Maintenance and inoculation of the tumor
2.2. Maintenance and inoculation of the tumor cells
To induce facial carcinoma in rats, Walker-256B lineage AR-13324 were used as previously described (Kopruszinski et al., 2018). The cells were maintained in vivo by weekly intraperitoneal (ip) passages. To induce the facial cancer, the animals were anesthetized with a solution of xy-lazine/ketamine (7.5/60 mg/kg, respectively, ip) and injected sub-cutaneously (sc) with a suspension of 2 × 106 Walker-256B cells in 100 μL into the right vibrissal pad. Control animals (sham) were simi-larly treated with 100 μL of vehicle (phosphate-buﬀered saline, PBS). The day of inoculation of the Walker-256B cells was considered day 0. From the total animals used for the behavior experiments, less than 5% did not develop tumor at the facial region. These animals were excluded of the experiments.
All drugs evaluated in the present study were diluted in 0.9% NaCl (i.e. saline), and administered locally, through sc injection into the rat’s right upper lip. Lidocaine hydrochloride (Cristália, Sao Paulo, Brazil) was administered at 1 mg/50 μL, morphine hydrochloride (Sigma Archives of Oral Biology 97 (2019) 231–237
Aldrich, San Francisco, USA) at 30 μg/50 μL, the peptidic endothelin ETA and ETB receptor antagonists BQ-123 and BQ-788 (American Peptides, San Francisco, USA) were injected alone or in combination at 20 μg/50 μL, whereas the non-peptidic dual endothelin ETA and ETB receptor antagonist bosentan monohydrate (Actelion Pharmaceuticals, Allschwill, Switzerland) was administered at 10 or 30 μg/50 μL. Controls animals received a 50 μL injection of saline. The choice of drug doses were based on the literature (Chichorro, Zampronio, Cabrini, Franco, & Rae, 2009, 2010; Kopruszinski et al., 2018; Remeniuk et al., 2015).
2.4. Heat Stimulation
Heat sensory thresholds of the vibrissal pad area were assessed as previously reported (Chichorro et al., 2009). Briefly, each animal was lightly restrained by the experimenter while a ∼50 °C radiant heat source was positioned at 1 cm from the surface of the right vibrissal pad. The response latency to display either head withdrawal or vig-orous flicking of the snout was recorded and adopted as a behavioral index of heat sensitivity. A 20 s cut-oﬀ time was used to prevent tissue damage.
2.5. Spontaneous grooming evaluation
Evaluation of spontaneous facial grooming was performed as pre-viously described (Kopruszinski et al., 2018). In brief, rats were sub-mitted to individual evaluation of time spent performing spontaneous facial grooming across a 10-minutes session conducted between 8:00-11:00 h. Facial grooming behavior was defined as bilateral cleaning of the face, at the vibrissae region, with the front paws (Hidaka et al., 2011).
2.6. Conditioned place preference
Evaluation of ongoing/tonic nociception relief using the condi-tioned place preference (CPP) paradigm was performed as previously described (King et al., 2009). The CPP apparatus consist of three chambers designated as two conditioning chambers, with each com-partment measuring 36 × 25 × 40 cm, with distinct visual and tactile cues, i.e. diﬀerent-patterned walls (one chamber with black walls and the other one with striped walls) and texturally distinct floors (black chamber with smooth floor and stripped chamber with textured floor), respectively, separated by a neutral chamber measuring 36 × 25 × 10 cm, Polycistronic mRNA was brightly lit (LED light 100 lx). The CPP paradigm consists in three steps, pre-conditioning or baseline, con-ditioning and test. The pre-conditioning step, defined as the first day of CPP paradigm, animals were located into the neutral chamber with free access to all chambers, and left to explore the chambers for 15 min. Exploratory behavior was filmed and recorded, with the baseline of the time-spent in each chamber analyzed. To avoid preference for one of the chambers on the baseline, rats that spent more than 80% or less than 20% in one of the conditioning chambers were removed from the study. Drug pairing was assigned to the conditioning chambers using an unbiased CPP design, with equivalent mean times across the two con-ditioning chambers. Drug assignment was counterbalanced across the conditioning chambers according to the amount of the time spent in each chamber during the baseline step, i,e, half of the animals were paired to the drug treatment in one of the conditioning chambers for all groups and the other half were paired to the drug treatment on the opposite conditioning chamber. A two conditioning days CPP protocol was chosen based on the drug route treatment, whereby the con-ditioning step occurred across two consecutive days, as detailed below. On the test day, rats were once again placed into the neutral chamber with free access to all chambers, and allowed to explore the apparatus for 15 min. Time spent in each compartment was analyzed to determine whether drug treatment produced CPP, indicated by an increased time