Animals
Throughout the present study, healthy adult male Wistar rats weighing 240-270 g were maintained in a laboratory under a 12:12 h light-dark cycle (lights on at 07:00 h) at a controlled ambient temperature (22 ± 0.5°C) with free access to food and water. Experiments were performed between 10:00 h and 14:00 h. Veterinary Ethics Committee of the Faculty of Veterinary Medicine of Urmia University approved all protocols used in the present study.
Drugs
Histamine dihydrochloride, 2-pyridylethylamine dihydrochloride (2-PEA), mepyramine hydrochloride, dimaprit dihydrochloride, ranitidine hydrochloride and naloxone hydrochloride purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. All drugs were dissolved in sterile normal saline 30 min before intra-VLOC and intra-PAG microinjection.
Study Protocol
The rats were divided into sham and CCI groups. On the first day, all rats underwent surgery on the sciatic nerve. Sham group, with no manipulation of sciatic nerve, had a surgery only on skin and muscles. Six days later (day 7), VLOC and vlPAG of all rats were cannulated. Sham and CCI groups were programmed to receive intra-VLOC and intra-PAG microinjections of normal saline and test drugs. On days 14 and 17 after CCI induction, nociceptive and aversive behaviors were recorded, respectively. The timeline and experimental procedures used in the present study have been presented in Fig. 1.
Chronic Constriction Injury of the Sciatic Nerve
The CCI model of neuropathic pain was induced according to Bennett and Xie (1988). Initially, rats were anaesthetized with intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (8 mg/kg) solution. A small incision was then made at the left mid-thigh level. The left sciatic nerve trunk was exposed by blunt dissection through the biceps femoris. After removing the sciatic nerve surrounding connective tissue, four chromic catgut ligatures (5-0) were loosely tied around the nerve trunk with about 1 mm spacing at nearly 1 cm distal to its trifurcation. A typical minor twitching of the relevant muscles was observed during the nerve constriction. Following surgery, the rats were allowed to recover for one week before implantation of guide cannulas.
Intra-cerebral Guide Cannula Implantation
Six days after sciatic nerve surgery (day 7), the rats were anesthetized again with intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg) and placed in a stereotaxic apparatus (Stoelting, Wood Lane, IL, USA). Thereafter, the right and left sides of VLOC and PAG were bilaterally implanted with cannulas at the following coordinates (Paxinos and Watson, 1986): 3.2 mm anterior to the bregma, 2.2 mm left and right sides of the midline and 4.1 mm below the top of the skull (VLOC) and 7.8 mm posterior to the bregma, 2.3 mm left and right sides of the midline and 5.7 mm below the top of the skull with a 15° angel (PAG). The cannulas were then fixed to the skull using screw and dental acrylic. A 29-gauge, 13 mm stylet was inserted into each cannula to keep them patent prior to microinjection. At least 7 days were allowed for recovery from microinjection protocol.
Intra-cerebral Microinjection of Test Drugs
The rats were designed to receive normal saline, 2-PEA, dimaprit, mepyramine, ranitidine and naloxone into the VLOC. Naloxone was also administered into the PAG before intra-VLOC microinjection of 2-PEA and dimaprit. Microinjections of the-above-mentioned drugs were performed using a 30-gauge, 14 mm needle attached to a 5 µl Hamilton syringe in a constant volume of 0.25 µl. To facilitate drug diffusion, the microinjection needle was held in place for another 45 s. Mechanical allodynia and aversion recording began 5 and 3 minutes after microinjection of antagonists (mepyramine, ranitidine and naloxone) and agonists (2-PEA, dimaprit), respectively. The drug doses used here were in accordance with previous studies (Erfanparast et al., 2015; Hamzeh-Gooshchi et al., 2015; Ghasemi et al., 2017; Salimi and Tamaddonfard, 2019).
Mechanical Paw Withdrawal Threshold Measurement
Von Frey filament test was used for evaluation of mechanical allodynia according to Dang et al. 2010. In brief, following certain intervals (20 min before and 0, 20, 40, 60 after microinjection), the filament number 13 equivalent to 20 g was applied to the plantar surface of the hindpaw (Taati and Tamaddonfard, 2018). After recording of positive and negative responses, paw withdrawal threshold 50% (PWT 50%) were calculated using the following formula (Chaplan et al., 1994): 50% threshold = 10(Xf+kd), where Xf is the value of the final von Frey filament used (in log units), k is the tabular value for the pattern of positive/negative responses presented at the appendix 1 of Chaplan et al. (1994) article, and d is the mean difference between stimuli in log units (0.155). Values of 0.4 or 20.0 g were assigned where continuous positive or negative responses were observed all the way out to the end of the stimulus spectrum, respectively.
Neuropathic Pain-Induced Aversion Measurement
On day 17 after CCI induction, place escape/avoidance paradigm (PEAP) was used to assess aversive dimension as described previously (Fuchs and McNabb, 2012; Salimi and Tamaddonfard, 2019). Rats were placed under the PEAP Plexiglas chamber (60×30×30 cm) that consisted of a dark side (painted black) and a light side (painted white) as well as a mesh floor to allow access to the animal’s hind paws. Immediately, the plantar surface of the hind paw was mechanically stimulated with a von Frey filament (number 15 equivalent to 65 g) every 15 s for a period of 30 min. The sciatic nerve-non-injured (right) hind paw was stimulated when the animal was in the light (white) side, whereas the sciatic nerve-injured (left) hind paw was stimulated when the animal was in the dark (black) side of the chamber. Time spent duration within white side of the chamber and the total number of line crosses from one side to the other were recorded. The latter was used as a general measure of locomotor activity (LaBuda and Fuchs, 2005; Salimi and Tamaddonfard, 2019).
Verification of the Intra-cerebral Microinjection Sites
At the end of each experiment (day 18), each sides of the VLOC and PAG were microinjected with 0.25 µl of methylene blue. After euthanizing, the brains were removed, and after giving a photo from the surface of the brain, placed in a formalin solution (10%). Seven days after, the brains were sectioned (50-100 µm) and viewed under a laboratory loup to observe the localization of the injected site according to the atlas of Paxinos and Watson (1986).
Data Analysis
Analyzing the obtained results was done using the Graph Pad Prism version 5 (Graph Pad Software Inc, San Diego, CA USA). The data obtained from PWT 50% were analyzed using two-way repeated measure analysis of variance (ANOVA) and then Bonferroni post hoc test. The obtained results from mean 50 % PWT, WSTS % and crossing number were analyzed by one-way analysis of variance (ANOVA) and post hoc Tukey’s test. All values are expressed as mean ± SEM. P < 0.05 was considered statistically significant.
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