ECN from Tussilago Farfara Attenuates Neuropathic Pain by Suppressing Oxidative Stress, Inflammatory and Pro-Apoptotic Proteins Expression in Surgical Model

7β-(3-ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid obtained from a natural origin (Tussilago farfara)has proved to be effective in minimizing various side effects associated with opioids and nonsteroidal anti-inflammatory drugs. The current study focused on investigating the effects of ECN on neuropathic pain induced by partial sciatic nerve ligation (PSNL) by mainly focusing on oxidative stress, inflammatory and apoptotic proteins expression in mice. Neuropathic pain was induced in mice by PSNL surgery performed on day 1 and ECN (1 and 10 mg/kg, i.p.), was administered once daily for 11 days, starting from the third day after surgery. ECN post-treatment was found to reduce hyperalgesia and allodynia in a dose dependent manner. ECN significantly reversed the severity of neuropathic pain by improving distress symptoms and survival rate. ECN remarkably reversed the histopathological abnormalities associated with oxidative stress, apoptosis and inflammation. Furthermore, ECN prevented the suppression of antioxidants (glutathione, glutathione-Stransferase, catalase, superoxide dismutase, NF-E2-related factor-2 (Nrf2), hemeoxygenase-1 and NAD(P)H: quinone oxidoreductase) by PSNL. Moreover, pro-inflammatory cytokines (tumor necrotic factor alpha, interleukin 1 beta, interleukin 6, cyclooxygenase-2 and inducible nitric oxide synthase) expression was reduced by ECN administration. Treatment with ECN was successful in reducing caspase-3 level consistent with the observed modulation of pro-apoptotic proteins. Additionally, ECN showed protective effect on the lipid content of myelin sheath as evident from FTIR spectroscopy which showed the shift of lipid component bands to higher values. Thus, antineuropathic potential of ECN might be due to inhibition of oxidative stress, inflammatory mediators and pro-apoptotic proteins.


Introduction
Neuropathic pain can result from the direct injury to various peripheral nerves via peripheral and central sensitization mechanisms as the pain stimuli are constantly produced which leads to impaired pain processing [1]. Neuropathic pain can arise from traumatic spinal cord injury, multiple sclerosis, stroke, persistent diabetes, lumbar disc syndrome, herpes infection, cancer and AIDS [2,3]. Neuropathic pain is characterized by burning and stabbing pain due to hyperactivity of nerve fibres leading to hyperalgesia (increased pain sensitivity) and allodynia (pain resulting from non-painful stimuli) [4,5]. Different experimental models of neuropathic pain have been developed to assess the analgesic potentials of newer pharmacotherapies [6]. PSNL is the most commonly used model of peripheral neuropathy [7]. PSNL model produces unilateral peripheral mononeurotherapy as observed in humans that can be modelled for causalgia (incessant burning pain) and regional pain syndrome in rodents [8].
Oxidative stress and inflammation plays a key role in the initiation and maintenance of neuropathic pain [9]. Oxidative stress leads to the activation of reactive oxygen species which as a result amplifies the pathogenesis of neuropathic pain [10]. It has been reported that underlying cause of oxidative stress mediated neuropathic pain is the under-expression of Nrf2, a transcription factor involved in regulation of genes that encodes antioxidant proteins and phase 2 detoxifying enzymes [11]. Nrf2 binding with the antioxidant responsive element (ARE) leads to the transcriptional activation of downstream genes such as NAD(P)H: quinone oxidoreductase (NQO1), glutathione-Stransferase (GST) and hemeoxygenase-1 (HO-1) [11]. Moreover, several studies have shown that overexpression of Nrf2 produces protective effect against oxidative stress [9]. Recently, the activation of inflammatory markers and apoptotic pathways in the pathogenesis of neuropathic pain have been demonstrated [12]. Expression of inflammatory mediators such as tumor necrotic factor alpha (TNFα), interleukin 1 beta (1L-1β), interleukin 6 (1L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) while pro-apoptotic protein such as (caspase 3) contributes in the induction and maintenance of neuropathic pain [13,14].
Phytochemicals have proved to be an effective Nrf2 activators inducing antioxidant proteins and phase 2 detoxifying enzymes [15]. Various studies have concluded that terpenoids possess neuroprotective effects [16]. A previous study revealed that a sesquiterpenoid, ECN isolated from Tussilago farfara exhibited remarkable neuroprotective activities [15]. The aim of present study was to assess the Nrf2 mediated anti-neuropathic activities of ECN against expression of oxidative stress proteins, inflammatory markers (COX-2, iNOS, TNF-α, 1L-1β, 1L-6) and pro-apoptotic protein (caspase-3) in PSNL-induced neuropathic pain in mice.

Effect of ECN on distress symptoms and survival rate
The highest mortality rate was observed in PSNL treated mice as compared to the normal control (P < 0.05). The ECN treatment groups displayed higher survival rate as compared to the PSNL treated group (P < 0.001) ( Figure 1A). The PSNL treated group showed highest distress score as compared to the normal group (P < 0.05). The ECN treatment groups displayed reduced distress scores as compared to the PSNL treated group (P < 0.001) ( Figure 1B). In brief, ECN groups showed reduced mortality rates and distress symptoms in comparison with PSNL treated group. ) treatment on distress symptoms. Total score of distress symptoms (dull/ruffled coat, change in temperament, reluctance to move) were recorded daily. Each criteria was scored from (0-3). Data shown is mean ± S.D. from 5 mice. (*) P < 0.05, ( * * ) P < 0.01, and ( * * * ) P < 0.001 indicates significant differences from the PSNL control group). (###) means comparison of normal group and PSNL control group with treatment groups.

Effect of ECN on neuropathic mechanical hyperalgesia, thermal hyperalgesia, mechanical allodynia and cold allodynia
In PSNL-induced hyperalgesia, post-treatment with ECN (10 mg/kg) significantly inhibited mechanical hyperalgesia on day 3 in a dose dependent manner as compared to PSNL control ( Figure  2A). Further evaluation of anti-inflammatory activity of drug was carried out by evaluating thermal hyperalgesia. Three days after injury, thermal hyperalgesia was significantly reduced in treatment groups as compared to PSNL-treated group. Post-treatment with ECN (10 mg/kg) significantly reduced the hyperalgesic responses such as paw licking and jumping ( Figure 2B). Seven days after injury, thermal and mechanical hyperalgesia were still present in neuropathic mice, whereas ECN and positive control reversed the nociceptive hypersensitivity. In PSNL-induced mechanical allodynia, post-treatment with ECN (10 mg/kg) significantly reduced mechanical allodynia as compared to PSNL-treated group ( Figure 2C). In PSNL-induced cold allodynia, post-treatment with ECN (10 mg/kg) inhibited cold allodynia as compared to PSNL-treated group ( Figure 2D). In brief, ECN reduced the nociceptive hypersensitivity because of the neuropathic lesion in a time and dosedependent manner.

Effect of ECN on neuropathic muscle and motor coordination
Weight lifting and inverted screen tests showed that ECN-treated groups did not produced any abnormal effect on muscle and motor function. There was no noticeable deficit in muscle as well as motor coordination (Figure 3). The effect on muscle coordination and motor coordination was measured at 0, 3, 7, 10 and 14 day after PSNL. The data were reported as the means S.D; (n=5 mice per group) of scoring average. The data was reported as the means ± S.D. (*) P < 0.05, (**) P < 0.01 and (***) P < 0.001 indicate significant differences from the PSNL control group. (###) means comparison of normal group and PSNL control group with treatment groups.

Effect of ECN on DNA disruption
PSNL control group showed augmentation of DNA damage in sciatic nerve and spinal cord tissue. Administration of ECN (10 mg/kg) significantly (P < 0.001) reduced DNA damage with a decrease in tail length and percentage DNA in tail as compared to the PSNL challenged group (Figure 4). and ***P < 0.001 represents a statistically significant variation from PSNL-induced group. (###) denotes comparison of normal control group and PSNL-induced group with ECN treated group. .

Effect of ECN on histological examination
H&E staining analysis of the sciatic nerve section showed that ECN (10 mg/kg) reduced the swelling of nerve fiber, degenerated myelin sheath, hypertrophy of satellite cells and necrosis at day 7 and 14 post PSNL surgery when observed under light microscope ( Figure 5). ECN treated groups showed reduction in myelin swelling, cell infiltration and necrosis of lumbar spinal cord section at day 7 and 14 post PSNL surgery in contrast to PSNL control when observed under light microscope ( Figure 6). Histopathological analysis of the coronal section of brain showed significant (P < 0.001) increase in thickness of dentate gyrus in treatment groups when quantified by Image J tool ( Figure  7).

Effect of ECN on inflammatory cytokines expression
The effect of ECN (10 mg/kg) was also investigated against various pro-inflammatory cytokines such as TNFα, IL-1β, IL-6, COX-2 and iNOS in the spinal cord and sciatic nerve tissues using quantitative RT-PCR. Neuropathic pain strongly induced expression of TNF-α, IL-1β, IL-6, COX-2 and iNOS, the treatment with ECN significantly (P < 0.001) decreased the mRNA expression levels of these inflammatory cytokines as compared to the PSNL-treated group ( Figure 8).

Effect of ECN on anti-oxidants expression
The mRNA expression levels of anti-oxidant enzymes and proteins such as Nrf2, HO-1 and NQO1 in spinal cord and sciatic nerve of mice were determined using quantitative RT-PCR. Oxidative stress leads to the activation of reactive oxygen species which as a result amplifies the pathogenesis of neuropathic pain. The present results showed that there was a significant (P < 0.001) up-regulation of Nrf2, HO-1 and NQO1 enzyme due to ECN and pregabalin treatment as compared to the PSNL-treated group ( Figure 9).

Effect of ECN on MDA production.
The MDA level in sciatic nerve, spinal cord and brain (prefrontal cortex and hippocampus) was determined on 7, 10 and 14 day after PSNL surgery. Upregulation of oxidative stress markers after nerve injury plays a major role in promoting pathogenesis of neuropathic pain. Treatment with ECN and pregabalin significantly (P < 0.001) decreased the MDA level as compared to PSNL control group ( Figure

Effect of ECN on NO production
Nitrite level was determined in brain (hippocampus, prefrontal cortex), spinal cord and sciatic nerve tissue on 7, 10 and 14 day post PSNL surgery ( Figure 11). Excess reactive oxygen species leads to the increased production of NO which further worsens the neuropathic pain. Treatment with ECN significantly (P < 0.001) reduced the PSNL induced nitrite production, hence this confirmed the antiinflammatory effect of drug.

Effect of ECN on SOD .
SOD level was determined in brain (hippocampus, prefrontal cortex), spinal cord and sciatic nerve tissue on 7, 10 and 14 day post PSNL surgery ( Figure 12). Increased expression of antioxidant proteins and enzymes can treat the neuropathic pain induced by oxidative stress. Treatment with ECN and pregabalin significantly (P < 0.001) increased the level of SOD antioxidant enzyme, therefore this confirmed the antioxidant effect of ECN.

Effect of ECN on GST.
The GST level in sciatic nerve, spinal cord and brain (prefrontal cortex and hippocampus) was determined on 7, 10 and 14 day after PSNL surgery ( Figure 14). Neuropathic pain cause significant increase in free radicals which as a result amplifies the pathogenesis of neuropathic pain. Treatment with ECN and pregabalin significantly (P < 0.001) increased the level of GST antioxidant enzyme as compared to the PSNL control group.

Effect of ECN on Catalase.
The catalase level in brain (prefrontal cortex and hippocampus), spinal cord and sciatic nerve was determined on 7, 10 and 14 day after PSNL surgery ( Figure 15). Increased expression of antioxidant proteins and enzymes can treat the neuropathic pain induced by oxidative stress. Treatment with ECN and pregabalin significantly (P < 0.001) increased the level of catalase as compared to the PSNL control group.

Effect of ECN on Kidney and Liver Functions
The side effect profile of drug on liver and kidney was evaluated through measurement of AST, ALT and creatinine concentrations in blood plasma. Daily treatment with ECN starting from day 3 did not changed plasma levels of AST and ALT and creatinine as compared to the PSNL control (Table 1). Values are expressed as the mean S.D (n= 5 mice/ group); a P 0.001 compared with the naive group; b P 0.001 compared with the PSNL control group.

Effect of ECN on Nrf2 and caspase-3 expression
Modulatory action of ECN was further strengthened by assessing the expressions of caspase-3 and Nrf2 by using immunohistochemistry. Caspase-3, a pro-apoptotic protein plays a vital role in the pathogenesis of neuropathic pain. The present result showed that ECN post treatment significantly (P < 0.001) decreased the immune-labelling of caspase-3 as compared to the PSNL control ( Figure 16A and 16C). The underlying cause of oxidative stress mediated neuropathic pain is the under-expression of Nrf2, a transcription factor involved in regulation of genes that encodes antioxidant proteins and phase 2 detoxifying enzymes. ECN post treatment significantly (P < 0.001) upregulated the expression of Nrf2 as compared to the PSNL control when observed under light microscope ( Figure 16B and 16 C).

Effect of ECN on myelin sheath of sciatic nerve
ECN exhibited protective effect on the lipid content of myelin sheath which shows its potential to treat nerve damage or neuropathy ( Figure 17). The change in unsaturated fatty acid lipid content is determined from the analysis of the olefinic band, arisen from HC=CH groups [17]. The wavenumber of this band was shifted significantly (P < 0.001) to higher value in the ECN treated group as compared to the PSNL control group ( Table 2). The CH2 antisymmetric (2927 cm -1 ), and the CH2 symmetric (2857 cm -1 ) stretching bands originates mainly from lipids. The CH3 antisymmetric (2959 cm -1 ) has equal contribution from lipids and proteins (Table 3) [18][19][20][21]. The wavenumber of these bands in ECN group was shifted significantly (P < 0.001) to higher value as compared to the PSNL control group (Table 2).

Discussion
Chronic pain initiated by a primary lesion or dysfunction in the nervous system and leads to prolonged changes in pain pathway structures (neuroplasticity) and abnormal processing of sensory information [22]. Chronic pain is a big challenge to the healthcare scientist [23]. PSNL model produces unilateral peripheral mononeurotherapy as observed in humans that can be modelled for causalgia (incessant burning pain) and regional pain syndrome in rodents. Numerous drugs are already in the market for the treatment of neuropathic pain [22,24]. The drugs for the treatment of neuropathic pain exhibited severe and serious side effects [22]. Therefore, there is a need to seek out new drugs with more safety and more efficacious profile.
In the present study an attempt was made to explore the potential effects of naturally isolated ECN in PSNL-induced model of neuropathic pain. PSNL model has been used clinically for the treatment of neuropathic pain [25]. Intraperitoneal administration of ECN (10 mg/kg) post-treatment once daily starting from day 3 after surgery, significantly attenuated mechanical hyperalgesia, thermal hyperalgesia, mechanical allodynia and cold allodynia. Decreased score of distress symptoms such as general health, changes in temperament, gait weakness and reluctance to move were observed in the treatment group as compared to the PSNL control group. High survival rate were seen in the treatment group as compared to the PSNL control group. In addition ECN (10 mg/kg) intraperitoneal administration from day 3 after surgery reversed histopathological abnormalities of PSNL-induced neuropathic pain. Our findings suggest that ECN had no disturbing effect on the muscle coordination and motor activity.
Earlier studies have reported the activation of pro-inflammatory cytokines and apoptotic markers in the induction and maintenance of neuropathic pain [26,27]. Upregulation of inflammation and apoptotic proteins after injury plays a crucial role in aggravating the neuropathic pain [28,29]. Previous findings reported that the up-regulation of inflammatory and apoptotic markers after nerve injury plays a major role in promoting pathogenesis of neuropathic pain [16,30]. In pathological conditions, excess reactive oxygen species and apoptosis leads to the increased production of proinflammatory cytokines which further worsens the neuropathic pain [31]. In the present study we found that pro-inflammatory (TNF-α, IL-1β and IL-6) and pro-apoptotic (caspase-3) proteins were elevated in the spinal cord and sciatic nerve of mice on day 7 and 14 after PSNL surgery. Treatment with ECN decreased the TNF-α, IL-1β, IL-6 and caspase 3 expression in contrast to PSNL group.
Recent findings suggests that iNOS releases NO and subsequently peroxynitrite which could lead to the production of pro-inflammatory cytokines and could participate in neuropathic pain [32]. Moreover, it has been demonstrated that increased production of iNOS and TNF-α could be responsible for the development of neuropathic pain [30]. In pathological conditions, large amounts of pro-inflammatory cytokines such as iNOS and TNF-α are released which further causes the production of reactive oxygen species (ROS) [12]. In the current study, ECN showed antiinflammatory effect by inhibiting the expression of iNOS and TNF-α.
Nrf2, one of the basic leucine zipper transcription factor that can bind to ARE-sequence has been shown to be important for protection against oxidative stress [33]. Many chronic neurological disorders such as multiple sclerosis and neuropathic pain are thought to involve oxidative stress as a factor contributing to the progression of disease [34]. Earlier studies reported that activation of Nrf2 pathway might be a contributing factor for the treatment of neuropathic pain [9]. It has been found that Nrf2 gene expression is vital for the maintenance and responsiveness of cell's defence system [12]. In the current study, we found that increased Nrf2 gene expression can treat the neuropathic pain induced by oxidative stress. Treatment with ECN increased the expression of Nrf2 and its downstream proteins such as HO-1 and NQO1 in treatment groups as compared to the PSNL group.
Previous studies have found that activation of apoptosis might be involved in the pathogenesis of neuropathic pain [12]. Apoptotic pathways leads to the activation of caspase-3 which leads to cell death [35]. Caspase-3, a pro-apoptotic protein plays a vital role in the pathogenesis of neuropathic pain [36]. Moreover it has been demonstrated that inhibition of caspase 3 would inhibit apoptosis and thermal hyperalgesia following chronic constriction injury [37]. In this study, caspase-3 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 18 August 2020 doi:10.20944/preprints202008.0389.v1 expression in the treatment group was reduced as compared to the PSNL control group. This shows the protective effect of ECN in neuropathic pain. Quantitative DNA damage in sciatic and spinal cord tissues were analysed with the help of comet assay. DNA disruption was identified by DNA relocation out of the nucleus and into the tail of comet. DNA strand breakage can be due to the enhanced generation of ROS leading to free radical formation which might be the reason for the alteration and breakage of double helical strands causing cell death [38]. This DNA disruption was noticeably attenuated by ECN representing its neuroprotective potential. Moreover, coronal section of the brain tissue, longitudinal section of the sciatic nerve and transverse section of the lumbar spinal cord were stained with H&E and then observed under microscope. It was found that dentate gyrus of hippocampus in PSNL-challenged group was reduced in thickness as compared to the treatment group. In the same way increased signs inflammation was observed in PSNL-induced sciatic nerve and lumbar spinal cord demonstrating debilitating condition of PSNL control group. Although, such pattern was not seen in treatment group. This shows the efficient potential of ECN in treating peripheral neuropathy.
To elucidate the lipid content of myelin sheath of sciatic nerve in PSNL model, FTIR spectroscopy was used in this study since it enables efficient, rapid and simultaneous monitoring of small changes in biochemical components and processes in diseases or drug-induced pathological conditions [17,20]. Damage to myelin sheath via alteration in the biochemical make-up of the sciatic nerve tissue contributes to neuropathy. ECN exhibited protective effect on the lipid content of myelin sheath which shows its potential to treat nerve damage or neuropathy. The change in unsaturated fatty acid lipid content is determined from the analysis of the olefinic band, arisen from HC=CH groups. In the current study, the wavenumber of this band was shifted significantly to higher value in ECN treated groups as compared to the PSNL control group.

Plant material
ECN was isolated from dried buds of T. farfara (mentioned compound was received from Prof. Yeong Shik Kim, Seoul National University, South Korea) and identified by comparison with spectral data ( 1 H NMR and 13 C NMR), as previously reported in the literature [39]. The extraction and purification of ECN was reported by our team elsewhere [39].

Chemicals and reagents
Pregabalin was purchased from Sigma-Aldrich, Germany. Ketamine (Ketarol, 500 mg/10ml) was purchased from Global Pharmaceutical, Islamabad, Pakistan. Pyodine was purchased from Brookes Pharma, Karachi, Pakistan. All the other chemicals used in the current study were of analytical grade. All the drugs described above were freshly prepared for the study, dissolved in 2% DMSO and diluted with 0.9% saline. Peripheral neuropathic pain was induced in mice by PSNL as previously described [16]. Briefly, mice were deeply anesthetized with ketamine (100 mg/kg i.p.), the left sciatic nerve was exposed after the incision of skin and the exposed skin was swabbed with a povidone iodine topical 10% w/v solution. The sciatic nerve was freed of the adhering tissue gently for about 7 mm, and four ligatures (chromic catgut 4.0) were loosely tied around approximately 1/3 and 1/2 diameter of the sciatic nerve. After performing partial nerve ligation, muscle and skin layer were at once sutured with thread, and topical antibiotic was applied. In sham-operated mice, an identical operation was performed, except that the sciatic nerve was not ligated. All surgical procedures were conducted under normal sterile conditions.

Drug treatment and PSNL model
Mice were randomly divided into 6 groups (n=10/group). The sample size was selected according to the previously established protocol [40]. Optimal administration doses were selected according to the results of the preliminary experiments. ECN (1 and 10 mg/kg) was administered i.p. to neuropathic mice once a day for 11 days, starting from the third day after surgery (Figure 18). Positive control (pregabalin 5 mg/kg) was also administered i.p. to neuropathic mice once a day for 11 days, starting from the third day after surgery. No treatment was given to normal, sham and PSNL control groups. 3 to 4 mice from each group were sacrificed on day 7, 10 and 14 after PSNL surgery for biochemical evaluations (Figure 18).

Behavioural experiments
The experimental animals were subjected for various behavioural studies for investigation of mechanical hyperalgesia, thermal hyperalgesia, mechanical allodynia, cold allodynia and muscle coordination carried out on the different time intervals of 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.

Distress symptoms and survival rate
The number of mice surviving in each group and mortality rates were recorded daily as previously described [41,42]. Animals were observed throughout the experiment to record distress symptoms. Distress symptoms included general health, changes in temperament, gait weakness and reluctance to move. Scoring was carried out according to criteria mentioned previously [41]. The number of mice surviving in each group and mortality rates were recorded daily.

Mechanical hyperalgesia
In order to monitor the mechanical hyperalgesia, Randall Sellito (IITC Life Science Inc. CA, USA) was used as per method previously described with slight modifications [43,44]. Before the start of the test, the mice were placed in a quiet room for 15-30 min to acclimatise with the environment. This test consists of evoking a hind paw flexion reflex with a hand hold force transducer and the force exerted is noted on the screen. The tip of the Randall Sellito was applied perpendicular to the plantar surface of the ipsilateral left hind paw and the pressure was increased gradually. The final reading of the Randall Sellito is the characteristic withdrawal of the paw and clear movement of the mice. The duration of paw withdrawal (PWD) was recorded with a cut-off latency of 15 s [45]. Mechanical hyperalgesia was evaluated before and after the initiation of the treatment. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.

Thermal hyperalgesia
Thermal hyperalgesia of the plantar surface of the ipsilateral left hind paw was assessed as described previously [46,47]. The temperature of the hot plate was kept at 50 ± 0.5°C. Once the animals were placed on the top of preheated hot plate, the paw licking was taken as a positive response. The paw withdrawal latency (PWL) and PWD were recorded with a cut-off latency of 35 s. Thermal hyperalgesia was evaluated before and after the initiation of the treatment. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.

Mechanical allodynia
In order to monitor the mechanical allodynia, a series of 9 von Frey filaments (0.4, 0.6, 1, 1.4, 1.8, 2, 4, 6 and 8 g) were used as per method previously described with slight modifications [48,49]. The filaments were applied to the plantar surface of the ipsilateral left hind paw and the applied force was increased gradually. Lifting or licking the paw was considered as a withdrawal response and the time taken to show a withdrawal response (PWL) three out of five times was considered positive. A cut-off latency of 15 s was imposed [45]. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.

Cold allodynia
In order to assess cold allodynia, acetone drop method was employed as per method previously described with slight modifications [50,51]. A 25 µ l volume of acetone was sprayed onto the midplantar surface of the ipsilateral left hind paw, using a blunt needle connected to a syringe without touching the paw. The duration of the withdrawal response (PWD) during 60 s of the acetone application to the plantar surface of the ipsilateral left hind paw was recorded. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery. In order to determine the sedative effect of ECN on muscle strength and motor co-ordination, Kodzeila's inverted screen was performed as described previously [52,53]. Each mouse was placed in the center of wire mesh screen and screen was inverted over 2 s, with a mouse's head declining first. Investigator was trained to hold the screen steadily 40-50 cm above a padded surface. Time was recorded using digital stop watch when the mouse fall off and score was assigned according to the protocol [54]. A cut-off latency of 60 s was imposed [55]. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.
For full assessment of motor deficit, weight lifting test was performed as described previously [56]. Each weight was prepared using a thin wire mesh to which a length of steel chain consisting of from 1 to 7 links, each weighing 13 g, was attached. Each mouse was held by the tail and successively allowed to grasp a series of increasing weights, with a rest of 10 s between each lift. A hold of 3 s is the criterion. Time was recorded using digital stop watch when the mice dropped the weight in less than 3 s. If it held the weight for 3 s then it was allowed to grasp the next heavier weight. Score was assigned to each mouse. Three consecutive readings were taken for statistical analysis. Test was performed 1 day before PSNL surgery and 3, 7, 10 and 14 days post PSNL surgery.

Biochemical experiments
4.6.1. Comet assay DNA damage in sciatic nerve and spinal cord tissues were assessed following 14 days post PSNL surgery by comet assay as described previously [38,57,58]. Small piece of sciatic nerve and spinal cord collected from mice after sacrifice were suspended separately in 1 ml of cold lysing solution i.e., Ca 2+ and Mg 2+ free Hanks' balanced salt solution (HBSS) in a micro centrifuge tube and then the tissues were homogenized separately. About 5-10 μl of the cell suspension was mixed in 0.5% low melting point agarose (LMPA), layered on the slides precoated with 1% normal agarose solution (NMA) and then covered with cover slip, retained for a duration of 10 min on an ice pack. After repeating this step twice, the slides were placed in lysing solution for 2 h at 4 °C. After electrophoresis slides were stained with 1% ethidium bromide and were examined under fluorescent microscope. The amount of DNA damage was analysed by CASP 1.2.3.b software. The tail length and % DNA in the tail was used to assess the amount of DNA damage.

Histopathological analysis
The Hematoxylin and Eosin (H&E) staining of sciatic nerve, lumbar spinal cord and brain tissues were performed following 7 and 14 days post PSNL surgery. The collected sciatic nerve, lumbar spinal cord and brain were fixed with 10% formalin solution, mounted in paraffin blocks, cut into 4 μm sections and observed under microscope (100X).

Quantitative real-time reverse transcriptase polymerase chain reaction
The quantitative RT-PCR was used to determine the effect of ECN (10 mg/kg) on the mRNA expression levels of inflammatory mediators (COX-2, iNOS, TNFα, IL-1β and IL-6) and anti-oxidant proteins (Nrf2, HO-1 and NQO1) following 7 and 14 days post PSNL surgery. Trizol Reagent was used to isolate the total RNA from the sciatic nerve and lumbar dorsal spinal cord (L4-L6) tissue of mice as described previously [46,59,60]. Briefly, GenDEPOT 0.2mL 8-strip tubes were used for quantitative PCR. 10ul each of forward primer and reverse primer along with 80ul DEPC-treated water (Sigma-Aldrich) and the fluorescent dye, SYBR green working solution was used. The reaction conditions were as follows 95°C for 5 min followed by 40 cycles at 95°C for 1 min (denaturation), then 55°C for 45 s, and lastly 72°C for 30 s (annealing and elongation). The optimal conditions, melting point, and the reaction specificity were determined beforehand. 7300 real-time PCR system software was used for analysis. Beta actin, a housekeeping gene, was chosen as an internal standard.

Determination of malondialdehyde (MDA)
To investigate the effect of ECN (10 mg/kg. i.p.) on oxidative stress marker, MDA level was quantified in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery as described previously [61,62]. The concentration of MDA, a marker of lipid peroxidation was analysed in the form of thiobarbituric acid reacting proteins (TBARS). 4.6.5. Determination of nitric oxide (NO) NO level in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery were determined by Griess assay according to the method previously described [46,63]. The concentration of NO was determined by using Griess reagent, 1% sulfanilamide and 2.5% phosphoric acid. The concentration of nitrite was determined by measuring absorbance at 540 nm. 4.6.6. Determination of superoxide dismutase (SOD) To investigate the effect of ECN (10 mg/kg. i.p.) on anti-oxidant marker, SOD level was quantified in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery according to the method previously described [33]. The SOD activity was measured by taking Tris-EDTA buffer (50 Mm, pH 8.5), pyrogallol (24 mM) and 10 µ l of sample in a total volume of 0.2 ml. The final readings were noted in triplicate at 420 nm.

Determination of glutathione (GSH)
GSH level was quantified in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery as described previously [38]. The reduced glutathione level was measured by mixing 0.1 ml of tissue homogenate obtained from each dilution and 2.4 ml from phosphate buffer solution. The final volume was adjusted to 3 ml by adding 0.5 ml DTNB solution. The absorbance was measured at 412 nm.

Determination of GST
GST level was quantified in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery as described previously [43]. The enzyme was quantified by its ability to conjugate GSH and CDNB. The level of GST was determined by mixing 0.1 ml of homogenate and 0.1 ml of CDNB. Finally 0.1 M phosphate (pH 6.5) buffer was added to make the final volume up to 3 ml. The absorbance was measured at 314 nm.

Determination of catalase (CAT)
Catalase level was quantitated in sciatic nerve, lumbar dorsal spinal cord (L4-L6), hippocampus and prefrontal cortex tissues of day 7, 10 and 14 post PSNL surgery according to previously described methodology [61]. The catalase activity was determined by taking 3 ml of H2O2-phospahte buffer in an experimental cuvette and by the rapid addition of 40 µ l of an enzyme extract. The final readings were noted in triplicate at 240 nm.

Analysis of renal and hepatotoxicity
The systemic toxicity of ECN (10 mg/kg, i.p) in blood was assessed by measuring the levels of alanine amino transferase (AST), aspartate amino transferase (ALT) and creatinine. The whole blood obtained on 14 days after PSNL surgery was centrifuged at 5000 rpm for 5 min to separate the serum from blood cells and the temperature was maintained at 4°C . The serum was used to determine the level of ALT, AST and creatinine. 4.6.11. Immunohistochemistry Immunohistochemical analysis of Nrf2 and caspase-3 were performed following 14 days post PSNL surgery according to the method already mentioned [61]. Briefly, lumbar dorsal spinal cord (L4-L6) tissues (paraffin-fixed) were deparaffinised and rehydrated through xylene and alcohol. The intracellular peroxidase was quenched by H2O2 (3%) in menthol and then incubated for 20 min with goat serum. The slides were incubated overnight long with the mouse anti-Nrf2 and mouse anticaspase 3 antibodies. After washing, the slides were incubated in biotinylated goat-anti-mouse secondary antibodies, then incubated with ABC reagents for 1 h. Further washed with phosphate buffer saline and stained with diaminobenzidine solution. Images were taken under light microscope. The relative expression of Nrf2 and caspase-3 proteins were measured using ImageJ tool. 4.6.12. Determination of sciatic nerve structural damage The sciatic nerves were purged and placed at -80 0 C on day 14 after PSNL for the spectroscopic analysis to check lipid content of myelin sheath in PSNL model. The samples were lyophilized and were used directly in the Fourier transform infrared (FTIR) spectrometer [64]. Moreover, the instrument was constantly removed from dry air to dismiss water vapours. The infrared spectra of sciatic nerve specimens were acquired in the FTIR spectrometer IR tracer (Shimadzu, Japan). The specimens were subjected to the range of 650 to 4000 cm -1 with a resolving power of 4 cm -1 . Essential FTIR was used for digital data manipulation. For the estimation of position of bands, the wavenumber values coinciding with the mid of weight were utilized.

Statistical analysis
Results were expressed as mean ± standard deviation (S.D). Sigma plot version 12.5 (statistical software) was used for the statistical analysis. Two-way analysis of variance (ANOVA) was applied following Dunnett's t-tests for the assessment of statistical significance amongst various treated groups. For the statistical significance the value of "P" less than 0.05 was selected as criterion of significance difference.

Conclusions
The present work suggests that ECN could treat PSNL-induced neuropathic pain through alleviation of distress symptoms, biochemical alteration in sciatic nerve morphology and modulation of expression of anti-oxidant proteins, pro-apoptotic proteins and pro-inflammatory cytokines represented in Figure 19. Unlike opioids and nonsteroidal anti-inflammatory drugs, minimal side effects and improved safety profile of ECN might contribute towards attenuating pathological condition of neuropathic pain. Thus, on the basis of present findings, it is concluded that ECN could be used in the treatment of PSNL induced peripheral neuropathy. Figure 19. Proposed mechanism of anti-neuropathic effect of ECN in PSNL model.