Background: Neuropathic pain is associated with severe chronic sensory disturbances characterized by spontaneous pain, increased responsiveness to painful stimuli (hyperalgesia) and pain perceived in response to non-noxious stimuli (allodynia). Morphine is effective treatment for neuropathic pain but produces tolerance on chronic use. The present study was designed to explore the anti-nociceptive and anti-hyperalgesic effect of grape seed extract using sciatic nerve ligation-induced neuropathic pain in rats.
Methods: Chronic constructive injury (CCI) was performed under anesthesia, on one side leg exposed by making a skin incision, and chromic gut ligatures were tied loosely around the sciatic nerve at 1 mm intervals. The treatment with grape seed proanthocyanidin extract (GSPE) (100 and 200 mg/kg, p.o.) was initiated on 7th day post-surgery and continued for next 14 days. Morphine (10 mg/kg, s.c.) alone and morphine in combination with GSPE (100 mg/kg, p.o.) were administered in CCI rats for 5 days starting from 7th day. On 3rd, 7th, 14th and 21st day, behavioral parameters (mechanical allodynia and thermal hyperalgesia) were assessed. Then the animals were killed on 22nd day and biochemical parameters [reduced glutathione (GSH), lipid peroxidation (LPO), catalase, nitrite, superoxide dismutase (SOD)] were assessed.
Results: Ligation of the sciatic nerve significantly induced mechanical allodynia and thermal hyperalgesia and induces oxidative stress (increase in LPO and nitrite) and decline of anti-oxidant enzyme levels (catalase, SOD, GSH) in sciatic nerve homogenate. GSPE (100 and 200 mg/kg, p.o.) attenuated all the behavioural and biochemical parameters. Morphine also significantly reversed the symptoms of neuropathic pain but produced tolerance after 5 days. Further, co-treatment of GSPE (100 mg/kg) with morphine (10 mg/kg, s.c.) in CCI rats significantly reversed the morphine tolerance and enhanced its anti-hyperalgesic effect as compared to the morphine-alone-treated group.
Conclusions: In the present set of experiments, GSPE showed a significant anti-hyperalgesic and anti-nociceptive effect in rats.
Pain is an unpleasant sensory and emotional experience, associated with actual or potential tissue damage, or described in terms of such damage or damaging stimuli  or “the neural processes of encoding and processing noxious stimuli” . It is generally classified into two types: inflammatory pain and neuropathic pain (NP). NP is defined as “pain initiated or caused by a primary lesion or dysfunction in the nervous system”. It is associated with severe chronic sensory disturbances characterized by spontaneous pain, increased responsiveness to painful stimuli (hyperalgesia) and pain perceived in response to non-noxious stimuli (allodynia). Intensive research has been focused on the mechanisms underlying NP. Indeed, peripheral nerve injury is associated with local neurogenic inflammation characterized by increased capillary permeability that is triggered by the activation of mast cells, followed by recruitment of neutrophils and macrophages [3, 4]. Recently investigators have emphasized the role of non-neuronal cells, particularly microglia of the spinal cord in the exaggerated pain states [5–7]. Accumulation evidence indicates that reactive oxygen species (ROS) play an important role in the peripheral and central sensitization of NP . Furthermore, selective inhibition of activation and/or recruitment of inflammatory cells, pharmacological modulation of oxidative stress and expression of pro-inflammatory cytokines ameliorates hypersensitivity following nerve injury .
Current treatment status in pain management is with conventional analgesics such as opioids . Thus, opioids are centrally acting analgesic agents, which produce analgesia through μ, κ and δ opioids receptors. Numerous studies have demonstrated that NP-induced hyperalgesia leads to a decrease in anti-nociception and to the early development of morphine tolerance [10, 11]. Conventional therapies for pain such as non steroidal anti-inflammatory drugs (NSAIDs) are associated with various adverse effects, including the tolerance and sedation, and have limited their therapeutic effectiveness in pain management . These drugs, however, provide a transient relief from NP in only a fraction of patients and often produce severe central nervous system (CNS)-related, dose-limiting side effects . Thus, there is an unmet need to understand disease pathogenesis, identify and characterize novel targets, and develop newer agents which act at one or more sites in the pathogenesis of NP.
Grape seed extract (GSE) is obtained from the waste product of wine and grape juice, consists of various polyphenolic compounds such as catechin, epicatechin, gallic acid and abundant of flavonoids and polyphenols which consist of about 80%–90% of proanthocyanidins, and is known as grape seed proanthocyanidin extract (GSPE). Polyphenols are the main compounds present in GPSE which are responsible for an anti-inflammatory action. GSPE has shown to inhibit the release of various excitatory cytokines [substance P, calcitonin gene related peptide (CGRP), cytokines] which cause NP and exhibits reduction in the expression of various soluble adhesion molecules. GPSE is also responsible for increasing the levels of endogenous anti-oxidants, i.e. superoxide dismutase (SOD), catalase and reduced glutathione (GSH). In addition to their free radical scavenging and anti-oxidant activity, proanthocyanidins have been reported to have antibacterial, antiviral, anticarcinogenic, anti-allergic and vasodilatory actions .
Therefore, based on the multiple mechanisms and activity reported for GSPE, the present study was designed to investigate the anti-hyperalgesic and anti-nociceptive potentials of GSPE against chronic constructive injury (CCI)-induced NP in rats.
Materials and methods
Wistar rats of either sex (150–250 g), obtained from central animal house of ISF College of Pharmacy, Moga, Punjab (India), were used in the present study. The animals were kept in polyacrylic cages and maintained under standard housing conditions (room temperature 22±20 °C and relative humidity of 60%–65%) with 12-h light/dark reverse cycle. The food in the form of dry pellets and water were made available ad libitum. The protocol was reviewed and approved by the “Institutional Animal Ethics Committee” ISFCP/CPCSEA/M9/Approval No. 170, and the animal experiments were carried out in accordance with the Indian National Science Academy Guidelines for use and care of animals.
Drugs and chemicals
Drugs such as GPSE were obtained from Biogen Extracts Pvt. Ltd. (Bangalore, India); GPSE was freshly prepared by solubilizing in 0.5% of carboxy methyl cellulose. Unless stated, all other chemicals and biochemical reagents of highest analytical grade quality were used.
Induction of chronic constrictive injury of sciatic nerve ligation
The mono-neuropathy was produced according to the method described by Bennett and Xie . Briefly, the rats were anesthetized using thiopental sodium (40 mg/kg i.p.) and the common sciatic nerve of the left hind paw was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris muscle. Proximal to the sciatic trifurcation, approximately 7 mm of nerve was freed and 4 ligatures of 4-0 silk suture were placed around the sciatic nerve with 1-mm interval. Great care was taken not to interrupt epineural blood flow during tying the ligature. After surgery, all animals received gentamycin (5 mg/kg, i.p.) to prevent sepsis.
On day 1, ligation of the sciatic nerve was done and behavior parameters were assessed before surgery. The symptoms of NP such as hyperalgesia appeared on 3rd day onward and reached steady state between 7th and 21st day post-sciatic nerve ligation, indicating the development and maintenance of stable allodynia and hyperalgesia. On 7th day 7 onward, GSPE (100 and 200 mg/kg, p.o.) was given to different groups . Further, morphine (10 mg/kg, s.c.) was administered for 5 days to produce morphine tolerance in CCI rats. Also, the combination of GSPE (100 mg/kg, p.o.) and morphine (10 mg/kg, s.c.) was given to different groups. On 3rd, 7th, 9th, 11th, 14th and 21st day, behavioral parameters such as mechanical allodynia and thermal hyperalgesia were assessed. Then animals were killed on 22nd day and biochemical parameters such as GSH, LPO, catalase, nitrite and SOD were assessed.
Animals were randomly divided into six groups of n=6 animals in each group.
Group 1 Vehicle treated
Group 2 CCI control
Group 3 CCI+GSPE (100 mg/kg p.o.)
Group 4 CCI+GSPE (200 mg/kg p.o.)
Group 5 CCI+morphine (10 mg/kg s.c.)
Group 6 CCI+GSPE (100 mg/kg p.o.)+morphine (10 mg/kg s.c.)
Assessment of behavioral parameters
Mechanical allodynia (Von Frey test):
The mechanical threshold for touch sensitivity was measured in the hind paw, using an automated apparatus for applying a device consisting of a steel rod against the plantar region of the paw by increasing force until the animal withdrew its paw. It was done using a dynamic plantar aesthesiometer (37400-002; UgoBasile, Comerio, Italy). The maximum value of force in grams (50 g) was previously fixed. The force was set at 50 g to prevent tissue damage, and the ramp speed was 0.5 g/s with cut-off set to 20 seconds. The paw withdrawal latency (PWL) was checked [17, 18].
Hyperalgesia to thermal stimulation was determined using a plantar test apparatus (37370-002; UgoBasile, Comerio, Italy) modeled as described by Hargreaves et al. . Rats were placed individually in Plexiglas cubicles mounted on a glass surface maintained at 25±2 °C. A thermal stimulus, in the form of radiant heat emitted from a focused projection bulb, which was located under the glass floor, was focused onto the plantar surface of the right hind paw, and PWLs were recorded. A cut-off latency of 20 s was imposed to avoid tissue damage [17, 18].
Estimation of lipid peroxidation:
Briefly, 0.5 mL of supernatant containing 0.5 mL Tris HCl was incubated at 37 °C for 2 h. After incubation, 1 mL of 10% trichloroacetic acid was added and centrifuged at 10,000 g for 10 min, and from the resulting supernatant, 1 mL of it was collected which was further followed by the addition of 1 mL of 0.67% thiobarbituric acid. Lastly, the tubes were kept in boiling water for 10 min. After cooling, 1 mL of double distilled water was added and absorbance was measured at 532 nm using a spectrophotometer (UV-1700 Pharma Spec, Shimadzu, Japan). Thiobarbituric acid reactive substances (TBARS) were quantified using an extinction coefficient of 1.56×105 M−1 cm−1 and expressed as nmol of malondialdehyde (MDA) per mg protein .
Estimation of reduced glutathione:
The concentration of reduced glutathione in the sciatic nerve was determined by the Ellman method . According to this method, 1 mL of supernatant was precipitated with 1 mL of 4% sulfosalicylic acid and cold digested at 4 °C for 1 h. The sample was centrifuged at 12,000 g for 15 min at 4 °C. To 1 mL of this supernatant, 2.7 mL of phosphate buffer (0.1 m, pH 8) and 0.2 mL of 5,5-dithiobis (2-nitrobenzoic acid) were added. The yellow color developed was read immediately at 412 nm using the spectrophotometer. The results were calculated using the molar extinction coefficient of chromophore (1.36×10 4 m−1 cm−1) and expressed as percentage of control .
Estimation of nitrite concentration:
The accumulation of nitrite in the supernatant, an indicator of NO production, was determined by colorimetric assay with Griess reagent (0.1% N-(1-naphthyl) ethylene diamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) . Equal volumes of supernatant and Griess reagent were mixed, and this mixture was incubated for 10 min at room temperature in the dark. Absorbance at 540 nm was measured with the spectrophotometer. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as a percentage of the vehicle-treated group.
Estimation of catalase activity:
Catalase activity was assayed by the method of Luck , in which the breakdown of hydrogen peroxide (H2O2) was measured at 240 nm. Briefly, the assay mixture consisted of 12.5 mM H2O2 in phosphate buffer (50 mM of pH 7.0) and 0.05 mL of supernatant from the tissue homogenate (10%), and the change in absorbance was recorded at 240 nm. The result was expressed as mM of H2O2 decomposed per milligram of protein/min .
Estimation of superoxide dismutase activity:
The assay system consisted of 0.1 mM of EDTA, 50 mM of sodium carbonate and 96 mM of nitro blue tetrazolium. In the cuvette, 2 mL of the above mixture, 0.05 mL of hydroxylamine and 0.05 mL of the supernatant were added and auto-oxidation of hydroxylamine was measured for 2 min at a 30-s interval by measuring the absorbance at 560 nm using a Perkin Elmer Lambda 20 spectrophotometer (Himedia Laboratories, Mumbai, India) .
Protein estimation was done by the Biuret method .
The results are expressed as mean±SEM. The behavioral data were analyzed by two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for multiple comparisons, whereas the biochemical data were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. In all tests, values with p<0.05 were considered to be statistically significant.
Effect of GSPE and morphine on CCI-induced mechanical hyperalgesia in rats
CCI of the sciatic nerve resulted in the significant development of noxious static mechanical hyperalgesia, indicated by a decrease in the left hind paw withdrawal threshold on 3rd, 7th and 21st day in CCI-induced rats as compared to the vehicle-treated group. Pretreatment with GSPE (100 and 200 mg/kg p.o.) started on 7th day significantly attenuated CCI-induced NP symptoms as compared to the vehicle-treated group (Figure 1). Further treatment with morphine (10 mg/kg s.c.) started on 7th day significantly attenuated the pain threshold as compared to CCI-induced rats, but it produced tolerance on 11th day, whereas co-treatment of GSPE (100 mg/kg p.o.) with morphine (10 mg/kg s.c.) significantly increased the anti-nociceptive effect and decreased the morphine tolerance as compared to morphine-alone treatment in CCI-induced rats (Figure 2).
Effect of GSPE and morphine on CCI-induced thermal hyperalgesia in rats
CCI of the sciatic nerve resulted in the significant development of noxious thermal hyperalgesia, indicated by a decrease in the left hind paw withdrawal threshold, on 3rd, 7th and 21st day in CCI-induced rats as compared to the vehicle-treated group. Pre-treatment with GSPE (100 and 200 mg/kg p.o.) significantly attenuated CCI-induced thermal hyperalgesia on 14th and 21st day (Figure 3). Further, morphine (10 mg/kg s.c.) treatment started on 7th day significantly attenuated the pain threshold as compared to CCI-induced rats, but it produced tolerance on 11th day. On the other hand, co-treatment of GSPE (100 mg/kg p.o.) with morphine (10 mg/kg s.c.) did not produce tolerance and also attenuated the pain threshold significantly as compared to the morphine-alone-treated group in CCI-induced rats (Figure 4).
Effect of GSPE and morphine on the LPO and nitrite level in CCI-induced neuropathic pain in rats
CCI of the sciatic nerve in rats resulted in a significant increase in the levels of MDA and nitrate as compared to the vehicle-treated group. GSPE (100 and 200 mg/kg p.o.) and morphine (10 mg/kg s.c.) pretreatment in CCI-induced rats significantly attenuated the MDA and nitrate levels in sciatic nerve homogenate. Further, GSPE (100 mg/kg p.o.) pretreatment with morphine (10 mg/kg s.c.) significantly potentiated their protective effect (decreased MDA and nitrite levels) as compared to GSPE-alone- and morphine-alone-treated groups in CCI-induced rats (Figures 5 and 6).
Effect of GSPE and morphine on the reduced GSH level in CCI-induced neuropathic pain in rats
CCI of the sciatic nerve in rats decreased the level of reduced glutathione. Pretreatment with GSPE (100 and 200 mg/kg p.o.) and morphine (10 mg/kg s.c.) in CCI-induced rats significantly attenuated the level of GSH in sciatic nerve homogenate, whereas combined treatment of morphine (10 mg/kg s.c.) and GSPE (100 mg/kg p.o.) significantly potentiated their protective effect (increased reduced glutathione) as compared to GSPE-alone- and morphine-alone-treated groups in CCI-induced rats (Figure 7).
Effect of GSPE and morphine on the catalase and SOD level in CCI-induced neuropathic pain in rats
Sciatic nerve ligation in rats resulted in a significant decrease in the level of catalase and SOD. Pretreatment with GSPE (100 and 200 mg/kg p.o.) and morphine (10 mg/kg s.c.) in CCI-induced rats significantly attenuated the level of catalase and SOD in sciatic nerve homogenate, whereas the combined administration of morphine (10 mg/kg s.c.) and GSPE (100 mg/kg p.o.) significantly potentiated their protective effect (increased catalase and SOD) as compared to GSPE-alone- and morphine-alone-treated groups in CCI-induced rats (Figures 8 and 9).
Neuropathic pain is a chronic and debilitating condition that affects millions of patients worldwide, with estimates of prevalence rates ranging from 1% to 8.9% in the general population depending on the global data . Currently various drugs are available for the treatment of neuropathic pain clinically, such as anti-depressants, anticonvulsants, sodium and calcium channel blockers, N-methyl-d-aspartic acid receptors antagonists and opioids, but these drugs provide a symptomatic relief of pain to only a fraction of patients . Also their efficacies are generally limited and side effects are common. In addition, oxidative stress has also been shown to play an important pathogenic role in the peripheral, central sensitization and subsequent development of NP . Thus, there is an unmet need to understand disease pathogenesis, identify and characterize novel targets, and develops newer agents which act at one or more sites in the pathogenesis of NP.
The present study was designed to explore the beneficial effect of GSPE in CCI-induced NP in rats. CCI was induced in Wistar rats by placing four loose ligatures around the sciatic nerve surgically . After the CCI, the rats exhibited abnormal posture of the injured hind paw, as well as repeated shaking and licking of the injured hind paw, suggesting the presence of spontaneous pain. This caused loss of various sensory functions which led to the significant development of cold allodynia, mechanical allodynia and thermal hyperalgesia . All these pieces of evidence correlate with the syndrome of NP. After the ligation of the sciatic nerve, excitation of transmission neurons occurs and axons get degenerated, which leads to demyelination of neurons, due to sensitization of nociceptors and ectopic excitability of afferent neurons, which reveals the development and maintenance of peripheral neuropathy.
CCI significantly caused oxidative damage as indicated by the rise in lipid peroxidation, nitrite concentration and decreased cellular anti-oxidant defense system such as glutathione and catalase activity . Increased production of ROS leads to changes in cellular structure and function by damaging the various cellular proteins and deoxyribonucleic acid. Thus, ROS such as superoxide, NO and peroxynitrite plays an important role in neuro-inflammatory and immune responses . The abnormal activation of inflammatory and immune mechanisms, particularly glial cell activation and subsequent release of pro-inflammatory cytokines, in the peripheral and central nervous system plays an important role in the development of NP.
In the present study, CCI of the sciatic nerve produced robust mechanical and thermal hyperalgesia in the ipsilateral paw, started within 3–5 days of the post-nerve injury and lasted for about 40–50 days before progressing into hypoalgesia . Similarly, ipsilateral PWLs in vehicle-treated CCI-control rats showed marked thermal and mechanical hyperalgesia from day 3 and reached the steady state between day 7 and day 21 post-sciatic nerve ligation, indicating the development and maintenance of mechanical and thermal hyperalgesia. The administration of GSPE (100 and 200 mg/kg p.o.) significantly reduced thermal and mechanical hyperalgesia in CCI rats.
Accumulating clinical and pre-clinical evidence clearly suggests that increased generation of advanced glycation end-products , mitochondrial dysfunction , activation of nuclear factor-κB, and generation of pro-inflammatory cytokines cause a subsequent rise in ROS which plays a major role in the development of NP . The increase in the free radical activity is the possible mechanism that is operating to modulate a remarkable pathophysiological phenomenon associated with nerve injury. Normally, the ROS are scavenged by the anti-oxidative enzymes, but when their level rises above the normal limit, they cause changes in anti-oxidative balance. Also, it was observed in the present study that CCI-control rats developed a significant increase in the levels of oxido-nitrosative stress, which cause a rise in LPO, TBARS, nitrate and reduction in the anti-oxidant potential that is SOD, catalase and glutathione. The administration of GSPE (100 and 200 mg/kg p.o.) significantly attenuates the reduction in all oxidative parameters such as LPO, TBARS and nitrate by rising anti-oxidant potential (i.e. SOD, catalase and glutathione).
GSE contains various essential constituents such as lipids, proteins, carbohydrates and polyphenols . Proanthocyanidins (POC) and procyanidins are potent natural anti-oxidants with anti-inflammatory activity . They are the most abundant phenolic compounds in grape seeds comprising dimers or trimmers of (+)-catechin, (−)-epicatechin, gallic acid and necessary flavonoids. Polyphenols flavonoids are useful constituents present in GPSE, which shows an anti-oxidant, anti-inflammatory and potent neuroprotective property . The most common and richest source in GSE is higher concentration of POC which has a greater degree of oxygen free radical scavenging potential activity. It has been suggested that GSPE is an effective therapy for oxidation-related diseases in animal models of tumors, atherosclerosis, gastric ulcers, neuropathic disorders and many other diseases . In our experimentation, morphine (10 mg/kg, p.o.) was given to CCI-induced rats, which produced tolerance after 5 days, and thus when GPSE (100 mg/kg p.o.) was given in combination with morphine from day 7, it produced an anti-tolerance effect against morphine-induced tolerance.
In summary, the present study demonstrates that proanthocyanidins and procyanidins are the natural anti-oxidants which by reducing the oxidative potential show a neuroprotective effect. The evidence is clear from the study and data are also supported by previous studies that GPSE inhibits the production of cytokines, infiltration of neutrophils and tumor necrosis factor due to which it is considered to be a useful therapy in neuropathic disorders such as neuropathic pain.
The present study concludes that the oral administration of GSPE (100 and 200 mg/kg) in the CCI model of rats showed the anti-nociceptive and anti-inflammatory effect by inhibiting the number of inflammatory pathways. GPSE scavenges free radical by raising the level of potential anti-oxidants such as SOD, catalase and glutathione. Therefore, GSPE can be used as an adjuvant therapy with available drugs for clinical implications. Additional studies are needed to demonstrated GPSE efficacy in human.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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