocyte activation was measured as an increase in the number of GFAPexpressing cells in the dorsal horn of the spinal cord of treated rats. GFAP cell density in superficial laminae of oxaliplatin-treated rats was slightly greater than control at day 28. Spinal astrocytes did not show
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3.7. Histologic and immunohistochemical evaluation of spinal glial 298 activities 299
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0.92 vs. 10.22 ± 0.95% area positively stained, p b 0.05). To investigate if PC affects myelination in sciatic nerves in oxaliplatin-treated animals, we performed immunostaining analysis for MPZ in comparable sections on day 28 of drug administration. MPZ immunoreactivity was decreased in the oxaliplatin-treated group (47.00 ± 1.64 vs. 34.58 ± 2.69% area positively stained, p b 0.05), which was comparably reversed by PC (34.58 ± 2.69 vs. 41.87 ± 1.89% area positively stained, p b 0.05).
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Fig. 3. Representative photomicrographs of sciatic nerve sections from each group. PC administration attenuated oxaliplatin-induced changes in the density of nerve fibers, nitrotyrosine and myelination (magnification 400×). Data are expressed as mean ± S.E.M. (#p b 0.05 or ##p b 0.01 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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In the present study, we demonstrated the protective effects of PC on oxaliplatin-induced peripheral neuropathy using behavioral tests, biochemical tests, and histopathological and immunohistochemical evaluations. Previous studies show that symptoms of oxaliplatin-induced peripheral neuropathy are characterized by pain, numbness, dysesthesia and thermal-related hyperalgesia, and these symptoms occur when 4 mg/kg of oxaliplatin is injected intraperitoneally to rats twice a week for 4 weeks [1,31]. Based on these results, we injected the same doses of oxaliplatin, in the same way. In the paw pressure test, PC administration attenuated the mechanical hyperalgesia induced by oxaliplatin injection. This effect was also observed in the thermal nociceptive threshold in the tail-flick test, suggesting that PC has a protective effect on oxaliplatin-induced thermal hyperalgesia. Oxaliplatin-induced peripheral neurotoxicity in the peripheral nerve shows several histological characteristics including demyelination and degeneration of nerve fibers, and decrease in the number of myelinated fibers [13]. These neuronal damages were also observed in our study. Sciatic nerves of the oxaliplatin-treated group showed axonal
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degeneration and decreased density of myelinated fibers. PC administration prevented these changes, maintaining the morphologic appearance of sciatic nerves similar to the control group. Although some morphological differences between the oxaliplatin group and the oxaliplatin + PC group were observed in the dorsal horns of spinal cords, oxaliplatin-induced decreases in the number of neuronal cell bodies were not prevented by PC administration. To further examine the effect of PC on oxaliplatin-induced glial activation on CNS, immunohistochemical evaluation using GFAP and Iba1 antibodies was performed. On day 28, a lower pain threshold was accompanied by effects on spinal microglia that involve a significant increase of the number of cells immunoreactive to Iba1. Conversely, the oxaliplatin-induced glial activation profile is quite different from what is depicted in trauma-induced pain models, where the inflammatory component is preeminent (Pacini et al., 2010) and microglia act as a promoter in the initial phase of pain facilitation (Marchand et al., 2005; Scholz and Woolf, 2007). In the present model, microglia and astrocytes do not seem to work together, at least during the late phase of spinal cord sensitization. Combined with our previous study showing that PC significantly attenuated the increased serum levels of proinflammatory cytokines [20], the present study showed PCameliorated oxaliplatin-induced microglial activation, meaning that proinflammatory mediator-related nociceptor sensitization could be prevented by PC administration. One important hypothesis that explains the mechanism of this neurotoxicity is the oxidative stress induced by chemotherapeutic agents. Previous studies evaluated the level of oxidative stress induced by chemotherapeutic agents by measuring several antioxidant enzymes and oxidative stress markers and concluded that oxidative stress is
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altered morphology and the number of GFAP-positive cells was not changed after PC treatment. Microglial activation was measured by the quantification of Iba1-positive cells in the spinal cord of treated rats. On day 28, oxaliplatin treatment produced significantly increased Iba1 staining in the dorsal horns of the lumbar spinal cord (10.00 ± 2.12 vs. 16.67 ± 4.27, p b 0.01). PC administration ameliorated the oxaliplatin-induced increase in the number of microglia (16.67 ± 4.27 vs. 11.80 ± 3.11, p b 0.05).
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Fig. 4. Representative photomicrographs of the dorsal horn of spinal cord from each group. In H&E staining of spinal cords (magnification 400×), the PC-administered group showed normal morphologic appearance, while the number of cells was not different from the oxaliplatin group. GFAP cell density in superficial laminae of oxaliplatin-treated rats was slightly greater than control at day 28, which did not alter morphology and the number of GFAP-positive cells after PC treatment. On the other hand, PC administration ameliorated the oxaliplatin-induced increase in the number of microglia. Data are expressed as mean ± S.E.M. (##p b 0.01 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Conflict of interest statement
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[10] [11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24] [25]
The authors declare that there are no conflicts of interest.
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In conclusion, PC administration ameliorated the oxaliplatininduced behavioral, biochemical and histopathological changes in rats. The PC-mediated effects in this study may be attributed to its antioxidant and neuroprotection properties. However, further study is needed to evaluate the effect of PC on several symptoms of oxaliplatin-induced peripheral neuropathy. To get more conclusive results, a larger number of rats in each group and more elaborate techniques are needed.
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Baldelli, et al., Neuroprotective effect of reduced glutathione on oxaliplatin-based chemotherapy in advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial, J. Clin. Oncol. 20 (2002) 3478–3483. S. Cascinu, L. Cordella, E. Del Ferro, M. Fronzoni, G. Catalano, Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in advanced gastric cancer: a randomized double-blind placebo-controlled trial, J. Clin. Oncol. 13 (1995) 26–32. R.J. Cersosimo, Oxaliplatin-associated neuropathy: a review, Ann. Pharmacother. 39 (2005) 128–135. K.A. Conklin, Chemotherapy-associated oxidative stress: impact on chemotherapeutic effectiveness, Integr. Cancer Ther. 3 (2004) 294–300. L. Di Cesare Mannelli, A. Pacini, L. Bonaccini, M. Zanardelli, T. Mello, C. Ghelardini, Morphologic features and glial activation in rat oxaliplatin-dependent neuropathic pain, J. Pain 14 (2013) 1585–1600. L. Di Cesare Mannelli, M. Zanardelli, P. Failli, C. 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Lapied, M. Pelhate, E. Gamelin, A possible explanation for a neurotoxic effect of the anticancer agent oxaliplatin on neuronal voltage-gated sodium channels, J. Neurophysiol. 85 (2001) 2293–2297. E.K. Joseph, X. Chen, O. Bogen, J.D. Levine, Oxaliplatin acts on IB4-positive nociceptors to induce an oxidative stress-dependent acute painful peripheral neuropathy, J. Pain 9 (2008) 463–472. Y.Y. Jung, Y. Nam, Y.S. Park, H.S. Lee, S.A. Hong, B.K. Kim, E.S. Park, Y.H. Chung, J.H. Jeong, Protective effect of phosphatidylcholine on lipopolysaccharide-induced acute inflammation in multiple organ injury, Korean J. Physiol. Pharmacol. 17 (2013) 209–216. C. Kerksick, D. Willoughby, The antioxidant role of glutathione and N-acetylcysteine supplements and exercise-induced oxidative stress, J. Int. Soc. Sports Nutr. 2 (2005) 38–44. L.A. Kottschade, J.A. Sloan, M.A. Mazurczak, D.B. Johnson, B.P. Murphy, K.M. Rowland, et al., The use of vitamin E for the prevention of chemotherapy-induced peripheral neuropathy: results of a randomized phase III clinical trial, Support Care Cancer 19 (2011) 1769–1777. H.S. Lee, B.K. Kim, Y. Nam, U.D. Sohn, E.S. Park, S.A. Hong, et al., Protective role of phosphatidylcholine against cisplatin-induced renal toxicity and oxidative stress in rats, Food Chem. Toxicol. 58 (2013) 388–393. J.O.W. Li, W. Li, Z.G. Jiang, H.A. Ghanbari, Oxidative stress and neurodegenerative disorders, Int. J. Mol. Sci. 14 (2013) 24438–24475. S. Li, T. Yan, J.Q. Yang, T.D. Oberley, L.W. Oberley, The role of cellular glutathione peroxidase redox regulation in the suppression of tumor cell growth by manganese superoxide dismutase, Cancer Res. 60 (2000) 3927–3939. P.A. Low, K.K. Nickander, H.J. Tritschler, The roles of oxidative stress and antioxidant treatment in experimental diabetic neuropathy, Diabetes 46 (Suppl. 2) (1997) S38–S42. K.P. Navder, E. Baraona, C.S. Lieber, Dilinoleoylphosphatidylcholine protects human low density lipoproteins against oxidation, Atherosclerosis 152 (2000) 89–95. R. Noor, S. Mittal, J. Iqbal, Superoxide dismutase—applications and relevance to human diseases, Med. Sci. Monit. 8 (2002) RA210–RA215. G. Ossani, M. Dalghi, M. Repetto, Oxidative damage lipid peroxidation in the kidney of choline-deficient rats, Front. Biosci. 12 (2007) 1174–1183. M.G. Repetto, G. Ossani, A.J. Monserrat, A. Boveris, Oxidative damage: the biochemical mechanism of cellular injury and necrosis in choline deficiency, Exp. Mol. Pathol. 88 (2010) 143–149. S. Ushio, N. Egashira, H. Sada, T. Kawashiri, M. Shirahama, K. Masuguchi, et al., Goshajinkigan reduces oxaliplatin-induced peripheral neuropathy without affecting anti-tumour efficacy in rodents, Eur. J. Cancer 48 (2012) 1407–1413. M.I. Waly, M.S. Al Moundhri, B.H. Ali, Effect of curcumin on cisplatin- and oxaliplatin-induced oxidative stress in human embryonic kidney (HEK) 293 cells, Ren. 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related to peripheral neuropathy [14,19]. Based on this theory, several agents and nutraceuticals with antioxidant properties have been tested in in vivo studies and clinical trials to assess their effectiveness against peripheral neurotoxicity induced by chemotherapy. To be effective, these agents must not only mitigate the drug-induced neurotoxicity, but must also preserve the anti-cancer effectiveness. The potential effect on the anti-tumor activity of the chemotherapy was not seen in small case series reporting reduction of neurotoxicity of a particular drug [3]. Considering these protective effects of various antioxidant agents against chemotherapy-induced peripheral neuropathy, PC was tested in the present study as a potential antioxidant. In the present study, PC administration prevented oxaliplatin-induced MDA level increases, which means that PC administration decreased the level of lipid peroxidation. Oxaliplatin injection caused an increase of nitrotyrosine, a marker for detecting oxidative stress [7], in sciatic nerves, which was ameliorated by PC administration. In addition to evaluating the level of oxidative stress markers such as MDA, host antioxidant system-related enzymes and chemicals, such as SOD, GPx and total GSH levels, were evaluated in the present study. SOD is an important enzyme in the host defense system against oxidative stress due to its antioxidant property [28]. Many studies have shown that excessive oxidative stress causes damage to enzymes including SOD and GPx, resulting in the loss of antioxidant defenses [5, 24,32]. GSH and GPx also play important roles in endogenous celldefense systems against oxidative stress. GPx is an essential antioxidant enzyme that converts hydrogen peroxide into water, and GSH is an important cofactor of GPx to function in this reaction [25]. Low levels of GPx and GSH have been correlated with free-radical-related disorders such as cancer, chronic disease and aging [21]. Recently, it appears that GSH is a promising drug for the prevention of oxaliplatin-induced neuropathy without significantly compromising the clinical activity of oxaliplatin. It was thought that the mechanism of the neuroprotective action of glutathione was the prevention of the accumulation of platinum metabolites [10]. Therefore, the ameliorative effects of PC might result from preventing the oxaliplatin-induced decreases in SOD, GPx and GSH levels.
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Contents lists available at ScienceDirect
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Sung Tae Kim a,1, Yoon Hee Chung b,1, Ho Sung Lee c, Su Jin Chung c, Jong Hyuk Lee c, Uy Dong Sohn a, Yong Kyoo Shin c, Eon Sub Park d, Hyoung-Chun Kim e, Ji Hoon Jeong c,⁎
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Article history: Received 27 November 2014 Received in revised form 9 February 2015 Accepted 13 March 2015 Available online xxxx
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Keywords: Oxaliplatin Phosphatidylcholine (PC) Peripheral neuropathy Oxidative stress Microglial activation
Department of Pharmacology, College of Pharmacy, Chung-Ang University, Seoul, 156-756, Republic of Korea Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea d Department of Pathology, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea e Neuropsychopharmacology and Toxicology Program, College of Pharmacy, Kangwon National University, Chunchon 200-701, Republic of Korea
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Aims: The present study was designed to investigate the therapeutic potential of phosphatidylcholine (PC) on oxaliplatin-induced peripheral neuropathy. Main methods: Male Sprague–Dawley rats were randomly divided into three groups: the control group, the oxaliplatin group (4 mg/kg, twice per week for 4 weeks) and the oxaliplatin + PC (300 mg/kg) group. To evaluate the effect of PC, we examined the thermal nociceptive threshold changes in oxaliplatin-induced peripheral neuropathy by conducting paw pressure, hot-plate and tail-flick tests. Additional experiments on the degree of oxidative stress in the sciatic nerves were performed by measuring the level of MDA, total glutathione (GSH), glutathione peroxidase (GPx) activity and superoxide dismutase (SOD) activity. We also used histopathological and immunohistochemical methods to observe neuronal damage and glial activation. Key findings: PC attenuated oxidative stress by increasing antioxidant levels. In histopathological evaluation, the PC administrated group maintained normal morphologic appearance of sciatic nerves, similar to the control group. In spinal cords, however, no significant difference between the oxaliplatin-alone group and the oxaliplatin + PC group was observed. In the immunohistochemical evaluation, PC administration ameliorated oxaliplatin-induced microglial activation. Significance: It is suggested that PC has a therapeutic potential against oxaliplatin-induced peripheral neuropathy due to its antioxidant property and modulation of microglial activities. © 2015 Published by Elsevier Inc.
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Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats
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1. Introduction
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Oxaliplatin is a third-generation platinum-based antineoplastic agent, which is commonly used in treating advanced colorectal cancer, and as adjuvant therapy in several types of cancer [12]. Although oxaliplatin has less ototoxicity and nephrotoxicity than other platinum-based chemotherapeutic agents, it causes acute and chronic peripheral neurotoxicity [1,13]. Acute neuropathy can be observed in almost all patients. This neuropathy occurs within hours of injection, and can be resolved within
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Abbreviations:PC, phosphatidylcholine; GSH, total glutathione; GPx, glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; PMSF, phenylmethylsulfonyl fluoride; TBA, thiobarbituric acid; GSSG, glutathione disulfide; GFAP, glial fibrillary acidic protein. ⁎ Corresponding author at: Department of Pharmacology, College of Medicine, ChungAng University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea. Tel.: +82 2 820 5688; fax: +82 2 826 8752. E-mail address: [email protected] (J.H. Jeong). 1 These authors contributed equally to this work and share the first authorship.
days [4,18]. On the other hand, chronic neuropathy is observed in 10–15% of patients after cumulative injection of oxaliplatin, which cannot be resolved easily [10,16]. It is one of the main reasons that patients do not continue their cancer treatments; therefore, it is important to protect cancer patients from chemotherapy-induced neuropathic pain. Developing effective treatments to attenuate peripheral neuropathy is difficult because knowledge about the mechanism of oxaliplatininduced neuropathy is still insufficient [13]. Many studies suggest that oxidative stress associated with oxaliplatin is a direct cause of neuropathy. It is generally known that chemotherapeutic agents generate reactive oxygen species (ROS) to induce apoptosis in cancer cells [11]. However, ROS also affects normal cells and tissues and may be associated with neurotoxicity. In particular, peripheral nerves can be physically damaged by demyelination, mitochondrial dysfunction, inflammation, and apoptosis [26]. Thus, levels of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and activities of mitochondrial enzymes are good biomarkers for determining neuropathy. A recent study suggests that the spinal cord and its subpopulation are directly
http://dx.doi.org/10.1016/j.lfs.2015.03.013 0024-3205/© 2015 Published by Elsevier Inc.
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Oxaliplatin (5 mg/ml) was purchased from the Sanofi-Aventis pharmaceutical company. Phosphatidylcholine was purchased from Lipoid GmbH (Phospholipon90G). MDA, SOD activity, total GSH and GPx activity assay kits were purchased from Biovision Inc. (San Francisco, CA, USA). All other essential chemicals were purchased from SigmaAldrich chemical Co. (St. Louis, MO, USA).
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Male Sprague–Dawley rats, 5 weeks old and weighing about 180 g, were purchased from Samtako Biotechnology (Osan, Republic of Korea). Animals were housed in standard laboratory conditions (22 ± 2 °C, light:dark cycle of 12:12 h), with food (Purina, Republic of Korea) and tap water ad libitum for 1 week before starting the experiment. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chung-Ang University, in accordance with the guidelines for the Care and Use of Laboratory Animals in Seoul, Republic of Korea.
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To determine the proper dosage of PC, we conducted preliminary experiments in which rats were orally administered several dosages of PC (200 mg, 300 mg or 500 mg/kg) and injected intraperitoneally with 4 mg/kg of oxaliplatin [1,31]. As a result, the PC dosage 300 mg/kg was chosen because it showed the most desired effect against oxaliplatin-induced neuronal damage and thermal & mechanical hypersensitivity. Rats were divided into three groups (n = 6 for each group): the control group was injected intraperitoneally with 0.9% saline twice a week for 4 weeks and orally administered distilled water five times a week for
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All behavioral tests were performed on days 0, 7, 14, 21, and 28. Each 135 test was conducted by the same researcher to minimize variation. 136 2.5. Paw pressure test
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Mechanical nociceptive threshold was assessed by using an analgesiometer (Ugo Basile, Varese, Italy). Briefly, rats were lightly restrained and mechanical pressure was applied to a hind paw of the rat. Pressure was constantly increased until withdrawal reflex was observed, then operators read the scale and recorded the force. The cutoff pressure was 180 g.
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4 weeks. The oxaliplatin group was injected intraperitoneally with oxaliplatin (4 mg/kg) twice a week for 4 weeks and orally administered distilled water. The oxaliplatin (4 mg/kg) + PC group was injected intraperitoneally with oxaliplatin twice a week for 4 weeks and orally administered PC (300 mg/kg) five times a week for 4 weeks. PC was suspended in distilled water (100 mg/ml).
The thermal nociceptive threshold was assessed using the hot-plate method. Rats were placed inside an acryl pipe with a hot-plate floor. The hot plate's temperature was kept constant at 49–50 °C. Once the painreflex behavior was observed, operators recorded the time (seconds). The cut-off time of the latency to pain-reflex behavior was 60 s.
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The thermal nociceptive threshold was measured in rats using a tailflick unit (Ugo Basile, Varese, Italy). Briefly, rats were lightly restrained and infrared heat stimulus was applied to the tail. When the animal flicked its tail due to thermal stimulus, a sensor detected it and automatically recorded the reaction time. The cut-off time of the latency to tailflick was 10 s.
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After the final behavioral tests (28 days after starting the experiment), the animals were anesthetized with ethyl ether. The right sciatic nerve was isolated and washed with 0.9% saline, then homogenized in lysis buffer containing a protease inhibitor and PMSF. After homogenization, the homogenate was incubated on ice for 1 h then sonicated. The obtained material was centrifuged (13,000 g for 15 min at 4 °C) and stored at − 70 °C, pending biochemical analysis. The left sciatic nerve was excised and fixed in 10% neutral formalin for histopathological examination.
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2.9.1. Lipid peroxidation Lipid peroxidation was determined by measuring MDA production using the lipid peroxidation assay kit (K739-100, Biovision). Briefly, sciatic nerve tissue homogenate was diluted with MDA lysis buffer and the total sample volume was 200 μl. A 600 μl of thiobarbituric acid (TBA) reagent was added to each sample and incubated at 95 °C for 60 min. The tube was cooled to room temperature in an ice bath for 10 min. After cooling, 300 μl of n-butanol was added and centrifuged (3 min at 16,000 g). n-Butanol was removed and the MDA–TBA adduct was placed into a 96-well plate and absorbance was measured at 532 nm. MDA content was calculated with MDA standards.
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damaged by oxaliplatin, and oxidative stress is the major reason for this neuronal damage [7]. To ameliorate oxaliplatin-induced neuropathy, various treatments have been suggested including acetyl-L-carnitine, vitamin E, vitamin C, glutathione, and amifostine [2,3,15]. Among these treatments, antioxidants (glutathione, N-acetylcysteine, and vitamin E) are known for their neuron-protecting actions, alleviating functional impairments of neurons [6,8,9,22]. However, their abilities to relieve pain have proven to be insufficient. There are several reasons that can explain this weakness: 1) irreversibility of established oxidative damage; 2) radical specificity of antioxidants; and 3) interference with oxidation-reduction signaling pathways [17]. Phosphatidylcholine (PC, 1,2-diacyl-sn-glycero-3 phosphocholine) is a major component of biological membranes and several studies suggest that PC has antioxidant effects and prevents lipid peroxidation [23,27]. In our previous study, the treatment with PC resulted in a significant attenuation on the increase in serum levels of TNF-α and IL-6, proinflammatory cytokines in lipopolysaccharide-induced acute inflammation in multiple organ injury, suggesting that PC may be a functional material for its use as an anti-inflammatory agent [20]. In addition, decreases in choline level are associated with oxidative damage, resulting in cellular injury and necrosis [30]. Therefore, we evaluated the protective effect of PC in rats by recording the thermal nociceptive threshold changes in oxaliplatin-induced peripheral neuropathy by conducting paw pressure, hot-plate and tail-flick tests. Then, we examined quantity of MDA, total GSH, glutathione peroxidase (GPx) activity and superoxide dismutase activity to determine the oxidative stress level. We used histopathological and immunohistochemical methods to observe neuronal damage and glial activation in sciatic nerves and lumbar spinal cords.
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GPx activity was determined using a glutathione peroxidase activity colorimetric assay kit (K762-100, Biovision). In this assay, GPx reduces hydroperoxide while oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG). The generated GSSH consumes NADPH (by glutathione reductase), which is reduced to GSH. The decrease of NADPH was measured at 340 nm, and the result was proportional to GPx activity.
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SOD activity was determined using a SOD activity assay kit (K335100, Biovision). In this assay, superoxide anion reacts with WST− 1 (water-soluble tetrazolium dye) and water-soluble formazan dye was produced. The rate of the reaction with a superoxide anion was proportional to xanthine oxidase (the enzyme that converts xanthine to uric
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Sections were made after fixing and incubating at room temperature for 1 h in a blocking solution. Tissues were incubated overnight at 4 °C in primary antisera against nitrotyrosine (05-233, Millipore, MA), myelin protein zero (MPZ, ab31851, Abcam, Cambridge, MA), GFAP (ab7260, Abcam, Cambridge) and Iba1 (ab15690, Abcam, Cambridge). Finally, the sections were washed in PBS and visualized with Dako REAL™ EnVision™ Detection System, Peroxidase/DAB, Rabbit/Mouse. Digital images of stained sections were obtained with an Olympus microscope (BX-50). Quantifications were conducted using the graphic editor software Paint.NET.
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The left sciatic nerve and lumbar spinal cord of rats from all groups were excised and fixed in 10% neutral formalin, then dehydrated in 70% ethanol. Each nerve was then embedded in paraffin. 5 μm sections were prepared from the paraffin block and stained by hematoxylin– eosin. Demyelination and degeneration of myelinated fibers in sciatic nerves were evaluated.
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The total GSH was determined using a glutathione detection kit (K251-100, Biovision). Each sample was added to a 96-well plate and assay buffer was added to each well to bring the total volume to 100 μl. Next, 2 μl of glutathione S-transferase reagent and 2 μl of monochlorobimane was added and the plate was incubated for 1 h at 37 °C. Total glutathione was measured by reading absorbance (Ex/Em = 380/461 nm).
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acid, forming a superoxide as a by-product) activity, and this activity was inhibited by SOD; thus, the formazan dye's color was reduced. SOD's inhibition activity was determined by reading the absorbance at 450 nm.
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Fig. 1. Effect of PC on body weight (A), mechanical nociceptive threshold (B) and thermal nociceptive threshold (C, D) in oxaliplatin-treated rats. Body weights (A), paw pressure (B), hotplate test (C) and tail-flick test (D) were measured on days 0, 7, 14, 21 and 28 (n = 6 in each group). Data are expressed as mean ± S.E.M. (#p b 0.05, ##p b 0.01 or ###p b 0.001 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Body weight changes of all groups are shown in Fig. 1A. On day 0, there were no significant differences in body weights between groups. Body weights of the oxaliplatin and oxaliplatin + PC groups were significantly lower than those of the control group on days 7, 14, 21 and 28 (p b 0.01 on days 7 and 14; p b 0.001 on days 21 and 28). However, there were no significant differences in body weights between the oxaliplatin group and the oxaliplatin + PC group.
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MDA levels were measured in each group to evaluate the level of lipid peroxidation. MDA levels of sciatic nerve tissue are shown in Fig. 2A. Oxaliplatin administration caused a significant increase in the MDA levels of sciatic nerve tissue compared with the control group (p b 0.001). The oxaliplatin + PC group showed significantly lower
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Mechanical nociceptive threshold changes in the paw pressure test are shown in Fig. 1B. No significant differences were observed on days 0, 7 and 14. Oxaliplatin significantly lowered paw withdrawal thresholds compared to the control group on days 21 and 28 (p b 0.01 on days 21 and 28). The oxaliplatin + PC group showed no significant difference compared to the oxaliplatin group on days 0, 7, 14 and 21;
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The thermal nociceptive threshold was evaluated by the hot-plate test and tail-flick test, and the results are shown in Fig. 1C and D, respectively. In the hot-plate test (Fig. 1C), paw withdrawal thresholds in the oxaliplatin group were significantly lower than those in the control group on days 21 and 28 (p b 0.01 on day 21; p b 0.001 on day 28). The oxaliplatin + PC group showed slightly higher thresholds than the oxaliplatin group, but no statistically significant differences were observed. In the tail-flick test (Fig. 1D), oxaliplatin administration significantly lowered the tail withdrawal thresholds compared with the control group on days 21 and 28 (p b 0.05 on day 21; p b 0.001 on day 28). The oxaliplatin + PC group showed a statistically significant increase of tail withdrawal thresholds compared with the oxaliplatin group on day 28 (p b 0.05).
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however, on day 28, paw withdrawal thresholds were significantly 245 higher than the oxaliplatin group (p b 0.05). 246
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Fig. 2. Effect of PC on levels of MDA (A), total GSH (B), GPx activity (C) and SOD activity (D) of sciatic nerve tissue in oxaliplatin-treated rats. Levels of MDA (A), total GSH (B), GPx activity (C) and SOD activity (D) were measured in rat sciatic nerves after final behavioral tests (n = 5 in each group). Data are expressed as mean ± S.E.M. (#p b 0.05, ##p b 0.01 or ###p b 0.001 vs. control group; *p b 0.05 or **p b 0.01 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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Oxaliplatin administration significantly decreased total GSH level and GPx activity in sciatic nerve tissue compared with the control group (Fig. 2B–D, p b 0.05 for tGSH; p b 0.01 for GPx; p b 0.001 for SOD). Significant increases in total GSH level, GPx and SOD activities were observed in the oxaliplatin + PC group compared to the oxaliplatin group (p b 0.05 for tGSH and GPx; p b 0.01 for SOD).
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Photomicrographs of sciatic nerves from each group after 28 days of oxaliplatin or PC treatment are shown in Fig. 3. In H&E staining, sciatic nerves of the control group showed no histological abnormalities, while oxaliplatin injection induced decreases in the density of nerve fibers. PC administration attenuated these oxaliplatin-induced decreases, resulting in a morphological appearance similar to the control group. To assess the extent of cellular injury in the peripheral nervous system of oxaliplatin-treated rats, we examined the expression of nitrotyrosine in the sciatic nerve by immunohistochemistry. Nitrotyrosine immunoreactivity was increased in the oxaliplatin-treated group (7.57 ± 0.43 vs. 15.67 ± 0.92% area positively stained, p b 0.01). These oxaliplatininduced increases were attenuated by PC administration (15.67 ±
H&E
Nitrotyrosine
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MPZ
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Control
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Representative micrographs of lumbar spinal cords from each group are shown in Fig. 4. In the H&E staining of spinal cords, the control group showed a normal appearance of the dorsal horn of the lumbar spinal cord. In the oxaliplatin group, neuronal cell bodies were increased and the number of cells was significantly decreased compared with the control group (10.67 ± 2.94 vs. 4.66 ± 1.37, p b 0.01). The PC-administered group showed normal morphologic appearance, while the number of cells was not different from the oxaliplatin group. To determine whether neurochemical reorganization in the spinal cord occurs following intraperitoneal oxaliplatin administration, we examined lumbar spinal cord sections using immunohistochemistry with antibodies against GFAP and Iba1 to label astrocytes and microglia, respectively. Astrocyte activation was measured as an increase in the number of GFAPexpressing cells in the dorsal horn of the spinal cord of treated rats. GFAP cell density in superficial laminae of oxaliplatin-treated rats was slightly greater than control at day 28. Spinal astrocytes did not show
Oxaliplatin
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3.7. Histologic and immunohistochemical evaluation of spinal glial 298 activities 299
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3.5. Effect of PC on antioxidant levels of sciatic nerve tissue in oxaliplatintreated rats
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0.92 vs. 10.22 ± 0.95% area positively stained, p b 0.05). To investigate if PC affects myelination in sciatic nerves in oxaliplatin-treated animals, we performed immunostaining analysis for MPZ in comparable sections on day 28 of drug administration. MPZ immunoreactivity was decreased in the oxaliplatin-treated group (47.00 ± 1.64 vs. 34.58 ± 2.69% area positively stained, p b 0.05), which was comparably reversed by PC (34.58 ± 2.69 vs. 41.87 ± 1.89% area positively stained, p b 0.05).
P
levels of MDA in the sciatic nerve tissue compared with the oxaliplatin group (p b 0.01).
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Fig. 3. Representative photomicrographs of sciatic nerve sections from each group. PC administration attenuated oxaliplatin-induced changes in the density of nerve fibers, nitrotyrosine and myelination (magnification 400×). Data are expressed as mean ± S.E.M. (#p b 0.05 or ##p b 0.01 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315
In the present study, we demonstrated the protective effects of PC on oxaliplatin-induced peripheral neuropathy using behavioral tests, biochemical tests, and histopathological and immunohistochemical evaluations. Previous studies show that symptoms of oxaliplatin-induced peripheral neuropathy are characterized by pain, numbness, dysesthesia and thermal-related hyperalgesia, and these symptoms occur when 4 mg/kg of oxaliplatin is injected intraperitoneally to rats twice a week for 4 weeks [1,31]. Based on these results, we injected the same doses of oxaliplatin, in the same way. In the paw pressure test, PC administration attenuated the mechanical hyperalgesia induced by oxaliplatin injection. This effect was also observed in the thermal nociceptive threshold in the tail-flick test, suggesting that PC has a protective effect on oxaliplatin-induced thermal hyperalgesia. Oxaliplatin-induced peripheral neurotoxicity in the peripheral nerve shows several histological characteristics including demyelination and degeneration of nerve fibers, and decrease in the number of myelinated fibers [13]. These neuronal damages were also observed in our study. Sciatic nerves of the oxaliplatin-treated group showed axonal
H&E
GFAP
Iba-1
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Control
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Oxaliplatin
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O
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Oxaliplatin + PC
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U
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4. Discussion
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degeneration and decreased density of myelinated fibers. PC administration prevented these changes, maintaining the morphologic appearance of sciatic nerves similar to the control group. Although some morphological differences between the oxaliplatin group and the oxaliplatin + PC group were observed in the dorsal horns of spinal cords, oxaliplatin-induced decreases in the number of neuronal cell bodies were not prevented by PC administration. To further examine the effect of PC on oxaliplatin-induced glial activation on CNS, immunohistochemical evaluation using GFAP and Iba1 antibodies was performed. On day 28, a lower pain threshold was accompanied by effects on spinal microglia that involve a significant increase of the number of cells immunoreactive to Iba1. Conversely, the oxaliplatin-induced glial activation profile is quite different from what is depicted in trauma-induced pain models, where the inflammatory component is preeminent (Pacini et al., 2010) and microglia act as a promoter in the initial phase of pain facilitation (Marchand et al., 2005; Scholz and Woolf, 2007). In the present model, microglia and astrocytes do not seem to work together, at least during the late phase of spinal cord sensitization. Combined with our previous study showing that PC significantly attenuated the increased serum levels of proinflammatory cytokines [20], the present study showed PCameliorated oxaliplatin-induced microglial activation, meaning that proinflammatory mediator-related nociceptor sensitization could be prevented by PC administration. One important hypothesis that explains the mechanism of this neurotoxicity is the oxidative stress induced by chemotherapeutic agents. Previous studies evaluated the level of oxidative stress induced by chemotherapeutic agents by measuring several antioxidant enzymes and oxidative stress markers and concluded that oxidative stress is
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altered morphology and the number of GFAP-positive cells was not changed after PC treatment. Microglial activation was measured by the quantification of Iba1-positive cells in the spinal cord of treated rats. On day 28, oxaliplatin treatment produced significantly increased Iba1 staining in the dorsal horns of the lumbar spinal cord (10.00 ± 2.12 vs. 16.67 ± 4.27, p b 0.01). PC administration ameliorated the oxaliplatin-induced increase in the number of microglia (16.67 ± 4.27 vs. 11.80 ± 3.11, p b 0.05).
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S.T. Kim et al. / Life Sciences xxx (2015) xxx–xxx
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H&E
GFAP
Iba-1
Control
10.67 ± 2.94
7.00 ± 1.63
10.00 ± 2.12
Oxaliplatin
4.66 ± 1.37##
7.25 ± 2.22
16.67 ± 4.27##
Oxaliplatin + PC
5.16 ± 1.33
6.60 ± 1.81
11.80 ± 3.11*
Fig. 4. Representative photomicrographs of the dorsal horn of spinal cord from each group. In H&E staining of spinal cords (magnification 400×), the PC-administered group showed normal morphologic appearance, while the number of cells was not different from the oxaliplatin group. GFAP cell density in superficial laminae of oxaliplatin-treated rats was slightly greater than control at day 28, which did not alter morphology and the number of GFAP-positive cells after PC treatment. On the other hand, PC administration ameliorated the oxaliplatin-induced increase in the number of microglia. Data are expressed as mean ± S.E.M. (##p b 0.01 vs. control group; *p b 0.05 vs. oxaliplatin group).
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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S.T. Kim et al. / Life Sciences xxx (2015) xxx–xxx
Conflict of interest statement
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[9]
[10] [11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
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[23]
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The authors declare that there are no conflicts of interest.
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In conclusion, PC administration ameliorated the oxaliplatininduced behavioral, biochemical and histopathological changes in rats. The PC-mediated effects in this study may be attributed to its antioxidant and neuroprotection properties. However, further study is needed to evaluate the effect of PC on several symptoms of oxaliplatin-induced peripheral neuropathy. To get more conclusive results, a larger number of rats in each group and more elaborate techniques are needed.
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Fail. 33 (2011) 518–523.
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related to peripheral neuropathy [14,19]. Based on this theory, several agents and nutraceuticals with antioxidant properties have been tested in in vivo studies and clinical trials to assess their effectiveness against peripheral neurotoxicity induced by chemotherapy. To be effective, these agents must not only mitigate the drug-induced neurotoxicity, but must also preserve the anti-cancer effectiveness. The potential effect on the anti-tumor activity of the chemotherapy was not seen in small case series reporting reduction of neurotoxicity of a particular drug [3]. Considering these protective effects of various antioxidant agents against chemotherapy-induced peripheral neuropathy, PC was tested in the present study as a potential antioxidant. In the present study, PC administration prevented oxaliplatin-induced MDA level increases, which means that PC administration decreased the level of lipid peroxidation. Oxaliplatin injection caused an increase of nitrotyrosine, a marker for detecting oxidative stress [7], in sciatic nerves, which was ameliorated by PC administration. In addition to evaluating the level of oxidative stress markers such as MDA, host antioxidant system-related enzymes and chemicals, such as SOD, GPx and total GSH levels, were evaluated in the present study. SOD is an important enzyme in the host defense system against oxidative stress due to its antioxidant property [28]. Many studies have shown that excessive oxidative stress causes damage to enzymes including SOD and GPx, resulting in the loss of antioxidant defenses [5, 24,32]. GSH and GPx also play important roles in endogenous celldefense systems against oxidative stress. GPx is an essential antioxidant enzyme that converts hydrogen peroxide into water, and GSH is an important cofactor of GPx to function in this reaction [25]. Low levels of GPx and GSH have been correlated with free-radical-related disorders such as cancer, chronic disease and aging [21]. Recently, it appears that GSH is a promising drug for the prevention of oxaliplatin-induced neuropathy without significantly compromising the clinical activity of oxaliplatin. It was thought that the mechanism of the neuroprotective action of glutathione was the prevention of the accumulation of platinum metabolites [10]. Therefore, the ameliorative effects of PC might result from preventing the oxaliplatin-induced decreases in SOD, GPx and GSH levels.
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[26]
[27] [28] [29] [30]
[31]
[32]
Please cite this article as: S.T. Kim, et al., Protective effects of phosphatidylcholine on oxaliplatin-induced neuropathy in rats..., Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.013
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