Chemicals

The authenticated dried powder of leaves of Rhus Tox (Family: Anacardiaceae) was obtained from Homeopathic Pharmacopoeia laboratory, Ghaziabad, Uttar Pradesh, India. Gabapentin was gifted by Mylan laboratories, India. Cytokine ELISA Ready SET-Go kits for TNF-α (Cat: 837324-22: Batch No. E09479-1645), IL-1β (Cat: 887013-22; Batch No. E09323-1645) and IL-6 (Cat: 837064-22: Batch No. E09358-1645) were purchased from e-Biosciences Incorporation, USA. Lipopolysaccharide (LPS) from Escherichia coli O55:B5 (Cat: L2880; Lot No. 025M4040V) was purchased from Sigma-Aldrich, USA.

Preparation of RT ethanolic extract and dilutions

The procedure prescribed in the monograph of Indian Homeopathic pharmacopoeia was followed for the preparation of RT extract and its ultra-dilutions except the characteristic successions used in preparation of homeopathic dilutions. Dried and coarse powder of RT leaves was pulverized. Exactly weighed (10 gm) powder was mixed with 100 mL of ethanol (70%) and kept in the glass jar for cold maceration up to 7 days with occasional shaking during each day36,54. On 8th day, mixture was filtered through Whatman filter paper and the filtrate was used as an alcoholic extract of RT. Various dilutions of extract in ethanol were prepared to obtain the final RT concentrations of 1 × 10−2, 1 × 10−4, 1 × 10−6, 1 × 10−8, 1 × 10−10, 1 × 10−12, 1 × 10−14, 1 × 10−16, 1 × 10−18, 1 × 10−20, 1 × 10−22, 1 × 10−24, 1 × 10−26, 1 × 10−28, 1 × 10−30, 1 × 10−32, 1 × 10−34, 1 × 10−36 (Fig. 7).

Figure 7 Schematics for the preparation of RT dilutions. Full size image

MTT cell viability assay

The cytotoxicity study of RT in LPS mediated ROS-induced U-87 cells was performed using MTT [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] viability assay as described earlier5,35. Approximately, 1 × 104 cells were seeded in triplicate in 96-well tissue culture plates and allowed to reach 80% confluence. The U-87 cells were treated with 500 ng/ml of LPS for 20 minutes to induce ROS. The LPS containing media was replaced with fresh media and the cells were treated with different concentrations of RT (1 × 10−2–1 × 10−36) for further 24 h. Then, MTT solutions (0.05 μg/μl) diluted in PBS was added to each well. The plates were incubated overnight at 37 °C to allow the formation of purple formazan crystals. Thereafter, detergent solution was added to each well to solubilise the crystals and incubated for 30 min at 37 °C. The intensity of formed color after dissolving the formazan crystals in DMSO was measured spectrophotometrically using a microplate reader (Berthold Technologies, Germany) at 570 nm. Each data point was performed in triplicate and all assays were executed thrice. The data were represented as the percent (%) viability against control.

Cell culture and treatment

The human glioblastoma cell line U-87 was maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml of streptomycin, 1.5 mM of L-Glutamine in the humidified atmosphere of 5% CO 2 at 37 °C. The U-87 cells were cultured in the culture flasks (75 cm2) and medium was changed at every alternate day. After 80% confluence, the media was replaced with fresh media containing LPS (500 ng/mL) for 20 min to induce the production of reactive oxygen species (ROS). Various concentrations of RT were added in LPS pre-treated cells for another 24 h prior to perform the next experiments. The concentration of ethanol in the final assay medium was less than 0.1%. The hydrogen peroxide (H 2 O 2 ) at the fixed concentration (10 μM) was treated for 30 min to produce ROS and used as positive control.

Determination of ROS, SOD, catalase activity and cytokines in U-87 cells

LPS pre-treated (500 ng/ml for 20 min) U-87 cells were treated with different concentrations of RT before the estimation of ROS. Flow cytometry analysis of ROS production by 2′-7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining were performed using flow cytometer (FACS Canto II, Becton & Dickinson, CA, USA) as described earlier by Eruslanov and Kusmartsev37 with some modifications. The H 2 O 2 was used as positive control for the generation of ROS. The concentration of SOD and catalase level in LPS-pretreated U-87 cells were measured using earlier reported methods38,39. The quantification of cytokines including TNF- α, IL-1β, IL-6, IL-10 in LPS-pretreated U-87 cells were executed in cell culture supernatants using 50 μg of protein by the commercially obtained ELISA kits as per the manufacturer’s instructions5,38.

Animals

Adult albino Wistar rats of either sex (170–220 g) were used for the present study. Animals were obtained from animal house facility of R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur. Animals were maintained in ventilated polypropylene cages under the standard conditions (25 ± 2 °C, 12 h light/ dark cycle) at the animal house facility of the institute. Animals were fed with standard pelletized feed (Nutrimix Std-1020) obtained from Nutrivet Life Sciences, Pune, India and water was provided ad libitum excluding the period of behavioral parameter evaluation. The study was approved by the Institutional Animal Ethical Committee (Approval No. IAEC/RCPIPER/2016-17/02) of the R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, India (Reg. No. 651/PO/ReBi/S/02/CPCSEA). All the experimental procedures involving the use of animals were carried out in accordance with the regulations laid down by Committee for the Purpose of Control and Supervision of Experimentation on animals (CPCSEA) constituted under the Prevention of Cruelty to Animals Act, 1960, Ministry of Environment and Forests, Government of India.

Induction of chronic constriction i njury (CCI) in rats

The surgery was performed to induce the CCI as described earlier by Chanchal et al.5. Briefly, the animals were anaesthetized with intraperitoneal administration of pentobarbital sodium (60 mg/kg). A blunt dissection through biceps femoris was executed to expose the common sciatic nerve of right hind limb at the middle of the thigh. Approximately, 5–7 mm of the nerve was freed off the adhering tissue proximal to the trifurcation of sciatic nerve and four ligatures (6.0 silk) were loosely tied around it about 1 mm apart. Following nerve ligation, the muscular and skin layers were instantly sutured and povidone-iodine solution was applied externally. The rats were kept in individual cages and allowed to recover29. The respective drug treatments were started on the next day after the surgery.

Drug treatment and groups

Animals were randomly divided into five groups, each consisting of 8 rats (n = 8). Group I: Normal control group of rats were orally administered once daily with 1 ml saline for 14 days. Group II: Sham operated group of rats were treated with 1 ml saline once daily for 14 days. Group III: CCI-induced neuropathy control group of rats orally received 1 ml saline once daily for 14 days. Group IV: CCI-induced neuropathy + RT treated group of rats orally received 0.1 ml of RT (1 × 10−12 dilution) with 1 ml of distilled water once daily for 14 days. Group V: CCI-induced neuropathy + gabapentin treated group of rats orally received Gabapentin (60 mg/kg/day, p.o.) suspended in 0.5% carboxymethyl cellulose (CMC) once daily for 14 days.

Experimental design

Subsequent to the induction of CCI, rats were habituated for 3 days. RT treatment was started on the next day after the CCI surgery. The thermal and mechanical allodynia were measured on Day-3, Day-7, Day-11 and Day-14 following the surgery by earlier reported methods5,40. Paw withdrawal latency (PWL) was recorded with the maximum cut off time of 20 sec. Right hind paw of each rat up to the ankle joint was immersed in warm water (40 ± 1 °C) and cold water (12 ± 1 °C) for the determination of thermal (warm and cold) allodynia. Mechanical allodynia was noted using electronic Von-Frey apparatus comprising of super-tip probes (2390 series, IITC Life Sciences Incorporation). Paw withdrawal threshold (PWT) was recorded with cut-off pressure of 30 gm. Rats were kept in polypropylene cages with metal mesh floor and acclimatized for approximately 10 min before the measurements. Mid-plantar surface of operated hind paw were probed with Von Frey filaments through the mesh floor, when the paw was in contact with floor. Each filament was applied to the planter surface until it just bent and kept in position for about 6–8 sec. Probes were applied in ascending order and the smallest filament which provoked paw withdrawal response was measured as threshold stimulus55.

On the 14th day after surgery, the animals were anesthetized with intraperitoneal injection of pentobarbital sodium (60 mg/kg). The body temperature of animal was maintained at 37 °C. Sciatic-tibial motor nerve conduction velocity (MNCV) was measured by the stimulation of sciatic and tibial nerves at the sciatic notch and tibial notch through the bipolar needle electrodes (Power Lab/ML856; AD Instruments, Australia) at the 0.10 Hz frequency, 0.1 ms duration and 1.5 V amplitude. After single stimulus the compound muscle action potential was measured from the first interosseous muscle of the hind-paw by unipolar pin electrodes. The recording was typical biphasic response with an initial M-wave which is a direct motor response owing to stimulation of motor fibers. The MNCV was calculated as the ratio of the distance (mm) between both sites of stimulation divided by the difference between proximal and distal latencies recorded in ms32,56.

Following the recording of MNCV, the rats were sacrificed using overdose of pentobarbital sodium. The injured sciatic nerve was isolated along with 1 cm segments on the both sides of CCI injury. The central 5 mm portion of the isolated nerve segment was processed for histological examination. The sections of 4 μm thickness were stained with haematoxylin and eosin. The stained sections were examined under the light microscope for structural alterations including fiber derangement, swelling of nerve fiber and presence of activated satellite cells and Schwann cells. Paraformaldehyde-fixed nerve tissues were dehydrated in ascending graded series of alcohol and embedded in paraffin. The specimens were cut into the sections of 4 μm thickness using microtome and stained with hematoxylin and eosin according to routine staining protocols. The stained sections were examined under the light microscope (Leica D1000, LED) for structural alterations like nerve fiber swelling, fiber derangement and presence of activated satellite cells and Schwann cells13,57.

Segments of sciatic nerve from the rats were process in ice chilled phosphate buffer (pH 7.4) to obtain the 10% homogenate. Homogenate was centrifuged at 2000 g for 20 min at 4 °C and aliquots were used for the determination of malondialdehyde (MDA)58, reduced glutathione (GSH)59, superoxide dismutase (SOD)38 and catalase39,60.

Nitric oxide (NO) was estimated using earlier reported method by Kumar et al.41 with some modifications. Briefly, 50 μl of tissue supernatant was mixed with 500 μl of Griess reagent and the absorbance was determined spectrophotometrically at 540 nm using Powerwave XS microplate spectrophotometer (Biotek, USA). Calibration curve was obtained by using Sodium nitrite as a standard. The concentration of NO was expressed in μM of NO per mg of protein.

The quantification of cytokines like TNF- α, IL-1β and IL-6 was determined in homogenate and using 50 μg of protein by the commercially obtained ELISA kits as per the manufacturer’s instructions5,38.