Animal preparation
Adult Wistar male rats, (weight 180–200 g, n=6/group) were bought from breeding colony of Baqiatallah University of Medical Sciences, Tehran, Iran. Animals were kept in well ventilated sterile Plexiglas cages in the animal houses of Baqiatallah University of Medical Sciences. Each cage contained equal number of rats (two rats in each cage). Animals were maintained under 12 h light/dark cycle in a room at 22–24 °C with free access to food and water. All experiments conducted in agreement with the National Institutes of Health Guide for Care and Use of Laboratory Animals, and was approved by the local ethical committee of Baqiatallah University of Medical Sciences, Tehran, Iran (Ethical code: IR.BMSU.REC.1400.033).
Experimental design and groups
In the current study, animals were divided into 3 groups (n=6 per group). These groups were as follows: [Group 1: sham group]; [Group 2: CCI group]; [Group 3: CCI+TC group]. Animals (in the CCI+TC group) received TC aqueous extract (40 mg/kg) [15] for 30 days via gavage needles once a day. Sham animals received saline 0.9% by gavage needle for the same period of time. Cold allodynia was assessed 1 day prior to neuropathic surgery (CCI model), and on days 2, 4, 7, 14 and 30 post-surgery, using acetone test (Figure 1). Anxiety-like behaviors were also assessed 1 day prior to neuropathic surgery (day -1), and on days 2 and 7 post-surgery, using EPM (Figure 1).
Induction of neuropathic pain (CCI surgery)
Neuropathic pain (CCI model) was induce, as it was previously introduced by Bennett and Xie [16]. Briefly, after anesthetizing the animals with chloral hydrate (350 mg/kg, i.p), the left body of sciatic nerve (1 cm) was exposed and then four loss ligatures (4/0 catgut) was tied around the nerve, about 1 mm apart, until a brief twitch in the hind limb was observed. In sham animals, only the left sciatic nerve was exposed, but not ligated.
Cold Allodynia (Acetone Test)
To assess cold allodynia in neuropathic rats, foot withdrawal (as a positive response) in response to acetone drop (acetone test) was evaluated [17]. Briefly, the rat was placed under a transparent Plexiglas chamber with a metal mesh floor. After 10 min accommodation period, a drop of acetone was applied with a syringe to the plantar surface of the left hind paw (ipsilateral to spinal nerve injury). Withdrawal of the paw or licking/shaking of the toes are considered as a positive response. The acetone was applied 5 times (every 5 min) to the left hind paw (neuropathic paw). The frequency of paw withdrawal was expressed as a percent as follows: (Number of positive response × 100)/(5 trials).
Anxiety-like behaviors (Elevated Plus Maze)
Assessment of anxiety-like behaviors performed using EPM test. The EPM is a cross-shaped platform consisted of two open and two closed arms (opposing each other). All arms communicate via a central zone, allowing rats to move freely into each arm. Rats were placed on the central zone, facing an open arm for 5 min. Then, their movements on the maze monitored for 5 min period with a camera. The percent of open arms spent time and also the percent of open arms entries were assessed as an index of anxiety-like behaviors. Less time spent into the open arms and less number of entrances into the open arms were in favor of anxiety [18].
Scarification of animals and brain tissue dissection
At the end of experiment (day 30), the animals were anesthetized with an intraperitoneal injection of 100/10 mg/kg of ketamine/xylazine mixture. After that, laparotomy was performed to isolate the complete brain tissue and kept at −80 °C for the evaluation of GSH, MDA, SOD and CAT as well as the Western blot assays.
Determination of protein levels in the brain
The method of Bradford was used to measure the protein levels of brain samples [19]. Bovine serum albumin (BSA; Sigma, Germany) was used as a standard.
Biochemical analysis
The activity of the brain catalase (CAT) enzyme was assessed according to method of Aebi in 1984 [20]. The activity of brain superoxide dismutase (SOD) was assessed based on the nitroblue tetrazolium (NBT) reduction by SOD [21]. Lipid peroxidation in the brain was assessed by measuring the malondialdehyde (MDA) content. The protocol follows that described by Yazdanparast et al. [22]. Brain glutathione (GSH) content was assessed by the method of Moron et al. [23].
Western blotting analysis for brain NGF and NF-kB protein expression
The brain tissues were subjected to homogenization in a RIPA buffer solution to which protease and phosphatase inhibitors were added. To remove the insoluble substances, the homogenates were subjected to centrifugation at 12,000× g for 25 min. Then, 5 µL of the supernatant was utilized for assessment of the concentration of the protein using a Bio-Rad Quick StartTM Bradford protein assay kit. Proteins from the tissue homogenates were subjected to a denaturation step using 4X Bio-Rad Laemmli sample buffer and then loaded on sodium dodecyl sulfate polyacrylamide gel. Then, the proteins were electrophoresed and transferred from the gel to nitrocellulose membranes. In order to block the membrane’s free sites, it was incubated in 5% Bio-Rad non-fat dried milk for an hour and finally washed before overnight incubation with the selected primary antibodies: NGF (catalog number: orb228196) and NF-kB (catalog number: GTX102090) at 4 °C with gentle agitation. The following step was the washing of the blots and incubation with secondary antibodies (rabbit, PZ5610) and GAPDH (ab181602). The reacted antigens were visualized by enhanced chemiluminescence by a commercial detection kit. The densitometric analysis for the color intensity was measured by ImageJ software [24].
Statistical analysis
Statistical analyses were conducted with the SPSS software (version 24.0). Data are expressed as mean ± SEM. The two-way ANOVA used to understand if there is an interaction effects between the two independent variables (groups and time) on the dependent variable (cold allodynia and anxiety like behaviors). Then, we use one-way ANOVA to compare mean difference of between groups, followed by the Tukey HSD post hoc test, and p<0.05 was considered significantly.