NeuroToxicology 41 (2014) 1–8

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NeuroToxicology

The neuroprotective effect of tropisetron on vincristine-induced neurotoxicity Anita Barzegar-Fallah a, Houman Alimoradi b, Saeed Mehrzadi c, Niloofar Barzegar-Fallah d, Adib Zendedel e, Ata Abbasi f, Ahmad Reza Dehpour c,g,* a

Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran Department of Pharmacology and Toxicology, University of Otago, P.O. Box 913, Dunedin, New Zealand Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran d Department of Pharmacology, Islamic Azad University of Pharmaceutical Sciences, Tehran, Iran e Institute of Neuroanatomy, Faculty of Medicine, RWTH Aachen University, Germany f Department of Pathology, Tehran University of Medical Science, Tehran, Iran g Experimental Medicine Research Center, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 July 2013 Accepted 6 December 2013 Available online 26 December 2013

Vincristine (VCR) peripheral neuropathy is a dose-limiting side effect. Several studies have shown that tropisetron, a 5-HT3 receptor antagonist, exerts anti-inflammatory and immunomodulatory properties. Current study was designed to investigate a suppressive effect of tropisetron on VCR-induced neuropathy and whether this effect exerts through the 5-HT3 receptor or not. Neuropathy was induced in rats by administration of vincristine (0.5 mg/kg, 3 intraperitoneal injections on alternate days) and in treatment group, tropisetron (3 mg/kg); m-chlorophenylbiguanide (mCPBG), a selective 5-HT3 receptor agonist (15 mg/kg); tropisetron (3 mg/kg) plus mCPBG (15 mg/kg); granisetron, another selective 5-HT3 receptor antagonist (3 mg/kg) were administered intraperitoneally 1 h prior to vincristine injection. Hot plate, open field tests (total distance moved, mean velocity and percentage of total duration of the movement) and motor nerve conduction velocity (MNCV) were performed to evaluate the sensory and motor neuropathy. Further, plasma levels of tumor necrosis factor-alpha (TNF-a) and interleukin-2 (IL-2) and the level of TNF-a in sciatic nerve were assessed as well as histological examination. In only VCR-treated rats hot plate latencies were significantly increased, total distance moved, mean velocity, total duration of the movement and sciatic MNCV significantly decreased compared with control. In tropisetron and tropisetron plus mCPBG groups, one injection of tropisetron prior to each VCR injection robustly diminished TNF-a and IL-2 levels, and also prevented mixed sensory-motor neuropathy, as indicated by less mortality rate, better general conditions, behavioral and electrophysiological studies. Moreover, pathological evidence confirmed the results obtained from other findings. But granisetron and mCPBG had no significant effect on the mentioned parameters. In conclusion, these studies demonstrate that tropisetron significantly suppressed VCR-induced neuropathy and could be a neuroprotective agent for prevention of VCR-induced neuropathy via a receptor-independent pathway. ß 2013 Elsevier Inc. All rights reserved.

Keywords: Vincristine Tropisetron Granisetron Peripheral neuropathy Neuroprotective

1. Introduction Vincristine (VCR), commonly prescribed chemotherapeutic agent, (Sandler et al., 1969) is being used in treatment of

* Corresponding author at: Pharmacology Department, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. Tel.: +98 2188973652; fax: +98 2166402569. E-mail addresses: [email protected], [email protected] (A.R. Dehpour). 0161-813X/$ – see front matter ß 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2013.12.002

various cancers especially Hodgkin’s lymphoma, non-Hodgkin’s lymphoma and leukemia either by itself or in combination with other antitumor agents (Bromberg, 2000; Pal, 1999). Unfortunately, neuropathic pain following by mixed sensorymotor neuropathy is a dose-duration dependent adverse effect of vincristine which often limits its clinical use and persists even after stopping its administration (Quasthoff and Hartung, 2002; Rosenthal and Kaufman, 1974; Sandler et al., 1969). The pathogenesis of peripheral neuropathy induced by vincristine is poorly understood in detail, but infiltration of immune cells such as macrophages and lymphocytes into the

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injured region and up-regulation of pro-inflammatory cytokines were suggested to play a critical role in this (Okamoto et al., 2001; Scholz and Woolf, 2007). This neuropathic pain is resistant to standard analgesics (Gilron et al., 2005; Ueda and Rashid, 2003). Therefore, from a therapeutic viewpoint, immune modulating agents or the inhibitory agents of inflammatory cytokines should be able to be effective therapeutic strategies for managing VCR-induced neuropathic injury. These agents could make VCR more acceptable and improve quality of life for patients during the treatment of cancer (Kiguchi et al., 2009). Tropisetron is used as an effective and well tolerated antiemetic treatment for chemotherapy-induced emesis. It is commonly administered without special precautions to all patients who receive chemotherapy regimens and also it remains effective during multiple chemotherapy courses (de Bruijn, 1992; Scuderi, 2003). Several human and animal studies have indicated that tropisetron, a highly selective 5-HT3 receptor antagonist, exerts immunomodulatory and anti-inflammatory properties (Fiebich et al., 2004). Moreover, evidence from clinical studies has shown that tropisetron demonstrates potent analgesic and antiphlogistic effects in patients with chronic inflammatory joint disease and soft tissue rheumatism (Muller et al., 2006), however the underlying mechanisms for these effects are largely unknown. In ex vivo model of isolated human peripheral blood monocytes, Fiebich et al. (2004) have shown that lipopolysaccharidestimulated secretion of both TNF-a and IL-1b was dosedependently inhibited by tropisetron. Moreover, we have recently reported notable anti-inflammatory properties for tropisetron in an embolic model of stroke in rat. Tropisetron significantly improved neurological deficits, diminished leukocyte transmigration into the brain, suppressed the inflammatory cytokine TNF-a, and also attenuated brain infarction and edema. But granisetron, another selective 5-HT3 receptor antagonist, had no effect on the mentioned parameters and it appears these effects are independent of its action on 5-HT3 receptors as concomitant administration of mCPBG, a selective 5HT3 receptor agonist, failed to reverse the beneficial effects of tropisetron (Rahimian et al., 2011). Vega et al. (2005) have shown that tropisetron inhibits antigeninduced proliferation of human peripheral T cells and production of interleukin-2 (IL-2). They also identified that calcineurin, a Ca2+/ calmodulin-dependent phosphatase, is one of the main targets for the inhibitory effect of tropisetron on T-cell activation. In the light of all previous evidences, we investigated the neuroprotective effects of pretreatment with tropisetron in preventing the VCR-induced neuropathy besides its antiemetic effect and examined whether 5-HT3 receptors could take part in its probable effects.

10 rats received VCR 1.5 mg/kg + granisetron 3 mg/kg; and finally (4) 10 rats received VCR 1.5 mg/kg + mCPBG 15 mg/kg; (5) 10 rats received VCR 1.5 mg/kg + mCPBG 15 mg/kg + tropisetron 3 mg/kg, and finally 8 rats as control group that received saline in the same program as VCR-treated animals. 2.3. Drug administration Vincristine sulfate (Sigma Chemical Co, St. Louis, MO) dissolved in saline was injected (ip) with the dosage calculated on daily body weight, on three alternative days at the dose of 0.5 mg/kg (total cumulative dose is 1.5 mg/kg). Tropisetron (Sigma, St. Louis, MO, USA), and granisetron (Sigma, St. Louis, MO, USA) were dissolved in saline and injected ip at the dose of 3 mg/kg 1 h prior to VCR injections. Tropisetron’s neuroprotective effects were evaluated at several doses from 1 to 5 mg/kg through a preliminary study by the authors (not published), and we found that the best dose is 3 mg/ kg. Our previous reports show the same results (Rahimian et al., 2011). Since, both tropisetron and granisetron antagonize the 5HT3 receptor with similar potency (Vega et al., 2005) and based on our previous study on the embolic model of stroke (Rahimian et al., 2011), therefore we have chosen this dose for current study. To completely novelize the effects of tropisetron and granisetron on 5-HT3 receptors, 15 mg/kg of mCPBG (Sigma, St. Louis, MO, USA), was dissolved in saline and injected to the animal intraperitoneally half an hour prior to tropisetron injections. 2.4. Survival study and general toxicity In order to assess general toxicity including edema, cachexia, alopecia and mortality, animals were observed daily in all groups while the body weight was measured twice a week throughout the whole experiment (Pourmohammadi et al., 2012). 2.5. Behavioral examinations

2. Materials and methods

Potential effects of VCR on sensory nerve function were assessed by hot plate test. It was used once a day to assess the effect of VCR on sensory neuropathy from the first dose of VCR treatment to the last one. Animals were placed on a 52  0.2 8C heated plate (socrel Hot plate model DS37, Ugo Basile, Italy) and time spent until the first episode of heat sensitivity includes jumping, forepaw or hind paw licking. To evaluate the motor impairment induced by VCR, open field activity of rats were tested. Animals were placed into an area (diameter = 1.4 m) and locomotion within the area was tracked over a 10 min period recorded using a high resolution monochrome camera and stored and analyzed with Ethovision software (v.8). The total distance moved (cm), mean velocity (cm/s) and total duration of the movement (percent) were calculated (Alimoradi et al., 2012; Pourmohammadi et al., 2012).

2.1. Ethics

2.6. Electrophysiological examination

This study was performed according to the guidelines of the US national institute of health (NIH publication no. 85.23, revised 1985) guides for the care of lab animals.

After the last behavioral tests, rats were anesthetized with pentobarbital sodium (50 mg/kg, ip; Sigma, St. Louis, MO, USA) dissolved in saline, body temperature was monitored and maintained within normal limits and then NCV in the left sciatic nerve was recorded as previously described using power lab (MLT 1030/D, AD Instruments, Power Lab Chart 5.5) and the same stimulating and recording pin electrodes (AD Instrument, pin) (Ja’afer et al., 2006; Kandhare et al., 2012; Pourmohammadi et al., 2012).

2.2. Animals Experiments were performed on 250–300 g male Sprague Dawley rats in pharmacology department of Tehran University of medical sciences. Animals were housed in a temperature and humidity controlled environment with 12-h light/12-h dark cycle. Food consists of normal rat chow and water ad libitum. Rats were randomly divided into 6 groups: (1) 10 rats received VCR 1.5 mg/ kg; (2) 10 rats received VCR 1.5 mg/kg + tropisetron 3 mg/kg; (3)

2.7. Biochemical estimations After the electrophysiological examinations, animals were deeply anesthetized by ip injection of sodium pentobarbital

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(100 mg/kg). Blood was collected from left ventricle for measurement of TNF-a and IL-2 levels in plasma. Moreover the sciatic nerve was isolated and its homogenate (10% w/v) was prepared with 0.1 M Tris–HCl buffer (pH 7.4) and phosphatebuffered saline (PBS), pH 7.4, for total protein and TNF-a estimation respectively (Muthuraman et al., 2011). 2.7.1. Estimation of plasma TNF-a and IL-2 levels Collected blood samples centrifuged at 3000 rpm and plasma levels of IL-2 and TNF-a were measured with the use of rat TNF-a, IL-2 enzyme-linked immunosorbent assay (ELISA) kits were from R&D systems (Minneapolis, MN) and procedure was followed according to the manufacturer instructions. 2.7.2. Estimation of total protein content Protein concentration in the sciatic nerve was estimated according to the method of Lowry et al. (1951), using bovine serum albumin (BSA) as a standard. 2.7.3. Estimation of TNF-a level in rat sciatic nerve The sciatic nerve homogenates were centrifuged at 10,000  g, at 4 8C for 10 min and the supernatants were immediately used for measurement of TNF-a protein levels in duplicate using a rat TNFa ELISA kit. The results were expressed as picograms of TNF-a per mg of total protein in the supernatant (Muthuraman et al., 2011). 2.8. Histological and morphometric studies For pathologic studies, sciatic nerve was carefully dissected from the proximal aspect of the thigh to the knee joint proximal to its point of division into common peroneal, tibial, and sural nerves and then fixed in 1.25% glutaraldehyde, 1% paraformaldehyde in 0.2 M phosphate buffer at pH 7.4. A laminectomy was then performed and the lumbar (L4-L5) DRG (dorsal root ganglion) were exposed and fixed in the same fixator. After dissecting the sciatic, the middle part of the nerve was harvested and preceded for paraffin embedding process. To this end, tissue specimens were embedded in paraffin (Merck, Germany), and 5 mm paraffin sections were transversely cut and were stained with hematoxylin and eosin (H and E) (Pourmohammadi et al., 2012). For morphometric studies, 3 sections from each animal were randomly chosen, number and diameter of axons (sciatic sections) and the number and diameter of A (large) and B (small) type cells were measured (total number of section for each group is 18) using an image evaluation program (Optika, Italy). A type cells were characterized as those cells with one large intensely stained central nucleus, and the cytoplasm appears granular. In the periphery of the cytoplasm of some of the cells, an unstained area can be seen. B nucleus containing multiple peripherally located nuclei and a homogeneous cytoplasm are marked characteristics of B type cells. In some cells, the central cytoplasm of B cells is only lightly stained, while the outer parts are intensely stained (Hosseini et al., 2011).

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(40%) rats in VCR + mCPBG group died, but no death was detected in VCR + tropisetron or VCR + tropisetron + mCPBG group indicating that tropisetron completely diminished mortality rate. In VCR, VCR + granisetron and VCR + mCPBG groups, VCR caused looseness and reduction in amount of stool, alopecia and abnormal posture consisting of foot and head drop. However animals treated with VCR + tropisetron or VCR + tropisetron + mCPBG showed fewer signs of general toxicity. A significant difference in body weight loss was observed between in only VCR, VCR + granisetron and VCR + mCPBG groups versus saline group (P < 0.05, data not shown). No significant body weight changes were determined in VCR + tropisetron and VCR + tropisetron + mCPBG groups in comparison with saline group (P > 0.05, data not shown). 3.2. Effects of tropisetron on behavioral studies 3.2.1. Hot plate Hot plate latencies after the 7th day of study significantly increased in VCR-treated animals compared with saline group (P < 0.001). Latencies in VCR + Grani group and VCR + mCPBG were also significantly higher than saline group (P < 0.05 and P < 0.01 respectively), while no significant change in latencies was observed in VCR + tropisetron and VCR + tropisetron + mCPBG group versus saline group (P > 0.05; Fig. 1). 3.2.2. Open field VCR caused gait disturbance, as total distance moved, mean velocity and percentage of total duration of the movement (good indicators of abnormal locomotion activity) (Pourmohammadi et al., 2012) were significantly reduced in VCR group compared with saline group (P < 0.01, P < 0.001 and P < 0.01 respectively). The same results were detected in VCR + granisetron and VCR + mCPBG groups. While there was no significant difference in these parameters between VCR + tropisetron or VCR + tropisetron + mCPBG group and saline group (P > 0.05; Fig. 2a–c). Interestingly, besides gait disturbance, animals received VCR could not change their path during movement and their spontaneous

2.9. Statistical analysis The results are reported as mean  S.E. The statistical analyses were performed using one way analysis of variance (ANOVA) by SPSS (v.20). Group differences were calculated by post hoc analysis using Tukey test. For all tests, differences with values of P < 0.05 were considered significant. 3. Results 3.1. Effects of tropisetron on survival and general toxicity of rats After the third injection of VCR, three rats (30%) from only VCRtreated group, two rats (20%) from VCR + granisetron group and 4

Fig. 1. Hot plate response of rat treated with vincristine (VCR; 1.5 mg/kg) with or without tropisetron (Tropi) and granisetron (Grani) co-treatment. A significant increase in latencies was observed between single VCR or VCR + Grani (3 mg/kg) group and saline group (P < 0.001 and P < 0.05 respectively). In addition latencies in rats treated by VCR (1.5 mg/kg) + mCBPG (15 mg/kg) was significantly different from than that of saline treated animals (P < 0.01), while there was no significant difference between VCR + Tropi (3 mg/kg) or VCR + Tropi + mCPBG and saline groups (P > 0.05). Values are given by mean  S.E.M. *P < 0.05, **P < 0.01, ***P < 0.00l compared with saline group.

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Fig. 2. (a) Total distance moved within the open field area (diameter 1.4 m) was assessed over 10 min. It was significantly altered in VCR (vincristine), VCR + Grani (granisetron) and VCR + mCPBG compared with saline group. Total distance moved in VCR + Tropi and VCR + Tropi + mCPBG groups was not different from saline group. (b) Mean velocity of animals was also measured and it was significantly decreased in VCR, VCR + Grani and VCR + mCPBG groups compared with saline group. Mean velocity of animals in VCR + Tropi (tropisetron) and VCR + Tropi + mCPBG groups nearly was similar to saline group. (c) Percentage of total duration of the movement of animals within 10 min. Animals treated with only vincristine had less activity compare with saline group. Pretreatment of vincristine with tropisetron but not granisetron significantly normalize this impaired locomotion activity. In addition total duration of movement in animals treated by VCR + mCPBG was similar to VCR group while no significant differences was measured between VCR + Tropi + mCPBG group and saline group. (d) Spontaneous exploratory activity as measured in an open field area is altered in rats that suffer from neuropathy. It visually can be understood that vincristine (VCR) caused gait disturbance and disturbs animal’s motor function. Tropisetron pretreatment ameliorated motor impairment induced by VCR while granisetron pretreatment had no marked effects on this disorder. Values are given by mean  S.E.M. *P < 0.05, **P < 0.01, ***P < 0.00l compared with saline group.

exploratory activities were reduced and tropisetron pretreatment was able to normalized this abnormality (Fig. 2d). 3.3. Effects of tropisetron on motor nerve conduction velocity Fig. 3 shows sciatic MNCV was significantly reduced in VCR, VCR + granisetron and VCR + mCPBG groups in comparison with saline group (P < 0.001, P < 0.01 and P < 0.05 respectively). By contrast, in VCR + tropisetron and VCR + tropisetron + mCPBG groups, sciatic MNCV had no significant difference with saline group (P > 0.05, Fig. 3).

(P > 0.05, Fig. 4a and b). The plasma level of TNF-a and IL-2 were significantly increased in animals of VCR + mCPBG group (P < 0.001 and P < 0.05 respectively), while no significant changes were detected in VCR + tropisetron + mCPBG group (P < 0.05, Fig. 4a and b). Moreover, Fig. 4c shows level of TNF-a in sciatic nerve of animals treated by vincristine, VCR + granisetron, VCR + mCPBG significantly increased compared with saline group (P < 0.001). Although the level of TNF-a in sciatic of animals treated with VCR + tropisetron or VCR + tropisetron + mCPBG was significantly higher than saline treated rats (P < 0.05), there was a significant difference between VCR group and VCR + tropisetron or VCR + tropisetron + mCPBG group (P < 0.01; Fig. 4c).

3.4. Effects of tropisetron on concentration of TNF-a and IL-2 3.5. Effects of tropisetron on histopathologic findings A significant increase in the plasma level of TNF-a and IL-2 was determined in VCR group compared with saline group (P < 0.001). Pretreatment of vincristine with tropisetron, not granisetron, was able to ameliorate this and there was no significant difference in the levels of TNF-a and IL-2 between saline and VCR + Tropi groups

Light microscopic examinations of sciatic and DRG neurons sections in saline group were normal (Figs. 5A and 6A). But histologic sections from sciatic nerve of VCR-treated rats showed severe neural degenerative changes including nerve fibers atrophy

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Fig. 3. Sciatic motor nerve conduction velocity (MNCV) values recorded from rat treated with vincristine (VCR) with or without tropisetron (Tropi) and granisetron (Grani) co-treatment. MNCV significantly decreased in VCR, VCR + Grani and VCR + mCPBG groups versus saline group while it had no significant difference in VCR + Tropi and VCR + Tropi + mCPBG groups compared with saline group. Values are given by mean  S.E.M. *P < 0.05, **P < 0.01, ***P < 0.00l compared with saline group.

and degenerative fibers, the numbers of the axons per square millimeter (axonal density (n/mm2)) are significantly higher in VCR-treated group in comparison to saline (P < 0.05) which represented smaller/atrophic axons (Fig. 5B; Table 1). Moreover, in sections from DRG of VCR group, neurons tend to be small and more basophilic and with more and larger vacuoles, and also sections has more B type cells compare with saline (Fig. 6B). Table 1

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shows the diameter of A type cells in VCR group significantly decreased (P < 0.001), while the diameter of B type cells significantly increased compared with saline group (P < 0.05), pretreatment of vincristine with tropisetron abolished these effects and sections of VCR + tropisetron-treated rats showed no marked histopathologic changes, and approximately there is no evidence of degenerative changes (Fig. 5C). As it is presented in Table 1, the diameter of axons in this group is not significantly different from saline group. Fig. 6C and Table 1 show that tropisetron was able to attenuate vincristine damages to the DRG of the animal (size of cells are similar to saline group). Sciatic nerves and DRG neurons in VCR + granisetron group showed the same morphologic changes, and no recognizable differences between VCR + granisetron and VCR groups were detected (Figs. 5D and 6D). The same results were observed from the diameter of cells (Table 1). The animals which were given VCR + mCPBG show pathologic damages to both sciatic nerve and DRG neurons. Axonopathy and demylination and no significant remylination in sciatic nerve were observed compared with saline group (Fig. 5E). But VCR + Tropi + mCPBG group, sciatic sections showed less pathological and demylinated axons in comparison to VCR + mCPBG group (Fig. 5F), and the data of Table 1 approves this. As well in DRG, the pathological evidence of neuropathy in VCR + mCPBG group includes the more number of B type cells (Fig. 6E and F). Table 1 presents that diameter of A type cells significantly decreased compared with saline group. 4. Discussion The results in the present study showed for the first time that tropisetron pretreatment ameliorates VCR-induced nerve injury in rat. Tropisetron (3 mg/kg), given 1 h prior to VCR injection,

Fig. 4. The effect of pretreatment with tropisetron (Tropi) or granisetron (Grani) on vincristine (VCR)-induced alterations in the plasma level of TNF-a (a) and IL-2 (b). Mean concentrations are significantly different between groups (***P < 0001). Values are given by mean  S.E.M. *P < 0.05, **P < 0.01, ***P < 0.00l, ##P < 0.001 compared with VCR group.

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Fig. 5. Representative photographs of the sciatic nerve sections of rats treated with following groups: (A) saline group shows normal myelinated nerve fibers and normal axon morphology. (B) VCR-treated group shows focal areas of demyelination and degeneration of the nerve fibers. Note the higher population of schwan cells (dark blue cells) in this group. (C) VCR + tropisetron (Tropi) group shows more normal appearance of myelinated nerve fibers with larger caliber. (D) VCR + granisetron group demonstrates areas of demyelination which have smaller axons (yellow arrow) compared with the normal fibers (red arrow), and decrease in the caliber of nerve fibers. (E) VCR + mCPBG group shows demyelination. (F) VCR + tropisetron + mCPBG group demonstrates a marked decrease of demyelination. Scale bar: 20 mm. (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

improved the animals’ general condition and completely diminished mortality rate. It significantly reduced behavioral, electrophysiological scores and pathological, morphometric changes. While granisetron, another selective 5-HT3 receptor antagonist, with the same dosage did not exert any effects on the assessed parameters in comparison to the control group. Moreover, using a specific 5-HT3 agonist, mCPBG, at a dose which can fully neutralize 3 mg/kg of tropisetron or granisetron demonstrated that tropisetron (not granisetron) has its neuroprotective effects; therefore it could suggest a 5-HT3 receptor-independent protective

mechanism of tropisetron against vincristine-induced nerve injury. The same results obtained from previous studies on the embolic model of stroke (Rahimian et al., 2011) and human T cells (Vega et al., 2005) confirm this idea. Clinically, the vincristine-induced mixed sensory-motor neuropathy is the limiting factor of use in patients treated with vincristine and clinical recovery of sensory loss and motor defects is usually incomplete (Authier et al., 2003). In vitro and in vivo studies have indicated that repeated systemic injection of vincristine damages Schwann cells and DRG

Table 1 Morphometric data on sciatic nerve and dorsal root ganglion from the study groups; n = 18 for each group (three sections from each animal). Values are given by mean  S.E.M.

Diameter of axon Axonal density (n/mm2) Diameter of A type cells Diameter of B type cells * **

P < 0.05. P < 0.01. P < 0.00l.

***

Saline

VCR

VCR + Tropi

VCR + Grani

VCR + mCPBG

VCR + Tropi + mCPBG

2.50  0.13 9533  962 39.05  0.99 22.26  0.59

1.71  0.16* 21,500  2306* 24.30  1.38*** 25.70  0.98 *

2.18  0.20 12,883  1066 34.42  1.23 23.46  0.45

1.67  0.15* 21,500  2137* 27.82  1.51*** 26.13  1.04 *

1.65  0.21* 24,833  1409** 29.26  0.85 24.10  0.87

2.34  0.18 14,050  1148 31.79  1.63*** 22.74  1.34

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Fig. 6. Cross sections of dorsal root ganglion neurons (DRG) from control (A), rats treated with vincristine (VCR) alone (B), VCR + tropisetron (Tropi) (C) VCR + granisetron (Grani) (D), VCR + mCPBG (E) and VCR + tropisetron + mCPBG (F). There are two types of cells in DRG neurons: A type or B type. The nucleus of A cells has one large intensely stained central nucleus, and the cytoplasm appears granular (red arrow in B). B cells are characterized by a nucleus containing multiple peripherally located nuclei, and cytoplasm is more homogeneous and more intensely stained than that of A cells (yellow arrow in B) (Hosseini et al., 2011). Histologic sections from DRG neurons in saline group were normal. But in VCR, VCR + Grani and VCR + mCPBG groups, neurons tend to be small and more basophilic and with more and larger vacuoles. In the other words, DRG neurons in these groups have more B type cells and they are larger than that of saline group. Pretreatment with tropisetron abolished these effects to the DRG neurons, as can be seen from C and F (H&E Scale bar: 20 mm). (For interpretation of the references to color in this text, the reader is referred to the web version of the article.)

neurons by reduction of laminin in Schwann cells, neurite retraction in the DRG (Konings et al., 1994a,b) and prevention from development of the normal conformation of myelin (Djaldetti et al., 1996). In general, damaged Schwann cells produce and release the inflammatory cytokines (Shamash et al., 2002; Thacker et al., 2007) and chemokines such as monocyte chemoattractant protein-1 (MCP-1) that induces macrophage chemotaxis in the injured region (Tofaris et al., 2002; White et al., 2005). As a result of chemotaxis, macrophages invade into the lesion site in the peripheral nervous system (PNS) and release inflammatory cytokine IL-6, which causes neuroinflammation, leading to neuropathic pain such as hyperalgesia and allodynia (Hu and McLachlan, 2002; Kiguchi et al., 2009; Marchand et al., 2005; Scholz and Woolf, 2007). Besides, by IL-2 secretion from T-cells infiltrated into the injured nerve, macrophages are activated and release a variety of pro-inflammatory cytokines and mediators including TNF-a, IL1b, nitric oxide and reactive oxygen species. Overall these events create more nerve damage (Kleinschnitz et al., 2006; Pavlick et al., 2002). Moreover, the upregulation of TNF-a in the PNS and central nerve system (CNS) plays a critical role in the development and maintenance of neuropathic pain (Scholz and Woolf, 2007).

In the present study, VCR administration caused general toxicity (looseness and decrease in amount of stool, alopecia and abnormal posture consisting of foot and head drop) in rats and a significant difference in body weight loss. In addition, electrophysiological, behavioral and pathological tests were used to demonstrate axonal degeneration. According to our findings, a significant decrease in sciatic MNCV in sciatic nerve in VCR group was observed. Furthermore, in behavioral studies, hot plate test was performed to assess the degree of thermal hyperalgesia and it showed a significant increase in hot plate latency following neuropathic pain and sensory neuropathy in only VCR treated group. Also total distance moved, mean velocity and percentage of total duration of the movement, as good indicators of abnormal locomotion activity were clearly attenuated. Tropisetron is used as an effective and well tolerated antiemetic treatment for chemotherapy-induced emesis. It is commonly administered without special precautions to all patients who receive chemotherapy regimens and also it remains effective during multiple chemotherapy courses (de Bruijn, 1992; Scuderi, 2003). In vitro and in vivo studies have shown that tropisetron exerts immune modulatory and anti-inflammatory properties

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(Fiebich et al., 2004). It also inhibits IL-2 gene transcription and proliferation in antigen-stimulated human T cells via blockade of calcineurin/NFAT-dependent signaling pathway (Vega et al., 2005). Calcineurin is a widely distributed Ca2+-calmodulin dependent protein phosphatase (Klee et al., 1998) that plays a key role in calcium-dependent death pathways in thymocytes (Waring and Beaver, 1996) and neural tissues (Ankarcrona et al., 1996) and participates in an apoptotic death pathway activated by TNF (Kantrow et al., 2000). The raising of intracellular calcium level following nerve injury leads to calcineurin activation and dephosphorylation of NFAT and its translocation to the nucleus. Finally these events culminate in release of several cytokines, including IL-2, IL-4 and TNF and neuronal cell apoptosis (Kantrow et al., 2000). Since, calcineurin plays an important role in neural damage and has been suggested to be a new molecular target for tropisetron; inhibition of its activation by tropisetron represents one possible mechanism for neuroprotective effects of this drug (Vega et al., 2005). As in this study, we observed a significant reduction in plasma levels of TNF-a and IL-2, it is likely that these effects result from inhibition of calcineurin. Further, in the present investigation, tropisetron pretreatment significantly showed fewer signs of general toxicity and resulted in prevention of mixed sensory motor neuropathy, as indicated by less mortality rate, better general condition, and impairment in behavioral and electrophysiological studies. Moreover, pathological studies are consistent with the results obtained from other findings. 5. Conclusion Taken together, our results show for the first time that tropisetron pretreatment significantly suppresses VCR-induced neuropathy. They suggest that tropisetron, a well tolerated antiemetic in chemotherapy, could be a specific safe neuroprotective agent for prevention of neuropathy in patients treated with VCR. References Alimoradi H, Barzegar-Fallah A, Mohammadi-Rick S, Asadi F, Delfan B. Pretreatment of CAV combination chemotherapy with tropisetron shows less cardio and neurotoxicity side effects in rats. Journal of Clinical Toxicology 2012;S.6:2161-0495. Ankarcrona M, Dypbukt JM, Orrenius S, Nicotera P. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Letters 1996;394:321–4. Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F. A new animal model of vincristineinduced nociceptive peripheral neuropathy. Neurotoxicology 2003;24:797–805. Bromberg MB. Peripheral neurotoxic disorders. Neurologic Clinics 2000;18:681. de Bruijn KM. Tropisetron. A review of the clinical experience. Drugs 1992;43:11. Djaldetti R, Hart J, Alexandrova S, Cohen S, Beilin BZ, Djaldetti M, et al. Vincristine induced alterations in Schwann cells of mouse peripheral nerve. American Journal of Hematology 1996;52:254–7. Fiebich B, Akundi R, Lieb K, Candelario-Jalil E, Gmeiner D, Haus U, et al. Antiinflammatory effects of 5-HT3 receptor antagonists in lipopolysaccharide-stimulated primary human monocytes. Scandinavian Journal of Rheumatology 2004; 33:28–32. Gilron I, Bailey JM, Tu D, Holden RR, Weaver DF, Houlden RL. Morphine, gabapentin, or their combination for neuropathic pain. The New England Journal of Medicine 2005;352:1324–34. Hosseini A, Abdollahi M, Hassanzadeh G, Rezayat M, Hassani S, Pourkhalili N, et al. Protective effect of magnesium-25 carrying porphyrin-fullerene nanoparticles on degeneration of dorsal root ganglion neurons and motor function in experimental diabetic neuropathy. Basic & Clinical Pharmacology & Toxicology 2011;109:381–6. Hu P, McLachlan E. Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat. Neuroscience 2002;112:23–38.

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