Users Online: 476

Home Print this page Email this page Small font sizeDefault font sizeIncrease font size

Home | About us | Editorial board | Search | Ahead of print | Current issue | Archives | Submit article | Instructions | Subscribe | Contacts | Login 
     

   Table of Contents      
ORIGINAL ARTICLE
Year : 2014  |  Volume : 4  |  Issue : 4  |  Page : 214-223

Neuroprotective effect of ethyl pyruvate in vincristine and cisplatin induced neuropathic pain


1 Department of Pharmacology, All India Shri Shivaji Memorial Society's College of Pharmacy, Pune, Maharashtra, India
2 Department of Pharmacology, Indira Gandhi Government Medical College and Hospital, Nagpur, Maharashtra, India

Date of Submission20-Mar-2014
Date of Acceptance10-Apr-2014
Date of Web Publication22-Aug-2014

Correspondence Address:
Varsha J Bansode
Department of Pharmacology, All India Shri Shivaji Memorial Society's College of Pharmacy, Kennedy Road, Pune 411 001, Maharashtra
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.139402

Rights and Permissions
   Abstract 

Aim of Study: The aim of the present work was to screen synthetic compound Ethyl Pyruvate (EP) for its potential use in chemotherapeutic drugs induced neuropathic pain. Materials and Methods: In vincristine induced neuropathy model, Vincristine sulfate (50 μg/kg i.p.) for 10 consecutive days was administered to induce neuropathy in rats with EP for 14 days. In cisplatin induced neuropathy model, cisplatin was administered at a dose of (2 mg/kg i.p.) twice a week for 5 weeks with EP for 5 weeks daily in this model. The effect of ethyl pyruvate in tail heat immersion, paw pressure and acetone drop tests were performed to assess the degree of spinal thermal sensation, mechanical hyperalgesia and cold allodynia, respectively. Vacuolar changes, necrosis and cellular infiltration of sciatic nerve were assessed histopathologically. The levels of various antioxidants were determined to assess oxidative stress. Results: In vincristine and cisplatin induced neuropathy models, co-administration of EP 100 mg/kg significantly attenuated reduction of nociceptive threshold in tail heat immersion test and threshold in paw pressure along with the increased score in the acetone drop test. EP 100 mg/kg significantly attenuated reactive changes in histopathology and increase in oxidative stress. Conclusion: The observed neuroprotective effect can be attributed to the antioxidant property of EP. Therefore, with support from the literature and data in hand it seems quite evident that EP 100 mg/kg exerted its beneficial effects in vincristine and cisplatin induced peripheral neuropathic pain.

Keywords: Antioxidant, cisplatin, ethyl pyruvate, neuropathic pain, vincristine


How to cite this article:
Bansode VJ, Vyawahare NS, Munjal NB, Gore PN, Amrutkar PS, Sontakke SR. Neuroprotective effect of ethyl pyruvate in vincristine and cisplatin induced neuropathic pain. Int J Nutr Pharmacol Neurol Dis 2014;4:214-23

How to cite this URL:
Bansode VJ, Vyawahare NS, Munjal NB, Gore PN, Amrutkar PS, Sontakke SR. Neuroprotective effect of ethyl pyruvate in vincristine and cisplatin induced neuropathic pain. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2020 Jan 22];4:214-23. Available from: http://www.ijnpnd.com/text.asp?2014/4/4/214/139402


   Introduction Top


One of the most challenging areas of cancer pain management is managing chemotherapy-induced peripheral neuropathy (CIPN) and associated pain. Chemotherapy-associated pain is becoming more common as more effective chemotherapies enhance survival rates and as the number of neurotoxic chemotherapeutic agents increase. The neurotoxicity of the older agent's vincristine and cisplatin is well-known. These agents may cause a usually sensory neuropathy with increased risk to chemotherapy associated damage compared to motor nerves. Therefore, patients often experience sensory symptoms such as numbness, tingling, or burning sensations. [1]

Based on extensive literature demonstrating that platinum-based cancer chemotherapies causes mitochondrial dysfunction [2],[3] and also evaluated that the vincristine evoked neuropathic pain has been associated with impaired mitochondrial function. [4] Increased Reactive Oxygen Species (ROS) are the main mitochondrial mechanisms implicated in oxidative stress that triggers apoptosis. [5],[6]

Pyruvate is the anionic form of a simple alpha-keto acid, plays a key role in intermediary metabolism as a product of glycolysis and the starting substrate for the tricarboxylic acid (TCA) cycle. Pyruvate is also an important endogenous scavenger of certain reactive oxygen species (ROS). Pharmacological administration of pyruvate has been shown to improve organ function in animal models of oxidant-mediated cellular injury. Ethyl pyruvate (EP) is a simple derivative of the endogenous metabolite, pyruvic acid. [7] It has been already reported for antioxidant and neuroprotective activity but its use in neuropathic pain is remained yet to be determine.


   Materials and methods Top


Animals

Wistar albino rats of either sex were used. They were maintained at 25 ± 2°C and relative humidity of 45 to 55% and under standard environmental conditions (12 h light: 12 h dark cycle). They were allowed to take specified amount of standard laboratory feed (Amrut feed, Pune) and water ad libitum. All the experimental procedures and protocols used in this study were reviewed and approved by the Institutional Animal Ethical Committee (IAEC) of AISSMS College of Pharmacy, Pune, constituted under Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), approval no. (CPSEA/IAEC/PC-02/05-2K11) Ethical guidelines were strictly followed during all the experiments.

Chemicals and drugs

Accurately weighed quantity of ethyl pyruvate (Anand Agencies, Pune) was dissolved in distilled water to prepare the appropriate stock solution i.e., ethyl pyruvate 50 mg/ml and ethyl pyruvate 100 mg/ml, respectively. Accurately measured quantity of vincristine (50 μg/ml) and cisplatin (2 mg/ml) was dissolved in saline to prepare the appropriate stock solutions. Ethyl pyruvate was given orally. Vincristine and cisplatin were injected by intraperitoneal route. Sterile saline was injected by subcutaneous route.


   Experimental protocol Top


Experimental induction of neuropathy by vincristine

Vincristine sulfate was dissolved in physiological saline to a stock concentration of 1 mg/ml (pH 4.5-5.2) and stored at 4-8 ºC, and then diluted daily in saline. Peripheral neuropathy was induced in rats by administration of vincristine sulfate (50 μg/kg i.p.) for 10 consecutive days. [8],[9]

Animals will be divided in four groups, each containing six animals. Two doses ethyl pyruvate 50 and 100 mg/kg were selected for the study.

Group I (Control group)

Rats were subjected to normal saline (1 ml/kg) for 10 consecutive days. Behavioral tests were performed to assess nociceptive threshold on different days i.e. day 0, 7 and 14 th . All the animals were weighed and sacrificed at the end of the 14 th day. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group II (Neuropathy induction control group)

Rats were subjected to vincristine sulfate (50 μg/kg i.p.) for 10 consecutive days. Behavioral tests were performed to assess nociceptive threshold on different days i.e. day 0, 7 and 14 th . All the animals were weighed and sacrificed at the end of the 14 th day. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group III (EP 50 mg/kg group)

Rats were subjected to vincristine sulfate (50 μg/kg i.p.) for 10 consecutive days and EP 50 mg/kg was co-administered for 14 days. All the animals were weighed and sacrificed at the end of the 14 th day. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group IV (EP 100 mg/kg group)

Rats were subjected to vincristine sulfate (50 μg/kg i.p.) for 10 consecutive days and EP 100 mg/kg was co-administered daily for 14 days. All the animals were weighed and sacrificed at the end of the 14 th day. Biochemical analysis with histopathological examination was carried out at the end of the study.

Experimental induction of neuropathy by Cisplatin

Cisplatin was administered intraperitoneally twice a week (Monday and Friday) at a dose of 2 mg/kg (Cumulative dose, 20 mg/kg) for 5 weeks. Before each injection, 2 ml of sterile saline solution was given subcutaneously to prevent renal damage via hyperhydration. To avoid acute effects, the injections were therefore given after the tests were performed. [10],[11]

Animals will be divided in four groups, each containing six animals. Two doses of ethyl pyruvate 50 and 100 mg/kg were selected for the study.

Group I (Control group)

Rats were subjected to normal saline (1 ml/kg) intraperitoneally for 5 weeks. Behavioral tests were performed to assess nociceptive threshold on different weeks i.e., on 0, 1 st , 3 rd and 5 th week. All the animals were weighed and sacrificed at the end of the 5 th week. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group II (Neuropathy induction control group)

Rats were subjected to cisplatin intraperitoneally twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 5 weeks and 2 ml saline subcutaneously. Behavioral tests were performed to assess nociceptive threshold on different weeks i.e., on 0, 1 st , 3 rd and 5 th week. All the animals were weighed and sacrificed at the end of the 5 th week. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group III (EP 50 mg/kg group)

Rats were subjected to cisplatin intraperitoneally twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 5 weeks and 2 ml saline subcutaneously. EP 50 mg/kg was co-administered for 5 weeks daily. Behavioral tests were performed to assess nociceptive threshold on different weeks i.e. on 0, 1 st , 3 rd and 5 th week. All the animals were weighed and sacrificed at the end of the 5 th week. Biochemical analysis with histopathological examination was carried out at the end of the study.

Group IV (EP 100 mg/kg group)

Rats were subjected to cisplatin intraperitoneally twice a week at a dose of 2 mg/kg (cumulative dose, 20 mg/kg) for 5 weeks and 2 ml saline subcutaneously. EP 100 mg/kg was co-administered for 5 weeks daily. Behavioral tests were performed to assess nociceptive threshold on different weeks i.e. on 0, 1 st , 3 rd and 5 th week. All the animals were weighed and sacrificed at the end of the 5 th week. Biochemical analysis with histopathological examination was carried out at the end of the study.


   Behavioral examination Top


Evaluation of hyperalgesia and allodynia by following methods

Assessment of thermal hyperalgesia

Tail-immersion (hot water) test

Spinal heat thermal sensitivity was assessed by the tail immersion test. The tail heat hyperalgesia was noted with the immersion of terminal part of the tail (1 cm) into the water, maintained at a temperature of 52.5 ± 0.5°C. The duration of the tail withdrawal reflex was recorded, as a response of heat thermal sensation and a cut-off time of 15 seconds was maintained. Shortening of the tail withdrawal time indicates the induction of hyperalgesia. [12]

Assessment of mechanical hyperalgesia

Paw pressure withdrawal test (Randall Salitto test)

Nociceptive flexion reflexes were quantified using the Digital Randall-Selitto apparatus (IITC Life Science, USA). Linearly increasing pressure with the cut-off of 250g to avoid tissue injury, was applied to the center of the hindpaw. When animal displayed pain by withdrawal of the paw, vocalization or overt struggling; the applied paw pressure was registered by an analgesia meter and expressed in mass units (grams). [13],[14]

Assessment of cold allodynia

Paw cold-allodynia (acetone drop test)

Spray 100 μL of acetone onto the surface of the paw, without touching the skin. The response of the rat to acetone was recorded for 20 seconds and was graded as a 4-point scale like 0 for no response; 1 for quick withdrawal, flick or stamp of the paw; 2 for prolonged withdrawal or repeated flicking; 3 for repeated flicking of the paw with licking of the paw. With a gap of 5 min, acetone was applied thrice to the hind paw and the individual scores noted in 20 seconds interval were added to obtain a single score over a cumulative period of 60 seconds. The minimum score was 0, while the maximum possible score was 9. [15]

Assessment of general toxicity

Body weight

The weight (gm) of the animals was noted on the first and last day of treatment and the change in the body weight were calculated.


   Biochemical estimations Top


Preparation of homogenate

Animals were sacrificed and the sciatic nerve was isolated immediately. The uniformity among the different nerve samples was maintained by taking the constant weight of the respective samples. The excised sciatic nerve homogenate (10% w/v) was prepared with 0.1 M Tris-HCl buffer (pH 7.4). The tubes with homogenate were kept in ice water for 30 minutes and centrifuged at 4°C (2500 rpm, 10 min). The supernatant of homogenate was separated, and employed for estimations of extent of lipid peroxidation, (MDA), the levels of reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) enzymes. [16],[17]

Assay of lipid peroxidation

The estimation of lipid peroxidation in the sciatic nerve was done by measuring the thio-barbituric acid reactive substances by the method of Boehme et al. [18] The absorbance was measured spectrophotometrically at 532 nm. The concentration was expressed in terms of nM of MDA/gm of tissue.

Assay of reduced glutathione levels

The estimation of reduced glutathione levels (GSH) in the sciatic nerve was done by the method of Ellman GL. [19] The absorbance was measured spectrophotometrically at 412 nm. The concentration was expressed in terms of μgm of GSH/gm of tissue.

Estimation of catalase activity

The estimation of catalase (CAT) activity in the sciatic nerve was done by the method of Luck H. [20] The change in absorption was measured at 240 nm for 2-3 min. The concentration was expressed in μM of H 2 O 2 /gm of tissue/min.

Estimation of superoxide dismutase activity

The estimation of superoxide dismutase (SOD) activity in the sciatic nerve was done by the method of Misra and Fridovich. [21] The change in absption was measured at 480 nm for 3 min, with 60 seconds interval. The concentration was expressed in units/gm of tissue.


   Histopathological examination Top


Histology samples of sciatic nerve were paraffin embedded, cut to 4 μm thickness and stained by the hematoxylin and eosin method before examination. Sections were observed under light microscope (×400) for axonal degeneration and histopathological changes.

Statistical analysis

The results are expressed as mean ± SEM. Statistical analysis was done using GraphPad InStat software. Comparison between the groups were made with one way analysis of variance (ANOVA) followed by Dunnett's test.


   Results Top


Evaluation of effect of EP against vincristine induced peripheral neuropathy in Wistar albino rats

Effect of EP treatment on thermal hyperalgesia

Tail immersion (hot water) test

As shown in [Figure 1], the neuropathic induction control group showed significant (P < 0.01) duration dependent reduction in reaction time at 7 th and 14 th days of treatment respectively when compared with control group. The co-administration of EP 50 mg/kg not showed any significant increase in reaction time but, EP 100 mg/kg showed significant (P < 0.05) increase in the reaction time at 14 th day only [Figure 1].
Figure 1: Effect of EP treatment on thermal nociceptive threshold in vincristine induced peripheral neuropathy

Click here to view


Effect of EP treatment on mechanical hyperalgesia

Paw pressure withdrawal test

As shown in [Figure 2], mechanical hyperalgesia was evident in neuropathy induction control group since the paw pressure withdrawal threshold showed significant (P < 0.01) duration dependent reduction at 7 th and 14 th days of vincristine treatment respectively when compared with control group. The co-administration of EP 50 mg/kg showed significant (P < 0.05) increase in paw pressure withdrawal threshold at 14 th day only. But, EP 100 mg/kg showed significant (P < 0.05) increase in paw pressure withdrawal threshold at 7 th and 14 th days of treatment respectively as compared to neuropathy induction control group [Figure 2].
Figure 2: Effect of EP treatment on paw pressure withdrawal thresholds in vincristine induced peripheral neuropathy

Click here to view


Effect of EP treatment on cold allodynia

Acetone drop test

As shown in [Figure 3], the allodynia score was significantly (P < 0.05) increased at 14 th day in neuropathy induction control group than that of control group. The co-administration of EP 50 mg/kg not showed any significant (P < 0.05) effect but, EP 100 mg/kg modulate increased allodynia score significantly (P < 0.05) at 14 th day only as compared to neuropathy induction control group [Figure 3].
Figure 3: Effect of EP treatment on allodynia score in vincristine induced peripheral neuropathy

Click here to view


Assessment of general toxicity

Effect of EP treatment on body weight

As shown in [Figure 4], after 14 days of vincristine treatment, significant (P < 0.01) decrease in the body weight was observed as compared to control group. The co-administration of EP 50 mg/kg showed significant (P < 0.05) increase in the body weight after 14 days of treatment. The co-administration of EP 100 mg/kg showed significant (P < 0.01) increase in the body weight after 14 days of treatment [Figure 4].
Figure 4: Effect of EP treatment on body weight in vincristine induced peripheral neuropathy

Click here to view


Histopathology of sciatic nerve

As shown in [Figure 5], representative photographs showing vacuolar changes, necrosis (black arrow), cellular infiltration (blue arrow) and edema (white arrow) H and E stain sections of sciatic nerve under 400X. (A) Control group, Pathological grade-0 (B) Vincristine induction group, Pathological grade- ++++ (C) Vincristine induction + EP 50 mg/kg, Pathological grade- +++ (D) Vincristine induction + EP 100 mg/kg, Pathological grade- +.

Rats subjected to vincristine treatment showed morphological alterations like necrosis of neurons, cellular infiltration and edema along with nerve fibre dearrangement. Co-administration with EP 100 mg/kg significantly attenuated these reactive changes [Figure 5].
Figure 5: Effect of EP on vincritine induced histopathological changes in the sciatic nerve of rats

Click here to view


Biochemical parameters of sciatic nerve homogenate

As shown in [Table 1], a significant (P < 0.01) increase in MDA level along with decrease in GSH concentration, decrease in CAT concentration, and decrease in SOD concentration was found in neuropathy induction control group treated with vincristine as compared to control group. The co-administration with EP 50 and EP 100 mg/kg for 14 days significantly attenuated increase in MDA level along with decrease in GSH concentration and decrease in CAT concentration dose dependantly. The co-administration with EP 50 and EP 100 mg/kg for 14 days significantly (P < 0.01) attenuated decrease in SOD concentration [Table 1].
Table 1: Effect of EP on oxidative markers in sciatic nerve homogenate against vincristine induced peripheral neuropathy in rats


Click here to view


Evaluation of effect of EP against cisplatin induced peripheral neuropathy in Wistar albino rats

Effect of EP treatment on thermal hyperalgesia

Tail immersion (hot water) test

As shown in [Figure 6], the neuropathic induction control group showed significant (P < 0.01) duration dependent reduction in reaction time at 3 rd and 5 th week of cisplatin treatment, respectively. The co-administration of EP 50 mg/kg showed significant (P < 0.05) increase in reaction time at 5 th week only. The co-administration of EP 100 mg/kg showed significant (P < 0.05) and (P < 0.01) increase in reaction time at 3 rd and 5 th week respectively as compared to neuropathy induction group [Figure 6].

Effect of EP treatment on mechanical hyperalgesia
Figure 6: Effect of EP treatment on thermal nociceptive threshold in cisplatin induced peripheral neuropathy

Click here to view


Paw pressure withdrawal test

As shown in [Figure 7], mechanical hyperalgesia was evident in neuropathy induction control group since the paw pressure withdrawal threshold showed significant (P < 0.01) duration dependent reduction at 3 rd and 5 th week respectively when compared with control group. The co-administration of EP 50 mg/kg showed significant (P < 0.05) increase in the paw pressure withdrawal threshold at 5 th week only. The co-administration of EP 100 mg/kg showed significant (P < 0.05) and (P < 0.01) increase in the paw pressure withdrawal threshold at 3 rd and 5 th week, respectively [Figure 7].
Figure 7: Effect of EP treatment on paw pressure withdrawal thresholds in cisplatin induced peripheral neuropathy

Click here to view


Effect of EP treatment on cold allodynia

Acetone drop test

As shown in [Figure 8], the allodynia score was significantly (P < 0.01) increased at 5 th week in neuropathy induction control group than that of control group. The co-administration of EP 50 mg/kg modulate increased allodynia score significantly (P < 0.05) at 5 th week only. The co-administration of EP 100 mg/kg modulate increased allodynia score significantly (P < 0.05) and (P < 0.01) at 3 rd and 5 th week respectively as compared to neuropathy induction control group [Figure 8].
Figure 8: Effect of EP treatment on allodynia score in cisplatin induced peripheral neuropathy

Click here to view


Assessment of general toxicity

Effect of EP treatment on body weight

As shown in [Figure 9], in neuropathy induction control group with cisplatin treatment observed significant (P < 0.01) decrease in the body weight after 5 weeks as compare to control group. The co-administration of EP 50 mg/kg showed significant (P < 0.05) increase in the body weight after 5 weeks of treatment. The co-administration of EP 100 mg/kg showed significant (P < 0.01) increase in the body weight after 5 weeks of treatment [Figure 9].
Figure 9: Effect of EP treatment on body weight in cisplatin induced peripheral neuropathy

Click here to view


Histopathology of sciatic nerve

As shown in [Figure 10], representative photographs photograph showing vacuolar changes, necrosis (black arrow), cellular infiltration (blue arrow), edema (white arrow) and demyelination (yellow arrow ) H and E stain sections of sciatic nerve under 400X. (A) Control group, Pathological grade-0 (B) Cisplatin induction group, Pathological grade- ++++(C) Cisplatin induction + EP 50 mg/kg, Pathological grade- +++ (D) Cisplatin induction + EP 100 mg/kg, Pathological grade- ++.
Figure 10: Effect of EP on cisplatin induced histopathological changes in the sciatic nerve of rats

Click here to view


Rats subjected to cisplatin showed morphological alterations like necrosis of neurons, cellular infiltration and edema along with nerve fibre dearrangement. Treatment with EP 100 mg/kg significantly attenuated these reactive changes [Figure 10].

Biochemical parameters of sciatic nerve homogenate

As shown in [Table 2], a significant (P < 0.01) increase in MDA level along with decrease in GSH concentration, decrease in CAT concentration, and decrease in SOD concentration was found in neuropathy induction control group treated with cisplatin as compared to control group. The co-administration with EP 50 and EP 100 mg/kg for 5 weeks significantly attenuated increase in MDA level along with decrease in GSH concentration and decrease in CAT concentration dose dependantly. The co-administration with EP 50 and EP 100 mg/kg for 5 weeks significantly (P < 0.01) attenuated decrease in SOD concentration [Table 2].
Table 2: Effect of EP on oxidative markers in sciatic nerve homogenate against cisplatin induced peripheral neuropathy in rats


Click here to view



   Discussion Top


More patients are experiencing excellent outcomes of chemotherapy with vincristine and cisplatin as anticancer drugs with prolonged survival. On the other hand, increasing numbers of patients are unable to complete full treatment because of the development of chemotherapy induced peripheral neuropathic pain. Long-term management of pain is therefore becoming one of the most challenging aspects of treatment for neurologists and oncologists. The recognizing and managing the symptoms of neuropathic pain and maintaining quality of life are important factors.

Ethyl pyruvate (EP) is a simple derivative of the endogenous metabolite, pyruvic acid. As ethyl pyruvate is previously proved as antioxidant in various models, [22],[23] it may be helpful in reducing neuropathic pain in animals. In light of this the present investigation was planned to evaluate the effect of EP in chemotherapeutic drugs induced neuropathic pain.

Vincristine has been widely employed for the management of number of cancers. However, its clinical application has been limited due to painful sensorimotor neuropathy, observed in about half of the patients on vincristine treatment.[24],[25] Vincristine-evoked neuropathic pain has been associated with impaired mitochondrial function. Increased reactive oxygen species (ROS) are the main mitochondrial mechanisms implicated in oxidative stress that triggers apoptosis. Also, it was shown that the mitochondrial electron transport chains (mETC) have contributed major proportion in ROS production in mitochondria. [26]

Co-treatment with EP 50 mg/kg significantly attenuated mechanical hyperalgesia but it failed to show any improvement in thermal hyperalgesia and cold allodynia. However, co-treatment with EP 100 mg/kg showed significant (P < 0.01) improvement in mechanical hyperalgesia but, significant (P < 0.05) improvement in heat hyperalgesia and cold allodynia. Vincristine induced alterations in pain perception in response to noxious as well as non-noxious stimuli, was attenuated by EP 100 mg/kg significantly suggesting that EP 100 mg/kg may be employed to limit the painful untoward effects associated with chemotherapy treatment.

Body weight of animals treated with vincristine was significantly reduced within 14 days. This effect was significantly attenuated by EP 50 and 100 mg/kg treatment dose dependently.

Histopathology is an important tool to evaluate the protective effect of the drugs acting on the damaged or necrotic cells produced by the induction of neuropathy. Crucially, axonal degeneration has been reported due to these events, suggesting that cellular oxidant and inflammatory mediators play a key role in the pathogenesis of painful neuropathy under in vivo conditions. [27],[28],[29]

Histopathological examination of sciatic nerves gave the evidence of the neuropathy induced neuronal damage with increase in edema, vacuolar changes and necrosis significantly in neuropathy induction group. Co-treatment with EP 50 mg/kg and EP 100 mg/kg significantly exhibited marked protection by reversing the reactive changes, neuronal damage, and vacuolar changes in sciatic nerve produced by vincristine in dose dependant manner. The results suggest that scavenging of free radical in vivo; prevent the pathological changes in sciatic nerve as supported by histopathological findings. Hence, from the above discussion we can say that EP 100 mg/kg exerted significant protective effect against vincristine induced neuropathy.

Oxidative stress due to overproduction of reactive oxygen species may impair normal cellular processes and ultimately resulted in cellular death. Recent reports have suggested that ROS play an important role in a variety of types of persistent pain. [30] In addition, oxidative stress is an important determinant of degenerative and painful pathological conditions in peripheral nerve fibers.[30],[31]

Peripheral nervous system has a rich source of lipids and may be the predominant target of free radical mediated lipid peroxidation. Elevated levels of lipid peroxidation in sciatic nerve of vincristine administered rats are one of the characteristic features of neuropathy. [32] Increased concentration of thiobarbituric acid reactive substances was observed in sciatic nerve of vincristine treated rats. Our study showed that co-administration of EP at a dose of 50 and 100 mg/kg for 14 days tend to restore the sciatic nerve peroxides back to near normal levels in dose dependent manner.

GSH acts as an antioxidant and its level has been previously found to reduce in vincristine neuropathy. [33] In present study, we have also observed a significant decrease in GSH levels in sciatic nerve of vincristine treated rats. The decrease in GSH levels represents increased utilization due to oxidative stress. Co-administration of EP 50 and 100 mg/kg showed increase in GSH levels in dose dependant manner.

CAT and SOD are major scavenging enzymes that remove toxic free radicals in vivo. Previous studies have reported that the activity of CAT and SOD is low in vincristine induced neuropathy. [16],[34] Reduced activity of CAT and SOD in sciatic nerve has been observed in sciatic nerve homogenate of neuropathy induced rats which may result in a number of deleterious effects due to the accumulation of free radicals. Co-administration of EP resulted in the activation of CAT and SOD returning to near normal levels.

The observed neuroprotective effect can be attributed to the antioxidant property of EP. Therefore, with support from literature [35] and data in hand it seems quite evident that EP exerted its beneficial effects in vincristine induced peripheral neuropathic pain probably by virtue of its anti-oxidative properties like inhibition of superoxide anion generation, scavenging of ROS, Inhibition of redox-mediated cellular damage by non enzymatic scavenging of H 2 O 2 and other reactive intermediates. [36]

Cisplatin is a widely used and effective anticancer drug but, peripheral neurotoxicity remains a severe clinical restriction and a major dose-limiting factor of cisplatin therapy. [37] Cisplatin predominantly affects the sensory nerve bodies, which are located in the sensory root ganglia. This may be due to the absence of the blood-nerve barrier of this part of the nervous system, resulting in a higher accumulation inside the sensory nerve body. The clinical feature is that of an axonal sensory neuropathy is cumulative and dose-dependent. [38]

Apoptosis has been observed in Dorsal root ganglia (DRG) following cisplatin treatment both in vitro as well as in vivo and is correlated with increased platinum-DNA binding in the DRG neurons. [39] It has been shown that mitochondrial dysfunction is one of the routes triggering neuronal death. Based on extensive literature demonstrating effects of platinum-based cancer chemotherapies on mitochondrial dysfunction. [40] There is also evidence that oxidative stress may be involved in cisplatin neuropathy. [41]

Co-treatment with EP 50 and 100 mg/kg significantly increased the paw withdrawal threshold in mechanical hyperalgesia, response time in thermal hyperalgesia and decreased allodynia score in cold allodynia, in dose-dependent manner. Cisplatin induced alterations in pain perception in response to noxious as well as non-noxious stimuli, was attenuated by EP 50 and 100 mg/kg significantly in dose-dependent manner.

Cisplatin-induced weight loss, already reported by other authors, may be due to gastrointestinal toxicity or by lessened ingestion of food. [42] Body weight of animals treated with cisplatin was significantly reduced after 5 weeks. This effect was significantly attenuated by EP 50 and 100 mg/kg treatment dose-dependently.

Previous reports have suggested that histopathological changes were observed after cisplatin induction. Histopathological examination of sciatic nerves showed that increase in edema, vacuolar changes and demyelination was significant in neuropathy induction group. [43] Co-treatment with EP 50 mg/kg and EP 100 mg/kg significantly attenuated these histopathological changes in dose dependant manner. The results suggested that scavenging of free radical prevent the pathological changes in sciatic nerve as supported by histopathological findings.

Cisplatin is known to generate oxygen free-radicals that induce membrane lipid peroxidation with subsequent extensive tissues damage. It was also observed that after cisplatin administration, level of GSH, CAT and SOD in body was decreased which an evidence of oxidative stress. [44]

Our study showed that co-administration of EP 50 and 100 mg/kg for 5 weeks restored the sciatic nerve peroxides back to near normal levels in dose-dependent manner. It was also found that EP 50 and 100 mg/kg inhibit oxidative damage of sciatic nerve in dose-dependent manner by increasing levels of GSH, CAT and SOD.

In this study, we have investigated the role of the EP as an antioxidant has defense mechanism in peripheral neurotoxicity induced by vincristine and cisplatin chemotherapy. In conclusion, the results of our study indicated that EP significantly offer protection against vincristine and cisplatin induced peripheral neurotoxicity and reduce incidence as well as intensity of neuropathic signs and symptoms.


   Conclusion Top


All of the above findings have suggested that EP 100 mg/kg is more effective in attenuation of thermal, mechanical hyperalgesia and cold allodynia in vincristine and cisplatin induced neuropathy. It was also found that the histopathological changes and oxidative stress occurred after neuropathy induction was significantly reversed by EP 100 mg/kg. The observed neuroprotective effect may be due to the antioxidant property of EP. However, further studies are required to elucidate the exact molecular mechanism of the neuroprotective effect of EP.

 
   References Top

1.Bhagra A, Rao RD. Chemotherapy-induced neuropathy. Curr Oncol Rep 2007;9: 290-9.  Back to cited text no. 1
    
2.Galli F, Piroddi M, Annetti C, Aisa C, Floridi E, Floridi A. Oxidative stress and reactive oxygen species. Contrib Nephrol 2005;149:240-60.  Back to cited text no. 2
    
3.Goodisman J, Hagrman D, Tacka KA, Souid AK. Analysis of cytotoxicities of platinum compounds. Cancer Chemother Pharmacol 2006;57:257-67.  Back to cited text no. 3
    
4.Siau C, Bennett GJ. Dysregulation of cellular calcium homeostasis in chemotherapy-evoked painful peripheral neuropathy. Anesth Analg 2006;102:1485-90.  Back to cited text no. 4
    
5.Won YJ, Yoo JY, Lee JH, Hwang SJ, Kim D, Hong HN. Erythropoietin is neuroprotective on GABAergic neurons against kainic acid-excitotoxicity in the rat spinal cell cultures. Brain Res 2007;1154:31-9.  Back to cited text no. 5
    
6.Abd El-Ghany RM, Sharaf NM, Kassem LA, Mahran LG, Heikal OA. Thymoquinone triggers anti-apoptotic signaling targeting death ligand and apoptotic regulators in a model of hepatic ischemia reperfusion injury. Drug Discov Ther 2009;3:296-306.  Back to cited text no. 6
    
7.Kenneth K, Fink M. The biochemical basis for the anti-inflammatory and cytoprotective actions of ethyl pyruvate and related compounds. Biochem Pharmacol 2010;80:151-9.  Back to cited text no. 7
    
8.Kaur G, Jaggi AS, Singh N. Exploring the potential effect of Ocimum sanctum in vincristine-induced neuropathic pain in rats. J Brachial Plex Peripher Nerve Inj 2010;5:3.  Back to cited text no. 8
    
9.Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F. A new animal model of vincristine-induced nociceptive peripheral neuropathy. Neurotoxicology 2003;24:797-805.  Back to cited text no. 9
    
10.Pisano C, Pratesi G, Laccabue D, Zunino F, Lo Giudice P, Bellucci A, et al. Paclitaxel and cisplatin-induced neurotoxicity: A protective role of acetyl-L-carnitine. Clin Cancer Res 2003;9:5756-67.  Back to cited text no. 10
    
11.Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F. An animal model of nociceptive peripheral neuropathy following repeated cisplatin injections. Exp Neurol 2003;182:12-20.  Back to cited text no. 11
    
12.Necker R, Hellon RF. Noxious thermal input from the rat tail: Modulation by descending inhibitory influences. Pain 1978;4:231-42.  Back to cited text no. 12
    
13.Kuhad A, Chopra K. Tocotrienol attenuates oxidative-nitrosative stress and inflammatory cascade in experimental model of diabetic neuropathy. Neuropharmacology 2009;57:456-62.  Back to cited text no. 13
    
14.Beyreuther B, Callizot N, Stöhr T. Antinociceptive efficacy of lacosamide in a rat model for painful diabetic neuropathy. Eur J Pharmacol 2006;539:64-70.  Back to cited text no. 14
    
15.Flatters SJ, Bennett GJ. Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain 2004;109:150-61.  Back to cited text no. 15
    
16.Muthuraman A, Jaggi AS, Singh N, Singh D. Ameliorative effects of amiloride and pralidoxime in chronic constriction injury and vincristine induced painful neuropathy in rats. Eur J Pharmacol 2008;587:104-11.  Back to cited text no. 16
    
17.Jain V, Jaggi AS, Singh N. Ameliorative potential of rosiglitazone in tibial and sural nerve transection-induced painful neuropathy in rats. Pharmacol Res 2009;59:385-92.  Back to cited text no. 17
    
18.Boehme DH, Kosecki R, Carson S, Stern F, Marks N. Lipoperoxidation in human and rat brain tissue: Developmental and regional studies. Brain Res 1977;136:11-21.  Back to cited text no. 18
    
19.Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.  Back to cited text no. 19
    
20.Luck H. Catalase In: Methods of Enzymatic Analysis. In: Bergmeyer HU, editor. New York: Academic Press; 1971. p. 885-93.  Back to cited text no. 20
    
21.Misra HP, Fridovich I. Superoxide dismutase activity was measured in dialyzed extracts with a modified epinephrine assay. J Biol Chem 1972;247:3170-5.  Back to cited text no. 21
    
22.Fink MP. Ethyl pyruvate: A novel anti-inflammatory agent. J Intern Med 2007;261:349-62.  Back to cited text no. 22
    
23.Das UN. Pyruvate is an endogenous anti-inflammatory and anti-oxidant molecule. Med Sci Monit 2006;12:RA79-84.  Back to cited text no. 23
    
24.Nozaki-Taguchi N, Chaplan SR, Higuera ES, Ajakwe RC, Yaksh TL. Vincristine-induced allodynia in the rat. Pain 2001;93:69-76.  Back to cited text no. 24
    
25.Authier N, Gillet JP, Fialip J, Eschalier A, Coudore F. A new animal model of vincristine-induced nociceptive peripheral neuropathy. Neurotoxicology 2003;24:797-805.  Back to cited text no. 25
    
26.Kaplan RS, Wiernik PH. Neurotoxicity of antineoplastic drugs. Semin Oncol 1982;9:103-30.  Back to cited text no. 26
    
27.Topp KS, Tanner KD, Levine JD. Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat. J Comp Neurol 2000;424:563-76.  Back to cited text no. 27
    
28.Fiori MG, Schiavinato A, Lini E, Nunzi MG. Peripheral neuropathy induced by intravenous administration of vincristine sulfate in the rabbit. An ultrastructural study. Toxicol Pathol 1995;23:248-55.  Back to cited text no. 28
    
29.Oliver AS, Firth G, McKeran RO. Studies on the intracerebral injection of vincristine free and entrapped within liposomes in the rat. J Neurol Sci 1985;68:25-30.  Back to cited text no. 29
    
30.Götz ME, Künig G, Riederer P, Youdim MB. Oxidative stress: Free radical production in neural degeneration. Pharmacol Ther 1994;63:37-122.  Back to cited text no. 30
    
31.Lewén A, Matz P, Chan PH. Free radical pathways in CNS injury. J Neurotrauma 2000;17:871-90.  Back to cited text no. 31
    
32.Feillet-Coudray C, Rock E, Coudray C, Grzelkowska K, Azais-Braesco V, Dardevet D, et al. Lipid peroxidation and antioxidant status in experimental diabetes. Clin Chim Acta 1999;284:31-43.  Back to cited text no. 32
    
33.Cohen G, Heikkila RE. The generation of hydrogen peroxide, superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dialuric acid, and related cytotoxic agents. J Biol Chem 1974;249:2447-52.  Back to cited text no. 33
    
34.Searle AJ, Willson RL. Glutathione peroxidase: Effect of superoxide, hydroxyl and bromine free radicals on enzyme activity. Int J Radiat Biol Relat Stud Phys Chem Med 1980;37:213-7.  Back to cited text no. 34
    
35.Kim JB, Yu YM, Kim SW, Lee JK. Anti-inflammatory mechanism is involved in ethyl pyruvate-mediated efficacious neuroprotection in the postischemic brain. Brain Res 2005;1060:188-92.  Back to cited text no. 35
    
36.Moro N, Sutton RL. Beneficial effects of sodium or ethyl pyruvate after traumatic brain injury in the rat. Exp Neurol 2010;225:391-401.  Back to cited text no. 36
    
37.Krarup-Hansen A, Fugleholm K, Helweg-Larsen S, Hauge EN, Schmalbruch H, Trojaborg W, et al. Examination of distal involvement in cisplatin-induced neuropathy in man. An electrophysiological and histological study with particular reference to touch receptor function. Brain 1993;116:1017-41.  Back to cited text no. 37
    
38.Santos NA, Bezerra CS, Martins NM, Curti C, Bianchi ML, Santos AC. Hydroxyl radical scavenger ameliorates cisplatin-induced nephrotoxicity by preventing oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria. Cancer Chemother Pharmacol 2008;61:145-55.  Back to cited text no. 38
    
39.Zhu C, Raber J, Eriksson LA. Hydrolysis process of the second generation platinum-based anticancer drug cis-amminedichlorocyclohexylamineplatinum (II). J Phys Chem B 2005;109:12195-205.  Back to cited text no. 39
    
40.McKeage MJ, Hsu T, Screnci D, Haddad G, Baguley BC. Nucleolar damage correlates with neurotoxicity induced by different platinum drugs. Br J Cancer 2001;85:1219-25.  Back to cited text no. 40
    
41.Won SJ, Kim DY, Gwag BJ. Cellular and molecular pathways of ischemic neuronal death. J Biochem Mol Biol 2002;35:67-86.  Back to cited text no. 41
    
42.Appenroth D, Fröb S, Kersten L, Splinter FK, Winnefeld K. Protective effects of vitamin E and C on cisplatin nephrotoxicity in developing rats. Arch Toxicol 1997;71:677-83.  Back to cited text no. 42
    
43.Stillman M, Cata JP. Management of chemotherapy-induced peripheral neuropathy. Curr Pain Headache Rep 2006;10:279-87.  Back to cited text no. 43
    
44.Mathew JE, Joseph A, Srinivasan K, Dinakaran SV, Mantri A, Movaliya V. Effect of ethanol extract of Sphaeranthus indicus on cisplatin-induced nephrotoxicity in rats. Nat Prod Res 2012;26:933-8.  Back to cited text no. 44
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and me...
    Experimental pro...
    Behavioral exami...
    Biochemical esti...
    Histopathologica...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed2114    
    Printed75    
    Emailed0    
    PDF Downloaded215    
    Comments [Add]    

Recommend this journal