In total, 152 male monoarthritic Sprague-Dawley rats (225 to 250 g) were used in this study. The experimental groups were constituted by six animals in each group. All animals were obtained from the facilities of the Faculty of Medicine of the University of Chile, held in a light-dark cycle of 12/12 hours, starting at 8:00 AM, food and water ad libitum. After each experiment, rats were killed by using an overdose of urethane (3 g/kg, intraperitoneal, i.p.)
The experiments were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals of National Institutes of Health (NIH)"  and the rules of the International Association for the Study of Pain (IASP) "Models of animal pain and ethics in experimental animals"  and "Ethical standards in research and management of pain." Furthermore, the experimental protocols were approved by the Bioethics Committee of the Universidad de Santiago de Chile.
Induction of monoarthritis
Monoarthritic rats were used as a model of chronic inflammatory pain. Monoarthritis was induced in rats of 120 to 150 g by the method described by Butler et al., . In brief, rats were inoculated with a volume of 50 μl of Freund adjuvant, in the right ankle joint. The adjuvant consisted of a solution of 60 mg of Mycobacterium butiricum, 6 ml of mineral oil, 4 ml of sodium chloride (0.9%), and 1 ml of Tween 80. Subsequently, this mixture was autoclaved at 120°C for 20 minutes and stored at room temperature until use. Before injection, the solution was homogenized by constant stirring. The injection of adjuvant produces a localized arthritic syndrome that becomes stable around the fourth week after inoculation, and establishes a persistent pain with hyperalgesia of the tibiotarsal joint, which is maintained for a period exceeding 2 months. Around 90% to 95% of the injected rats developed mechanical hyperalgesia. Monoarthritic rats were used between the fourth and the fifth weeks after induction of monoarthritis.
(±)-CPP (Tocris) was administered at single doses of 2.5, 7.5, 12.5, 25, 50, and 100 μg/10 μl. PPF (Sigma) was administered in repeated doses of 1, 10, 30, and 100 μg/10 μl, once daily for a period of 10 days. The two drugs were administered via i.t. injection in a volume of 10 μl and dissolved in saline; i.t. injection consists of administering the drug into the subarachnoid space between lumbar vertebrae L5 and L6 , by using a Hamilton syringe with a needle 26G × 1/2 inch'. The access to the subarachnoid space is evidenced by a slight movement in the tail of the rat as a result of the needle mechanical stimulation penetrating the meninges of the spinal cord. The daily PPF i.t. injection was done under brief halothane anesthesia (2 minutes).
To evaluate the antinociceptive effect of both drugs individually on monoarthritic rats, the vocalization threshold to mechanical stimulation (Randall-Selitto test) was used. The animals were separated in a first stage of experimentation into two groups: (a) intrathecal administration of (±)-CPP: 2.5, 7.5, 12.5, 25, 50, or 100 μg/10 μl (n = 6 for each dose); and (b) daily i.t. administration of increasing PPF concentrations of 1, 10, 30, or 100 μg/10 μl (n = 8 for each dose) for 10 days.
To evaluate the antinociceptive effect of the PPF and (±)-CPP combination, we conducted a second series of experiments. Both drugs were diluted in decreasing doses (1/3, 1/10, and 1/100) in relation to its ED30. Five groups were used:
1. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of ED30 of (±)-CPP was done (n = 6).
2. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/3 of ED30 of (±)-CPP was done (n = 6).
3. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/10 of ED30 of (±)-CPP was done (n = 6).
4. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/30 of ED30 of (±)-CPP was done (n = 6).
5. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/100 of the ED30 of (±)-CPP was done (n = 6).
Controls were provided by normal and monoarthritic rats receiving saline, as follows:
1. Normal group of the same age of monoarthritic rats, receiving i.t. injection of saline before testing (n = 6).
2. Monoarthritic saline group, pooled from saline controls for the (±)-CPP, PPF, and combined (±)-CPP/PPF series, receiving i.t. daily injection of saline for a period of 10 days, followed by an i.t. injection of saline at day 11, or a single injection at day 11 (n = 16). The three groups were pooled because they showed no significant differences in vocalization threshold between them at any time of testing.
This behavioral test consists of adding a continuous and increasing pressure with a taper ending in blunt tip on the posterior knee joint of the rat to generate a nociceptive behavior. The response is evidenced by a vocalization or withdrawal reflex of the limb in response to stimulation. The pressure on the joint is increased gradually (linearly) up to 570 g, a value that does not harm the animal. The equipment used for this test was called analgesiometer Ugo Basile. Each animal was tested 2 times at 5, 15, 30, and 60 min for monoarthritic rats treated with (±)-CPP or the combination of PPF and (±)-CPP, and at 15, 30, and 60 min for monoarthritic PPF-treated rats. After the experiment, all rats were killed with an overdose of urethane. Grams of pressure, which expresses rat nociceptive behavior, were saved for later analysis. The data were expressed as percentage change to baseline and were then averaged over the different groups and different times. Later, the area under the curve (AUC) was calculated, by using the Microcal Origin V 6.0 program, and the groups were compared statistically.
The evaluation of the interaction between both drugs was performed by using isobolographic analysis . The isobologram is a graphic method that consists of calculating the theoretic additive dose for each level of effect and their statistical comparison with the combination dose that produces the same effect experimentally. Equieffective doses of both drugs alone are needed to calculate the expected dose in a combination. To this end, we determined the dose that produces 30% of maximal effect (ED30) by using a linear regression analysis from the dose-response curve of six increasing doses of (±)-CPP and the previously mentioned for increasing doses of PPF. Once we obtained the ED30 of both drugs, a graph was constructed by placing in the y-axis of the ED30 point of (±)-CPP and the x-axis point of the ED30 of PPF. The union of two points by a straight line (isobolo), also known as a line of additivity or no interaction, helped to establish the type of interaction (synergism or antagonism) of both compounds. The interaction between both drugs was carried out by an administration of 1, 1/3, 1/10, 1/30, and 1/100 of the ED30 (±)-CPP, and PPF. The coadministration was performed through intrathecal PPF ED30 daily for 10 days. The antinociception was assessed on day 11 with the Randall-Selitto test and then followed by i.t. administration of ED30 (±)-CPP; antinociception was assessed by the same test. Then the ED30 of the association of both drugs (ED30 experimental), from a dose-response curve, was obtained by linear regression analysis. This dose was compared statistically with the dose that theoretically represents the simple addition of effects, obtained by the following formula:
Where R is the power ratio between the two drugs given alone, P1 is the proportion of the drug (PPF) in the mixture, and P2 is the proportion of drug 2 ((±)-CPP) in the mixture.
The graphic region in which is located the experimental value (ED30 experimental) in relation to the theoretic value (ED30 theoretic additivity) determines the type of interaction: If the value is located under the line of additivity and is statistically different from the theoretic value, the type of interaction is synergistic or supraadditive (effect greater than the sum of the individual effects of drugs); if located next to the line of additivity and not statistically different from the theoretic value, the interaction is simple additivity (equal effect of the sum of each drug); conversely, if the experimental value lies above the line of additivity and is statistically different from the theoretic nature of the interaction, it is subadditive or antagonistic. At the same time, we calculated the interaction index (I.I.) between the drugs, obtained from the following formula:
This index, when less than 1 corresponds to a synergistic interaction, when equal to 1, corresponds to an additive interaction, and when greater than 1 is an antagonistic interaction .
The results were expressed as mean percentage of antinociceptive effect ± standard error of the mean (SEM) for each experimental group, from baseline obtained before the injection of saline or each of the drugs under study, as appropriate. The quantification of the antinociceptive effect (%AE) of the drugs tested were calculated as a percentage change in AUC from baseline (basal) for each rat, and set a maximum pressure cut-off of 570 g in the Randall-Selitto, according to the following formula:
Where AUCpre and AUCpost are approximate integrals of the curves obtained by the method of trapezoids and pre-post drug injection, respectively, according to Eq. 1. The AUCdrug effect values are the integrals of the real effect of the drug. The antinociceptive effect (AE) was calculated according to Eq. 2, where the AUCcut-off corresponds to the area of maximum pressure possible on the animal.
To analyze the time-course of the antinociceptive effect of increasing doses of i.t. (±)-CPP and PPF, two-way ANOVA was performed. It allowed us to assess both intergroup comparisons (vocalization-threshold changes under different treatments) and intragroup comparisons (vocalization thresholds along the time), followed by the Bonferroni multiple comparisons test. To analyze the percentage antinociception obtained from the area under the time-course curves, one-way ANOVA was used, followed by Tukey-Kramer multiple comparisons test. To assess differences for the theoretic ED30 and experimental ED30, the two-tailed Student t test was used. All statistical analyses were performed with the Prism 3.0 software (GraphPad Software, Inc., San Diego CA, USA).