Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy

Neuropharmacology 113 (2017) 137e147

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Combination strategy of PARP inhibitor with antioxidant prevent bioenergetic deficits and inflammatory changes in CCI-induced neuropathy Prashanth Komirishetty a, d, Aparna Areti a, Ranadeep Gogoi b, Ramakrishna Sistla c, Ashutosh Kumar a, * a

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, India Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, India Pharmacology Division, Indian Institute of Chemical Technology (IICT), Hyderabad, India d Division of Neurology, Department of Medicine, University of Alberta, 2E3.26 Walter C Mackenzie, Health Sciences Center, Edmonton, AB, T6G 2B7, Canada b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 May 2016 Received in revised form 4 September 2016 Accepted 27 September 2016 Available online 3 October 2016

Neuropathic pain, a debilitating pain condition and the underlying pathogenic mechanisms are complex and interwoven amongst each other and still there is scant information available regarding therapies which promise to treat the condition. Evidence indicate that oxidative/nitrosative stress induced poly (ADP-ribose) polymerase (PARP) overactivation initiate neuroinflammation and bioenergetic crisis culminating into neurodegenerative changes following nerve injury. Hence, we investigated the therapeutic effect of combining an antioxidant, quercetin and a PARP inhibitor, 4-amino 1, 8-naphthalimide (4-ANI) on the hallmark deficits induced by chronic constriction injury (CCI) of sciatic nerve in rats. Quercetin (25 mg/kg, p.o.) and 4-ANI (3 mg/kg, p.o.) were administered either alone or in combination for 14 days to examine sciatic functional index, allodynia and hyperalgesia using walking track analysis, Von Frey, acetone spray and hot plate tests respectively. Malondialdehyde, nitrite and glutathione levels were estimated to detect oxidative/nitrosative stress; mitochondrial membrane potential and cytochrome c oxidase activity to assess mitochondrial function; NAD & ATP levels to examine the bioenergetic status and levels of inflammatory markers were evaluated in ipsilateral sciatic nerve. Quercetin and 4-ANI alone improved the pain behaviour and biochemical alterations but the combination therapy demonstrated an appreciable reversal of CCI-induced changes. Nitrotyrosine and Poly ADP-Ribose (PAR) immunopositivity was decreased and nuclear factor erythroid 2-related factor (Nrf-2) levels were increased significantly in micro-sections of the sciatic nerve and dorsal root ganglion (DRG) of treatment group. These results suggest that simultaneous inhibition of oxidative stress-PARP activation cascade may potentially be useful strategies for management of trauma induced neuropathic pain. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Chronic constriction injury Quercetin 4-ANI PARP Oxidative stress Bioenergetic crisis

1. Introduction Pain is an alerting signal in our biological system which elicits during the malformation of the physiological process whereas neuropathic pain is not a symptom, but is a disease itself which is defined as ‘pain initiated or resulted by a primary lesion or

* Corresponding author. Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Hyderabad, Balanagar, Hyderabad, Telangana, 500037, India. E-mail addresses: [email protected], [email protected] (A. Kumar). http://dx.doi.org/10.1016/j.neuropharm.2016.09.027 0028-3908/© 2016 Elsevier Ltd. All rights reserved.

dysfunction in the nervous system’ (Brooks and Tracey, 2005; Treede et al., 2008). Till date, it remains as a chronic debilitating condition that affects the quality of life and reduces individual productivity. Neuropathic pain associated with the peripheral nerve injury is well characterized by various sensory abnormalities producing pain sensations like spontaneous pain, allodynia and hyperalgesia (Bennett and Xie, 1988). Currently, neuropathic pain is managed clinically using various conventional medicines including antidepressants (amitriptyline, duloxetine, venlafaxine), anticonvulsants (gabapentin and pregabalin); opioids (morphine and fentanyl) (Taylor et al., 2008). However, overall some 10e30% of pain patients are responsive to these drugs which suffer dose

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limitations with respect to efficacy and side effects (dizziness, sedation, reduced appetite, sedation, tachycardia and weight gain) (Dray, 2008). Hence, the search for innovative therapies and treatment strategies targeting the root cause of pathology is required for better management of the disease and improve the quality of patient's life. A multidisciplinary approach that combines therapies, targeting the mechanism as well as clinical symptoms may be employed for effective management of neuropathy and neuropathic pain (Chong and Bajwa, 2003; Gilron and Max, 2005). It has been demonstrated in many previous studies that the combination of therapeutic interventions targeting different mechanisms may provide improved pain relief with fewer side effects. The accumulating evidence suggests the involvement of oxidative/nitrosative stress and PARP overactivation in the traumatic injury induced neuropathies (da Avila et al., 2012; Obrosova et al., 2004; Sandireddy et al., 2014). Oxidative/nitrosative stress during the peripheral injury depletes cellular antioxidant levels and damage the biomolecules through lipid peroxidation and DNA fragmentation (Valko et al., 2007). The resultant free radicals which are highly toxic metabolites must be detoxified by phase II enzymes like glutathione S-transferase, heme oxygenase-1 (HO-1), NADPHquinone oxidoreductase etc (Ryter et al., 2006). The nuclear factor erythroid 2erelated factor 2 (Nrf2) has been recognized to play a critical role in the upregulation of these enzymes (Negi et al., 2011). Reactive oxygen/nitrogen species like superoxide or peroxynitrite under such stress conditions activates nuclear factor-kB (NF-kB), which triggers the upregulation of inflammatory mediators like inducible cyclooxygenase (COX-2), nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-a) and interleukin (IL)-6 (Makarov, 2000). This causes perturbations in the NF-kB and Nrf2 axis resulting in the aggravation of oxidative/nitrosative stress and neuroinflammation in neuropathy (Yerra et al., 2013). PARP overactivation also has been proven to play a crucial role in the pathophysiology of trauma-induced neurodegeneration and other neuropathies like diabetic and chemotherapy induced neuropathies (Genovese et al., 2005b; Negi et al., 2010b; Ta et al.). Excessive free radicals damage DNA and in turn lead to overactivation of DNA repairing enzyme, PARP. Overtly activated PARP synthesize poly ADP-ribose units utilising high amount of NAD which indirectly results in ATP depletion (Jagtap and Szaba, 2005). This also causes excessive PARylation of the cellular proteins especially mitochondrial proteins lead to mitochondrial dysfunction. Evidence indicate that PARP overactivation causes loss of mitochondrial membrane potential and decreased electron transport chain complexes activity especially cytochrome c oxidase (complex IV) and ATP synthase (Lai et al., 2008b; Lim et al., 2015). PARP is also known to play a role in transcription regulation and activation of NF-kB, thus aids in the inflammation-induced neurodegeneration (Hassa and Hottiger, 2002). A lot of research with antioxidants is present in scientific literature, but even with potent antioxidants, the pathophysiological cascades initiated and amplified post nerve injury could not be contained completely. It leads to the notion that there may be some independent pathways which are not related to oxidative-nitrosative stress which can initiate PARP overactivation and may lead to nerve tissue damage as seen in the neuropathy patients. Failure of potent antioxidants in controlling DNA damage induced PARP over-activation; may hint towards the involvement of some other factor contributing to PARP overactivation (Negi et al., 2010a). Hence, we proposed to evaluate the potential of simultaneous targeting two of the premier, correlated pathways (oxidative/nitrosative stress and PARP overactivation) that may be considered as the crux of pathophysiological factors involved in the development of trauma-induced neuropathy and neuropathic pain. Considering the role of oxidative/nitrosative stress and PARP overactivation in peripheral neuropathy we have

targeted them using an antioxidant (quercetin) and PARP inhibitor (4-ANI) in experimental model of trauma induced neuropathy. Quercetin (3,5,7,30 ,40 -Penta hydroxyl flavone) is a bioflavonoid having many beneficial effects on human health, including cardioprotection, neuroprotection, antioxidant and anti-inflammatory activities which are attributed to its modulating actions on the pathways like NF-kB, MAPK, JNK and Nrf2 (Ji et al., 2015; Maalik et al., 2014). Quercetin has been shown to attenuate the neuroinflammation process and decrease the extent of the secondary injury after the spinal cord injury (o cevik et al., 2013). It exhibited antinociceptive effects in oxaliplatin and streptozotocin-induced diabetic neuropathy in rodents (Azevedo et al., 2013; Narenjkar et al., 2011). 4-amino 1, 8-naphthalimide (4-ANI), a potent PARP inhibitor has shown neuroprotection in animal models of acute cerebral ischemia and diabetic neuropathy (Sharma et al., 2008, 2009). The protective actions of 4-ANI are also well demonstrated in other experimental models like Parkinson's disease and focal cerebral ischemia at optimal doses (Kabra et al., 2004; Outeiro et al., 2007). Therefore, with the support of literature and experimental evidences we proposed the combination of quercetin and 4-ANI may produce beneficial effects in peripheral neuropathy. Hence, current studies were undertaken to explore the neuroprotective effect of combination therapy at minimal doses (quercetin at 25 mg/kg and 4-ANI at 3 mg/kg) in the neuropathic rats induced by chronic constriction injury (CCI) of the sciatic nerve.

2. Materials & methods 2.1. Drugs and chemicals All the chemicals including quercetin (purity > 99%) were procured from Sigma-Aldrich Co, USA unless until specified. NAD/ NADH kit, MITOISO 1 and Cytochrome c Oxidase Assay kits were also obtained from Sigma-Aldrich Co, USA. 4-amino 1, 8naphthalimide was purchased from Santa Cruz Biotech, USA. Tissue protein extraction reagent (T-PER) was purchased from Thermo Scientific, USA. ATP detection kit from Abcam, Cambridge, UK was used to estimate the levels of ATP. Immunohistochemistry (IHC) detection kit procured from PathnSitu Biotechnologies Pvt Ltd. Hyderabad, India was used for IHC experiments. Isoflurane was obtained from Raman & Weil Pvt. Ltd. (Mumbai, India). TNF-a and IL-6 ELISA kits were procured from eBiosciences, Inc. San Diego, CA, USA.

2.2. Experimental animals All the experimental protocols were duly approved by the Institutional Animal Ethics Committee (IAEC) and experiments were performed in accordance to prevailing guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Male Sprague-Dawley rats weighing 180e230 g were procured from National institute of nutrition (NIN), Hyderabad, India. All the animals were housed under optimal laboratory conditions, maintained on 12 h' light and dark cycle one week before experimentation and had a free access to food and water ad libitum. The study has been compiled with the internationally accredited guidelines and ethical regulations on animal research. All the experiments were performed before CCI to obtain basal readings and on 7th and 14th day post CCI to screen the pharmacological activity of treatment. All behavioural measures were obtained by an observer who was blinded to the group assignment to avoid bias.

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2.3. Induction of sciatic nerve injury by chronic constriction injury (CCI) Peripheral neuropathy in rats was developed by chronic constriction injury of sciatic nerve according to Bennett and Xie (Bennett and Xie, 1988). Surgeries were performed under gaseous anaesthesia by using a mixture of 4% isoflurane and oxygen. Using a chromic 4-0 gut (Ethicon, USA) suture four loose ligatures were tied loosely around proximal trifurcation part of the sciatic nerve with 1 mm of spacing. The ligatures were neither too tight to occlude the epineural blood flow nor too loose to overlap on each other and it was made by tightening ligature by moving up and down on a sciatic nerve. Later muscle and skin were closed layer by layer using 3-0 silk suture and iodine solution was applied superficially on skin. After completion of surgery 5% dextrose was administered and animals were recovered on a hot pad to maintain body temperature. In sham groups, an identical surgical procedure was performed, except that the sciatic nerve was not ligated. All the surgical procedures were carried out by the same person. 2.4. Drug treatment Rats were randomly assigned into 5 groups of 10 animals each. Groups were divided into sham control (SHAM), disease control (CCI) and three treated groups. The three treatment groups comprised of CCI rats treated with quercetin (25 mg/kg, p.o.) (CCI þ Q), CCI rats treated with 4-ANI (3 mg/kg, p.o.) (CCI þ ANI) and CCI rats treated with both drugs (CCI þ Q þ ANI) from day 1 after surgery to 14 days whereas sham control received distilled water and disease control received vehicle similar as treatment groups. 2.5. Anaesthesia, euthanasia and tissue sampling Animals were anaesthetized in a mixture of 4% isoflurane and oxygen and maintained at 1.5e2% isoflurane during surgeries. Core temperature was monitored and maintained (37 ± 1  C) using a rectal probe with the help of a homeothermic blanket. After 14 days of treatment, rats were euthanized with CO2 anaesthesia. Ipsilateral sciatic nerves and L4-L6 parts of DRG were dissected collected. The nerves & DRG which were to be used for immunohistochemistry were fixed in 10% neutral buffered formalin. For biochemical estimations, the nerves were homogenized in phosphate buffer and were used for various estimations. For protein expression studies the nerves were homogenized using T-PER and were estimated using western blotting analysis. 2.6. Functional assessment 2.6.1. Sciatic functional index (Walking track analysis) SFI was calculated by using walking track analysis according to the method described by Bian et al., to assess the functional changes of sciatic nerve damage by measuring the hind paw footprints (Bain et al., 1989). Then animal hind paws were dipped in Indian ink and they were then allowed to walk on a white paper in a walking track ending in a darkened cage. The measurements were taken from the footprints of rat as follows (i) print length (PL); longitudinal distance from the heel to the third toe (ii) toe spread (TS); horizontal distance from the first to the fifth toe, and (iii) intermediary toe spread (ITS); horizontal distance from the second to the fourth toe. All the three measurements were taken from both ipsilateral (I) and contralateral (C) sides. Several footprints were taken from each track and average values of three-foot prints were considered. From these values, various factors were calculated as follows to obtain the sciatic functional index (SFI). (i) Print length

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factor (PLF) ¼ (IPL-CPL)/CPL; (ii) Toe spread factor (TSF) ¼ (ITS CTS)/CTS; (iii) Intermediary toe spread factor (ITF)¼ (IIT- CIT)/CIT. The sciatic functional index (SFI) from these factors was calculated by using the Bain-Mackinnon-Hunter (BMH) formula; SFI ¼ 38.3  PLF þ 109.5  TSF þ 13.3  ITF - 8.8. An SFI value for the normal foot is 0 and total impairment is 100, which would result from a complete transection of the sciatic nerve. 2.7. Behavioural tests 2.7.1. Assessment of allodynia and hyperalgesia Animals were habituated to the experimental conditions prior to the experiment. Hot and cold plate tests were used to assess thermal hyperalgesia at (52  C ± 1  C) and (4  C ± 1  C) with a cutoff time of 15 s and 60 s respectively (Huang et al., 2004). The average of six consecutive readings was taken at an interval of 10 min. Mechanical allodynia was determined by using Von Frey hairs (Samitek, USA; 1, 2, 4, 6, 8, 10 and 15 g). All animals were acclimatized in perplex boxes on a mesh surface and Von Frey hairs were applied perpendicular to plantar surface from lower pressure to higher pressure. Sufficient force was applied to bend the filaments slightly for two to three seconds and a sudden withdrawal of paw with a paw licking sign was noted as a positive response. The test was done again for three times for each animal with 5 min interval and average force of the monofilament which evoked the paw reflex was considered as the paw withdrawal threshold (Decosterd et al., 1998). Cold chemical allodynia was assessed by applying 100 ml of acetone on the plantar surface of left hind paw by using bent gauge needle without touching paw surface to prevent damage and the responses were observed for 20 s and graded to 4point scale. No response (Score 0), sudden withdraw (score 1), repeated flicking (score 2), repeated flicking and licking (score 3) were given. Acetone was applied thrice on the hind paw with an interval of 5 min and the individual score noted in 20 s interval were added to get the single value over a cumulative period of 60 s. The minimum score obtained was 0 and the maximum score was 9 (Bardin et al., 2009). 2.7.2. Assessment of foot deformity Rat foot posture was observed for its deformity by placing on a plain surface. The foot deformity was scored based on its posture as follows: score 0 if paw is normal with fanned toes. Score 1 if the toe is ventroflexed; Score 2 if paw is everted so that only the internal edge of the paw touches the surface (Jaggi and Singh, 2010). 2.8. Biochemical estimations 2.8.1. Estimation of oxidative/nitrosative stress markers Animals were euthanized after 14 days of the experimental protocol. Immediately, ipsilateral sciatic nerves were collected. The samples were then homogenized using Tris-HCl (pH-7.4) buffer containing protease cocktail inhibitor (Sigma, USA) and the supernatant was collected after centrifugation at 12,000 rpm and 4  C for 15 min. These supernatants were used for further determination of protein content, malondialdehyde (MDA), nitrite and glutathione (GSH) levels. Total protein content of homogenates was estimated using protein assay kit (Bio-Rad, USA) and using bovine serum albumin (BSA) as a standard. The extent of lipid peroxidation was assessed by measuring 'Thiobarbituric Acid Reactive Substances' (TBARS) (Janero, 1990). Absorbance was measured at 532 nm and MDA levels were expressed as mmol/mg protein. Nitrite levels were estimated at 540 nm using the Griess reagent and total nitrite levels were expressed as nmol/mg of protein after protein normalization (Sastry et al., 2002). Glutathione (GSH) levels were calculated using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB/Ellman's reagent) at

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405 nm (Sedlak and Lindsay, 1968). The total quantity of GSH was calculated by means of a calibration curve, normalized to the protein concentration and expressed as mmol/mg. 2.8.2. Measurement of bioenergetic markers The NAD (total) & ATP levels were measured to indicate the extent of PARP overactivation. NAD (total) levels were measured at 450 nm in multiplate reader (Spectramax M4, Molecular Devices LLC, California, USA) according to the manufacturer's instructions (Sigma, St. Louis, MO). The NAD (total) levels were calculated by means of a calibration curve and normalized to the protein concentration and expressed as ng/mg of protein. ATP levels were estimated according to the manufacturer's instructions (Abcam, UK). ATP levels were measured at 570 nm using multiplate reader (Spectramax M4, Molecular Devices LLC, California, USA). The total quantity of ATP was calculated by means of a calibration curve, normalized to the protein concentration and expressed as mmol/gm protein (Komirishetty et al., 2016b). 2.8.3. Assessment of mitochondrial functions 2.8.3.1. Mitochondrial isolation and assessment of membrane potential (Jm). Mitochondria were isolated from the ipsilateral sciatic nerve using the MITOISO1 Mitochondrial Isolation Kit (Sigma, St. Louis, MO) according to manufacturer's instructions. The resulting pellet was suspended in storage buffer. The total protein content was estimated using protein assay kit (Bio-Rad, USA). Mitochondrial membrane potential was immediately determined using the JC-1 membrane potential assay kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer's instructions. The assays were conducted in a 2 ml reaction mixture containing JC-1 assay buffer and approximately 25 mg of mitochondrial fractions. The reaction was initiated with the addition of JC-1 Stain and fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 590 nm by using a multiplate reader (Spectramax M4, USA). The fluorescence observed at 590 nm shows the extent of formation of J-aggregates which indicates the Jm. 2.8.3.2. Assessment of cytochrome c oxidase activity in isolated mitochondria. The isolated mitochondrial fraction was estimated for cytochrome c oxidase activity which indicates the mitochondrial complex IV activity. The assay was performed using Cytochrome c Oxidase assay kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer's instructions. It is a colorimetric assay based on the observation i.e. decrease in the absorbance of ferricytochrome c at 550 nm due to its oxidation by cytochrome c oxidase. The activity of cytochrome c oxidase is expressed in Units/mg/min of protein. 2.8.4. Estimation of inflammatory markers (TNF-a & IL-6) Ipsilateral sciatic nerves were homogenized using Tris-HCl (pH7.4) containing protease cocktail inhibitor (Sigma, USA) and the supernatant was collected after centrifugation at 4000 rpm for 15 min at 4  C. TNF-a and IL-6 levels were estimated using commercially available rat specific enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions and the levels were calculated with the standard curve produced from the respective kit and expressed as pg/mg protein after normalizing the protein content.

antibodies to their antigenic sites in sections was amplified by using Poly Excel HRP/DAB Detection System (PathnSitu Biotechnologies Pvt Ltd, Hyderabad, India). The antigen-antibody reaction sites were visualized using 3,3-diaminobenzidine (DAB) for 5 min and subsequently, sections were counterstained with Mayer's modified hematoxylin (Sigma, USA). Then the dehydrated sections were mounted and images were taken under a microscope (Nikon Eclipse TE2000-E, USA). IHC was performed with 6 animal sections for each group. From each section 5 different regions were selected to obtain images. The qualitative expression (brown stain) was considered as positive. The average of the specific protein expressed positive cells were indicated as a percentage of (%) total cells (Komirishetty et al., 2016a). 2.10. Western blotting analysis Ipsilateral sciatic nerves were homogenized in T-PER (Thermo scientific, USA) containing 1% protease cocktail inhibitor and clear supernatants were used for determination of protein expressions. An equal amount of protein samples were separated on SDSpolyacrylamide gel (SDS-PAGE); the separated proteins were then transferred onto polyvinylidene difluoride (PVDF) membrane followed by blocking with blocking solution (3% BSA) for 1 h. Then the membrane was incubated overnight at 4  C with primary antibodies to iNOS, COX-2, Nrf2 and HO-1 (Santa Cruz Biotechnology, USA, 1: 1000 dilution), NF-kB and b-actin (Cell Signalling Technology, Beverly, MA, USA, 1:1000 dilution). The washed membranes were then incubated with a horseradish peroxidase (HRP) conjugated secondary antibody (1:5000 dilution) and visualized by enhanced chemiluminescence. The relative band densities were quantified by (Image J 1.36; Wayne Rasband, National Institute of Health, MD, USA) software. Equal loading of protein was confirmed by measuring b-actin expression. 2.11. Data analysis The data obtained were expressed as the mean ± standard error of mean (SEM). The data from the behavioural results were statistically analyzed by two-way analysis of variance (ANOVA) and data from the biochemical results were statistically analyzed by oneway analysis of variance using the GraphPad Prism Version-5.0 software. Statistical comparisons were made with “Bonferroni's Multiple Comparison Test”. Results with P values < 0.05 were considered as statistically significant. 3. Results 3.1. General behavioural observations Sham-operated animals did not show any abnormal behaviour, but after the 3rd day of the surgery, CCI operated rats showed behavioural changes like abnormal gait, posture, flicking and licking of the ipsilateral hind paw. Those rats that underwent surgery were observed to lift their ipsilateral hind paw as they could not bear their body weight on the ipsilateral side. The food intake of operated rats was found to be decreased slightly in the initial days of surgery which might be due to the development of ongoing pain and it was slowly normalized.

2.9. Immunohistochemistry (IHC) The sections of ipsilateral sciatic nerve and L4-L6 DRG were deparaffinized, rehydrated and were incubated with primary antibodies to nitrotyrosine, Nrf2 and PAR (Santa Cruz Biotechnology) in dilutions of 1:200 for 2 h at room temperature in a humidified chamber. Then sections were washed and binding ability of the

3.2. Effect of quercetin and 4-ANI alone and in combination on sciatic functional index The CCI of sciatic nerve impairs its function and to assess the hind limb function, the sciatic functional index was measured by using footprint analysis. In CCI rats, a significant (p < 0.001)

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functional impairment on 7th and 14th day was observed as compared to sham group. Administration of quercetin (25 mg/kg, p.o) and 4-ANI (3 mg/kg, p.o) for 14 days improved the functional index but not completely recovered the normal sciatic functional index. Whereas, combination therapy of quercetin and 4-ANI for 14 days showed significant (p < 0.001) development in the functional index as compared to the sham animals and improvement was found to be better than the monotherapy group's. 3.3. Effect of quercetin and 4-ANI alone and in combination on pain behaviour A significant (p < 0.001) development of thermal hyperalgesia, mechanical and cold chemical allodynia was observed in rats with CCI of the sciatic nerve by 7th and 14th day when compared to sham group. Combination therapy of quercetin and 4-ANI significantly (p < 0.001) increased the paw withdrawal latencies to cold and hot stimuli when compared to the sham group as well as monotherapy of quercetin and 4-ANI (p < 0.05). Quercetin monotherapy did not show any improvement but 4-ANI monotherapy significantly (p < 0.05) increased the paw withdrawal thresholds to Von Frey fibres. The same effect was observed in response to cold allodynia with the monotherapy of 4-ANI (p < 0.05). But the combination therapy reversed these CCI-induced changes significantly (p < 0.001) in CCI rats. Combination therapy significantly

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attenuated the pain behaviour in response to thermal hyperalgesia (p < 0.001), mechanical (p < 0.01) and cold chemical allodynia (p < 0.001) by 14th day when compared to the treatment effect of monotherapy groups. 3.4. Effect of quercetin and 4-ANI alone and in combination on foot deformity Chronic constriction injury resulted in a significant postural defect in terms of foot deformity when compared to near normal postural index of sham group. Monotherapy of quercetin and 4-ANI (25 & 3 mg/kg, p.o. respectively), corrected the chronic constriction injury-induced foot deformity. Whereas combination therapy for 14 days markedly improved paw postural defects in CCI rats when compared to CCI group and was found to be superior to monotherapy groups. 3.5. Effect of quercetin and 4-ANI alone and in combination on oxidative/nitrosative stress in CCI rats CCI of sciatic nerve significantly (p < 0.001) elevated the levels of MDA and nitrite when compared to sham animals. Monotherapy of quercetin & 4-ANI reduced lipid peroxidation and decreased nitrite in CCI rats. But the combination therapy significantly (p < 0.001) attenuated this oxidant-induced lipid peroxidation and nitrite

Fig. 1. Effect of quercetin and 4-ANI alone or in combination on functional and behavioural changes: (A) Footprints and SFI. (B) Cold (4  C ± 1  C) hyperalgesia, Heat (52.5  C ± 1  C) hyperalgesia, mechanical allodynia and chemical induced cold allodynia. (C) Foot deformity in terms of postural index and Pictures of ipsilateral paw postures. Results were expressed as mean ± SEM (n ¼ 8). a, SHAM Vs CCI; b, treatment (quercetin & 4-ANI and combination) Vs CCI; c, combination Vs quercetin alone; d, combination Vs 4ANI alone; 1, p < 0.05; 2, p < 0.01; 3, p < 0.001. (SHAM: sham operated, CCI: CCI operated, CCI þ Q, & CCI þ ANI: CCI rats treated with quercetin and 4-ANI at 25 and 3 mg/kg respectively. CCI þ Q þ ANI: CCI rats treated with both quercetin and 4-ANI at 25 and 3 mg/kg respectively).

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formation when compared to the CCI group as well as monotherapy (p < 0.01). Combination therapy also significantly (p < 0.001) restored the depleted levels of glutathione in CCI rats. 3.6. Effect of quercetin and 4-ANI alone and in combination on bioenergetic markers in CCI rats CCI of sciatic nerve significantly (p < 0.001) depleted the levels of NAD & ATP when compared to the sham animals indicating the PARP overactivation induced bioenergetic failure. Monotherapy of quercetin did not show significant improvement but treatment with 4-ANI resulted in significant (p < 0.05) restoration of NAD and ATP levels in the sciatic nerve of CCI rats. Combination therapy significantly (p < 0.001) restored NAD and ATP levels when compared to the effects of CCI and both monotherapy groups. 3.7. Effect of quercetin and 4-ANI alone and in combination on mitochondrial functions in CCI rats CCI of sciatic nerve resulted in significant (p < 0.001) loss of mitochondrial membrane potential and cytochrome c oxidase activity when compared to the sham animals indicating the oxidative stress and PARP overactivation induced mitochondrial dysfunction. Monotherapy of quercetin and 4-ANI slightly improved the

mitochondrial function but the combination therapy checked the loss of CCI-induced mitochondrial activity and significantly (p < 0.001) attenuated the mitochondrial dysfunction in CCI rats. Combination therapy also significantly (p < 0.01) improved the loss of mitochondrial membrane potential and complex IV activity in CCI rats when compared to the monotherapy with either quercetin or 4-ANI. 3.8. Effect of quercetin and 4-ANI alone and in combination on inflammatory markers in CCI rats The levels of TNF-a & IL-6 in the sciatic nerve were significantly (p < 0.001) elevated in disease control group compared to sham control animals. Monotherapy of quercetin and 4-ANI, significantly (p < 0.05) reduced the levels of both inflammatory markers when compared to disease control group. Combination therapy showed even better (p < 0.001) reduction in these inflammatory markers in CCI rats when compared to the treatment with monotherapy groups. 3.9. Effect of quercetin and 4-ANI alone and in combination on expression of nitrotyrosine, Nrf2 and PAR levels in CCI rats Expression levels of nitrotyrosine and Nrf2 indicate the extent of

Fig. 2. Effect of quercetin and 4-ANI alone or in combination on oxidative/nitrosative stress markers: (A) & (B) Expression of nitrotyrosine in ipsilateral sciatic nerve and DRG respectively. Bar graph represents average percentage of positive cells among the total number of nuclei. Photographs were taken at 400. Micron bar shows a length of 50 mm. (C) & (D) shows the levels of MDA and nitrite in ipsilateral sciatic nerve. Results were expressed as mean ± SEM (n ¼ 6). a, SHAM Vs CCI; b, treatment (quercetin & 4-ANI and combination) Vs CCI; c, combination Vs quercetin alone. d, combination Vs 4-ANI alone; 1, p < 0.05; 2, p < 0.01; 3, p < 0.001. (SHAM: sham operated, CCI: CCI operated, CCI þ Q, & CCI þ ANI: CCI rats treated with quercetin and 4-ANI at 25 and 3 mg/kg respectively. CCI þ Q þ ANI: CCI rats treated with both quercetin and 4-ANI at 25 and 3 mg/kg respectively).

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oxidative/nitrosative stress and PAR levels indicate the extent of PARP overactivation. Hence, the levels of nitrotyrosine, Nrf2 and PAR were examined in the sciatic nerve and DRG sections of all the groups. CCI animals showed a significant (p < 0.001) increase in the nitrotyrosine, PAR and significant (p < 0.001) decrease in Nrf2 immunopositivity. Treatment with quercetin significantly (p < 0.05) attenuated increased expression of nitrotyrosine and improved the Nrf-2 levels but no effect was observed on PAR expression. Whereas 4-ANI significantly (p < 0.05) reduced the nitrotyrosine and PAR levels but there was minimal effect seen on Nrf2 levels. Combination therapy significantly reduced the nitrotyrosine and PAR levels in both the tissues and also restored (p < 0.001) the Nrf-2 levels when compared to CCI group. Combination therapy has reversed the CCI-induced changes markedly when compared to the monotherapy suggesting the abrogation of oxidative/nitrosative stress & PARP overactivation following the nerve injury.

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3.10. Effect of quercetin and 4-ANI alone and in combination on the expression of antioxidant and inflammatory markers in CCI rats To confirm the antioxidant & anti-inflammatory effect of combination therapy in CCI rats, the expression of various markers were evaluated in the ipsilateral sciatic nerve. A significant (p < 0.001) rise in the levels of iNOS, COX-2 and NF-kB were observed in CCI rats when compared to the sham animals. The levels of antioxidant defences like Nrf2 & HO-1 were decreased significantly (p < 0.001) in CCI group. Treatment with quercetin decreased the levels of iNOS, COX-2 (p < 0.05) and NF-kB where Nrf2 & HO-1 levels (p < 0.05 & p < 0.05 respectively) were increased significantly which indicates its antioxidant potential. 4-ANI monotherapy did not alter the levels of Nrf2, HO-1 and iNOS whereas COX-2 levels were significantly (p < 0.05) decreased in CCI rats. But the combination therapy significantly (p < 0.001) attenuated all these changes in CCI rats. It also improved the CCI-induced changes better than the monotherapy indicating its potential therapeutic

Fig. 3. Effect of quercetin and 4-ANI alone or in combination on antioxidant defenses: (A) & (B) Expression of Nrf2 in ipsilateral sciatic nerve and DRG respectively. Bar graph represents average percentage of positive cells among the total number of nuclei. Photographs were taken at 400. Micron bar shows a length of 50 mm. (C) Representative western blots showing the protein expression levels and bar diagram shows the quantified western blot data of respective protein. (D) The levels of GSH in ipsilateral sciatic nerve. Results were expressed as mean ± SEM (n ¼ 6). a, SHAM Vs CCI; b, treatment (quercetin & 4-ANI and combination) Vs CCI; c, combination Vs quercetin alone. d, combination Vs 4-ANI alone; 1, p < 0.05; 2, p < 0.01; 3, p < 0.001. (SHAM: sham operated, CCI: CCI operated, CCI þ Q, & CCI þ ANI: CCI rats treated with quercetin and 4-ANI at 25 and 3 mg/kg respectively. CCI þ Q þ ANI: CCI rats treated with both quercetin and 4-ANI at 25 and 3 mg/kg respectively).

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Fig. 4. Effect of quercetin and 4-ANI alone or in combination on the consequences of PARP overactivation: (A) & (B) Expression of PAR in ipsilateral sciatic nerve and DRG respectively. Bar graph represents average percentage of positive cells among the total number of nuclei. Photographs were taken at 400. Micron bar shows a length of 50 mm. (C) & (D) shows the levels of NAD and ATP in ipsilateral sciatic nerve. Results were expressed as mean ± SEM (n ¼ 6). a, SHAM Vs CCI; b, treatment (quercetin & 4-ANI and combination) Vs CCI; c, combination Vs quercetin alone. d, combination Vs 4-ANI alone; 1, p < 0.05; 2, p < 0.01; 3, p < 0.001. (SHAM: sham operated, CCI: CCI operated, CCI þ Q, & CCI þ ANI: CCI rats treated with quercetin and 4-ANI at 25 and 3 mg/kg respectively. CCI þ Q þ ANI: CCI rats treated with both quercetin and 4-ANI at 25 and 3 mg/kg respectively).

effect. 4. Discussion The study results illustrated the CCI-induced sciatic functional impairment, augmented changes in pain sensation and induced various biochemical deficits. None of the available drugs only promise to cure the disease but they only provide symptomatic relief from ailing symptoms of various neuropathies (Dworkin et al., 2007). Every agent has distinct advantages, disadvantages and can interact with single or multiple targets in the biological system compared to the others. Hence, the clinical outcomes might be improved when we switch to combination therapy, rather than depending on a single agent (Raffa, 2001). A combination is much more efficient when the individual agents act through different mechanisms and provide a benefit of additive and or synergistic outcomes. By targeting multiple pathways combination therapy can provide effective treatment outcomes and with better safety profile. The American Geriatrics Society (AGS), American Medical Directors Association (AMDA) and World Health Organization (WHO) also recommended combination therapy of analgesics for the treatment of chronic painful conditions in elderly patients (Gilron and Max, 2005). Outcomes of the current study add to existing knowledge on the involvement of oxidative/nitrosative

stress & PARP in the underpinning pathogenesis of neuropathy and warrants further research efforts for better understanding of current combination therapies. Growing evidence also support the role of ROS/RNS and PARP in trauma-induced peripheral neuropathy (Cornelius et al., 2013; Genovese et al., 2005a; Lai et al., 2008a). Oxidative/nitrosative stress in addition to activation of PARP also activates the other derogatory cascades like NF-kB and MAPK signalling pathways which in turn perturb various other signalling cascades including Nrf2 pathway (Peralta-Leal et al., 2009; Yerra et al., 2013). PARP overactivation induced by oxidative/nitrosative stress after peripheral nerve injury is said to be bidirectional rather than being unidirectional, as PARP overactivation further contributes to the generation of ROS and associated oxidative stress (Obrosova et al., 2005). These changes may be due to PARP-induced mitochondrial dysfunction and activation of inflammatory cascades (Giansanti et al., 2010; Hong et al., 2006; Lai et al., 2008a; Sandireddy et al., 2014). Hence, concurrent targeting of oxidative/ nitrosative stress & PARP using an antioxidant and PARP inhibitor may serve as a potential regimen against trauma-induced peripheral neuropathy. This combination therapy is not only believed to tone down oxidative/nitrosative stress & PARP overactivation but also abrogates the other pathological consequences like neuroinflammation and bioenergetic failure. We used quercetin and 4-ANI to counteract the functional,

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Fig. 5. Effect of quercetin and 4-ANI alone or in combination on mitochondrial functions & inflammatory markers: (A) & (B) shows the mitochondrial membrane potential and complex IV enzyme activity in an ipsilateral sciatic nerve. (C) & (D) shows the levels of TNF-a & IL-6 in ipsilateral sciatic nerve. (E) Representative western blots showing the protein expression levels and bar diagram shows the quantified western blot data of respective protein. Results were expressed as mean ± SEM (n ¼ 6). a, SHAM Vs CCI; b, treatment (quercetin & 4-ANI and combination) Vs CCI; c, combination Vs quercetin alone. d, combination Vs 4-ANI alone; 1, p < 0.05; 2, p < 0.01; 3, p < 0.001. (SHAM: sham operated, CCI: CCI operated, CCI þ Q, & CCI þ ANI: CCI rats treated with quercetin and 4-ANI at 25 and 3 mg/kg respectively. CCI þ Q þ ANI: CCI rats treated with both quercetin and 4-ANI at 25 and 3 mg/kg respectively).

behavioural and biochemical deficits induced by CCI of the sciatic nerve in rats. These two agents when administered in monotherapy only reduced CCI-induced behavioural and biochemical deficits significantly but the combination therapy appreciably reversed the functional, behavioural and biochemical deficits in CCI rats when compared to the monotherapy (Figs. 1 and 2). Combination therapy improved the hind limb function which may be due to the inhibition of CCI-induced axon degeneration (Fig. 1A). Combination therapy also attenuated the behavioural changes including hyperalgesia, allodynia and postural defects. The possible mechanism responsible for protection against neuropathic pain might be the inhibition of related pathomechanisms induced by oxidative/ nitrosative stress and PARP overactivation. Neuroprotective effect of these antioxidants and PARP inhibitors has been well demonstrated to reverse the functional and behavioural alterations in experimental models of neuropathy (Komirishetty et al., 2016a; Negi et al., 2010a;). In the present study, we also evaluated the antioxidant effect of treatment by measuring biochemical parameters and protein expression studies. Combination therapy markedly attenuated the oxidant-induced lipid peroxidation and nitrite levels in the nerve (Fig. 2B). It also restored the levels of the glutathione levels in the sciatic nerve homogenates in CCI group (Fig. 3D). The treatment reduced the free radical induced DNA damage and reduced the expression of nitrosylated proteins in ipsilateral sciatic nerve and DRG (Fig. 2A). These findings indicate the potential of antioxidant effect of quercetin and 4-ANI as a combination. Earlier studies also proved the potential of quercetin against oxidative and nitrosative

stress in the diseased neuronal tissues (Dajas et al., 2015; Shi et al., 2013). Our findings on abrogation of expression of nitrosylated proteins and reduced lipid peroxidation by PARP inhibition are also in line with the previous studies (Arora et al., 2008; Negi et al., 2011). The combination treatment also improved the Nrf2/HO-1 levels in sciatic nerve and DRG in CCI rats (Fig. 3A and B). We have also evaluated the effect of treatment on CCI-induced PARP overactivation. The levels of NAD and ATP were partially improved by the monotherapy whereas the combination therapy notably restored their levels indicating the effective PARP inhibition and antioxidant activity (Fig. 4C and D). The potent PARP inhibitory action of combination therapy has been well explained by the reduced levels of PAR in sciatic nerve and DRG in CCI rats (Fig. 4A and B). Recent studies have shown that the PARP overactivation directly inhibit electron transport chain complex IV (cytochrome c oxidase) and poly-ADP-ribosylation of mitochondrial proteins may play a significant role in mitochondrial dysfunction and subsequent neuronal death (Lai et al., 2008a; Lim et al., 2015). The mounting evidence suggests that traumatic nerve injury is associated with bioenergetic and mitochondrial dysfunction due to repression of ATP synthase and reduced activity of electron transport chain activity (Areti et al., 2015; Lim et al., 2015). Therefore, we studied the effect of treatment on sciatic nerve mitochondrial functions i.e. mitochondrial membrane potential which indicates the extent of mitochondrial dysfunction and cytochrome c oxidase enzyme activity which is one of the target proteins of overactivated PARP. The monotherapy reversed the changes but the combination therapy normalized mitochondrial function comparable to normal sham

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Fig. 6. Schematic representation of neuroprotective mechanisms involved in the concurrent targeting of oxidative/nitrosative stress-PARP cascades in CCI-induced neuropathic pain.

control animals (Fig. 5A and B). Following a similar trend the combination therapy also restored ATP levels which hind towards effectiveness of the combination strategy on mitochondrial function (Fig. 4D). PARP-induced inflammatory cascades also contribute to the pathogenesis of peripheral neuropathy (Drel et al., 2010; Hassa and Hottiger, 2002). Especially, the neuroinflammation aggravated through NF-kB pathway lead to the expression of proinflammatory cytokines (IL-6 and TNF-a) and other enzymes (iNOS and COX-2) (Tak and Firestein, 2001). Hence, the effect of combination therapy on these inflammatory contributors has been evaluated in the sciatic nerve of CCI rats. Combination therapy significantly reduced the CCI-induced levels of TNF-a & IL-6 (Fig. 5C and D) which points towards its anti-inflammatory actions in addition to its antioxidant capabilities. The expression levels of NF-kB, iNOS and COX-2 also has been reversed notably by combination regimen in CCI rats (Fig. 5E). These findings further support the claim of antiinflammatory potential of combination therapy. 5. Conclusions In summary, the present experiments demonstrated that CCIinduced oxidative/nitrosative stress play a crucial role in the pathogenesis of neuropathy. The functional, behavioural and biochemical deficits were due to oxidant-induced damage, neuroinflammation and bioenergetic failure. These pathological consequences of nerve injury have been attenuated by the combination of quercetin and 4-ANI (Fig. 6). Our findings suggest that enhanced neuroprotective effects of combination therapy may be attributable to simultaneous inhibition of oxidative/nitrosative stress, PARP overactivation and neuroinflammation. Hence, the combination therapy could be suggested as a potent strategy that can be further pursued for the management of trauma-induced peripheral neuropathy and neuropathic pain. Disclosure Supported by National Institute of Pharmaceutical Education

and Research, Hyderabad and Department of Biotechnology Government of India, via grant BT/527/NE/TBP/2013. Conflicts of interest The authors also declare no conflicts of interest. Acknowledgement Authors acknowledge the financial support from Department of Pharmaceuticals, Ministry of chemical and fertilizers and NIPER Hyderabad for their support to carry out the study. Authors would also like to acknowledge DBT funded NE intertwine project (MED/ 2013/192) to Dr Ashutosh Kumar and Dr Ranadeep Gogoi. References Areti, A., Ganesh, Y.V., Komirishetty, P., Kumar, A., 2015. Potential therapeutic benefits of maintaining mitochondrial health in peripheral neuropathies. Curr. neuropharmacol. 14 (6), 593e609. Arora, M., Kumar, A., Kaundal, R.K., Sharma, S.S., 2008. Amelioration of neurological and biochemical deficits by peroxynitrite decomposition catalysts in experimental diabetic neuropathy. Eur. J. Pharmacol. 596, 77e83. Azevedo, M.I., Pereira, A.F., Nogueira, R.B., Rolim, F.E., Brito, G.A.C., Wong, D.V.T., Lima-Janior, R.C.P., de Albuquerque Ribeiro, R., Vale, M.L., 2013. The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy. Mol. pain 9, 1. Bain, J.R., Mackinnon, S.E., Hunter, D.A., 1989. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plastic Reconstr. Surg. 83, 129e136. Bardin, L., Malfetes, N., Newman-Tancredi, A., Depoortere, R., 2009. Chronic restraint stress induces mechanical and cold allodynia, and enhances inflammatory pain in rat: relevance to human stress-associated painful pathologies. Behav. brain Res. 205, 360e366. Bennett, G.J., Xie, Y.K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87e107. Brooks, J., Tracey, I., 2005. Review: from nociception to pain perception: imaging the spinal and supraspinal pathways. J. Anat. 207, 19e33. Cevik, O., Erosahin, M., sener, T.E., Tinay, I., Tarcan, T., setinel, s., sener, A., Toklu, H.Z., sener, G., 2013. Beneficial effects of quercetin on rat urinary bladder after spinal cord injury. J. Surg. Res. 183, 695e703. Chong, M.S., Bajwa, Z.H., 2003. Diagnosis and treatment of neuropathic pain. J. pain symptom Manag. 25, S4eS11. Cornelius, C., Crupi, R., Calabrese, V., Graziano, A., Milone, P., Pennisi, G., Radak, Z., Calabrese, E.J., Cuzzocrea, S., 2013. Traumatic brain injury: oxidative stress and

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