br C M Kopruszinski et al br in seconds
C.M. Kopruszinski et al.
in seconds spent in the drug-paired chamber as compared to pre-con-ditioning time and post-conditioning vehicle-paired chamber time, with positives scores indicating preference.
2.7. Behavioral analysis protocols
The eﬀects of local treatment with lidocaine, morphine (used as control drugs) or with endothelin receptor antagonists on tumor-in-duced heat hyperalgesia was assessed in diﬀerent cohorts of rats, on day 6 post-inoculation. A total of 120 rats were used for this experiment. Baseline responsiveness to heat stimulation was evaluated solely on the right (ipsilateral) vibrissal pad prior (BL) to inoculation of tumor Z-VAD-FMK and again on day 6 (D6) to verify tumor-induced heat hyperalgesia. Following confirmation of heat hyperalgesia on day 6, bosentan, BQ-123, BQ-788, BQ-123 plus BQ-788 in association, lidocaine, morphine or vehicle were all administered locally into the right upper lip. Heat hyperalgesia was re-evaluated at 30 min and again every hour up to 4 h after drug or vehicle treatment. The eﬀect of the same treatments was assessed 30 min after their administration on tumor-induced increased spontaneous grooming in diﬀerent cohorts of animals on day 6 post tumor-inoculation. A total of 213 rats were used for this experiment.
In addition, CPP to local bosentan, lidocaine or morphine treat-ments was evaluated in separate cohorts of rats, on days 3–6 post tumor-inoculation. The pre-conditioning day occurred on day 3 after tumor cell inoculation as described above. The two conditioning days (days 4 and 5 after tumor cells inoculation) were divided in morning and afternoon sections. In the morning, rats were injected locally with vehicle and immediately confined to the appropriate pairing chamber for 30 min. Four hours later (in the afternoon), the same animals were injected locally with bosentan, lidocaine or morphine and immediately confined into the opposite pairing chamber for 30 min. The test day took place on day 6 after tumor cell inoculation. A total of 33 rats were used for this experiment. The timeline of the experimental procedures is illustrated in Fig. 1.
2.8. Statistical analysis
Data are presented as mean ± S.E.M. and n represent the number of rats analyzed. Heat hyperalgesia time-course data for drug eﬀects were analyzed using two-way ANOVA followed by Tukey post-hoc tests. For spontaneous grooming and CPP data, the eﬀects of treatment were analyzed by two-way ANOVA followed by Tukey post-hoc test between baseline (pre-conditioning) and test (post-conditioning) values. All data were analyzed with GraphPad Prism® 6. A statistically diﬀerence was set to a probability level of 0.05. Error bars are S.E.M.
3.1. Eﬀect of local endothelin receptor blockade on tumor-induced heat hyperalgesia
Tumor-bearing rats developed heat hyperalgesia (i.e. a decrease in response latency relative to baseline), which was attenuated by local control treatments, lidocaine and morphine. Both treatments sig-nificantly reduced tumor-induced heat hyperalgesia within 30 min of treatment, and the response latencies returned to pre-lidocaine and pre-morphine values by 1 and 2 h post-administration, respectively (Fig. 2A, p < 0.05 versus sham rats treated with vehicle and p < 0.05 verustumor-bearing rats treated with vehicle). Sham animals treated with lidocaine or morphine did not show any diﬀerence in response latencies when compared to vehicle-treated controls at any time-point (Fig. 2A, p > 0.05).
On day 6 post inoculation, local treatment with BQ-123 or BQ-788, either alone or in combination, failed to modify the heat hyperalgesia induced by tumor cell inoculation (Fig. 2B, p < 0.05 versus sham rats treated with vehicle). In contrast, similar treatment with the non- Archives of Oral Biology 97 (2019) 231–237
Fig. 1. Experimental protocols timeline. A. Heat stimulation: on day 0, baseline to facial heat stimulation was collected followed by the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, facial heat stimulation measurement was performed to confirm the development of heat hyperalgesia. Treatment was performed subcutaneously (right upper lip) and facial heat stimulation was assed hourly. B. Spontaneous facial grooming: on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissal pad. On day 6 after inoculation, treatment was performed sub-cutaneously (right upper lip) and spontaneous facial grooming was assessed for 10 min after the treatment. C. Conditioned place preference (CPP): on day 0, animals were submitted to the inoculation of tumor cells into animal’s right vibrissae pad. On day 3, drug-free animals were placed into CPP apparatus with free access to all three chambers for baseline collection. On days 4 and 5, rats were exposed to two conditionings days. In the mornings (AM), animals were treated with vehicle and conditioned to a designed chamber, with no access to the other chambers. In the afternoons (PM), animals were treated with drug and conditioned to the opposite chamber to the morning section, also with no access to the other chambers. On day 6, drug-free animals were submitted to the test day, with free access to all three chambers, for the evaluation of conditioned place preference.