Permission was obtained from the Institutional Animal Ethics Committee (695/IAEC/12;16/12/2013). Sprague–Dawley rats (male, weight about 250 g, n = 68) were obtained from the animal facility of All India Institute of Medical Sciences, New Delhi, India. Animals were randomly distributed into various testing groups. Food and water were provided ad libitum. Optimum environmental conditions like temperature (22°C–25°C) and light/dark cycles (12 hr/12 hr) were maintained throughout the experimental period. All experimental procedures were conducted under the guidelines of International Association for the Study of Pain [22].

The procedure of paw incision has been standardized earlier [23]. Under isoflurane anaesthesia, the plantar surface of the right paw was cleaned with povidone-iodine. A 1 cm long midline incision was made on the skin, starting one-half inch from the proximal end of the heel. The underlying fascia and muscle were also incised. Subsequently, the wound was sutured with 4–0 nylon and knots were placed on the lateral side. Finally, the rat was transferred to a warm recovery chamber. Postincisional nociception by various behavioral tests was determined at specific time intervals after incision (2 hr, 8 hr, and every 24 hr up to day 7). The suture was removed on day 2.

Lyophilised Protein C (P2200–1 mg; Sigma-Aldrich, St. Louis, MO) was dissolved in physiological saline at a concentration of 10 μg/10 μL. Protein C 10 μg was administered into the wound using a sterile micropipette just before suturing, and left undisturbed for 30 seconds (Fig. 1). Prior to this, skin edges were pulled up by forceps to form a pocket in order to prevent loss of the drug [10]. The drug was administered only once during the entire experimental procedure. The control group received saline.

The assay of COX activity was only determined in the control group. Animals were euthanized by inhalation of carbon dioxide. Skin and muscle tissue from the site of incision (a 5–6 mm block of tissue around the incision site) were isolated by a scalpel blade. The tissue samples (n = 4) at each time point (basal and post-incision 2 hr, 8 hr, as well as days 1 and 3) were pooled and then processed, using a COX activity kit as per manufacturer’s instructions (Cat. No. 760151; Cayman Chemicals, Ann Arbor, MI) to determine total COX along with the activity of COX-1 and COX-2 individually. The test was carried out in a 96 well enzyme-linked immunosorbent assay (ELISA) microplate. Absorbance was taken at 590 nm using an ELISA plate reader (iMark^TM Microplate Absorbance reader; Bio-Rad Laboratories, Hercules, CA). Final activity was measured in nM/min/mL (U/mL).

The procedure of the collection of paw tissue was the same as for the estimation of COX activity. Again, it was only evaluated in the control group. However, tissue samples were not required to be pooled. The concentration of LTB4 was quantified by a competitive LTB4 ELISA kit (Cat. No. 520111; Cayman Chemicals) as per manufacturer’s instructions. Absorbance was taken at 420 nm (iMark^TM Microplate Absorbance reader; Bio-Rad Laboratories). The final concentration was expressed in pg/mL.

The non-invasive component detection analysis tail-cuff system was used to measure heart rate and blood pressure (Kent Scientific Corporation, Torrington, CT) [24]. Only the control group was used for this study. This system uses volume pressure recording to measure the flow of the blood in the tail. One day prior to the experiment, rats (n = 6) were put in restrainers with darkened nose cones in a warm environment for 30 minutes. This was done twice during the day. Two cuffs were put on the tail, an occlusion and a volume-pressure recording cuff, on the day of recording. Values of blood pressure (systolic and diastolic) and heart rate were displayed in the accompanying software. Readings were taken under basal condition and at 2 hours and days 1, 3, and 5 after incision (1,000–1,200 hr). The rats were housed individually. The average invasive systolic and diastolic blood pressure in the caudal venous artery is 95.9 ± 12.9 mmHg and 73 ± 13.7 mmHg, respectively, whereas the mean heart rate is 305 beats/min [25].

Each animal (n = 6) belonging to the control group was habituated in individual cages for 2 days to note their average intake of food and water. Standard food pellets (50 g) were weighed in an electronic balance and kept daily in the food dispenser of the cage. The dispenser was closed from the outside to prevent extraneous loss. The remains of the food pellets at the bottom of the cage were collected every day and weighed along with the remaining pellets. Also, 100 mL of water was provided. The daily consumption of food and water was assessed every 24 hours for 7 days following the incision.

This was performed before (basal) and at specific time intervals after incision (2 hr, 8 hr, and days 1–4 for guarding and days 1–7 for evoked pain behavior) by the same observer. The observer was blinded to the drugs administered to the animals.

According to an earlier described protocol, rats were kept on a wire mesh platform (wire mesh with an 8 × 8 mm pore size) and covered with plastic (16 × 16 × 16 cm) enclosures [26]. The acclimatization period was 30 minutes. Thereafter, the position of the hind paws was observed for one hour. This time was divided into 12 periods of 5 minutes each. During the first minute of each 5 minutes period, scores were given based upon the following: paw completely off the mesh = 2; paw lightly resting on the mesh without weight bearing = 1; and paw firmly on the mesh = 0. Finally, the total scores were added for each paw and the score of the normal paw was deducted from the incised paw to obtain the cumulative pain score. Scores were between −24 and +24, with lower scores indicating less nociception. The guarding score was determined in the basal condition, as well as at 2 and 8 hours after paw incision and then every 24 hours until day 4.

Mechanical allodynia was determined by the sequential administration of von Frey filaments (North Coast Medical Inc., San Jose, CA) according to the “Up-down method” [27]. Von Frey filaments of different sizes (3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93, and 5.18) were applied for 7–8 seconds to the paw in a perpendicular manner from below the mesh. The filament exerts a pre-defined amount of pressure varying between 0.4 and 15 g. Normally the animals are able to bear the maximum pressure (15 g), but after incision, mechanical hypersensitivity was manifested as paw withdrawal. An interval of 2 minutes was maintained between two consecutive applications. The value of mechanical allodynia was expressed as a 50% withdrawal threshold (g). Allodynia was determined under the basal condition, 2 and 8 hours after paw incision, and then every 24 hours until day 7.

Thermal hyperalgesia was assessed by the Hargreaves method (Plantar Test apparatus; UGO Basile, Gemonio, Italy) [28]. It was evaluated just after allodynia. Rats were placed on a glass platform and covered by Perspex enclosures. Prior to recording, rats were allowed to acclimatize for 15 minutes. The source of radiant heat was kept beneath the platform and was manually directed towards the surgical site in the paw. The baseline latency period was between 8 and 10 seconds. The cut-off value was 20 seconds. Each rat was tested 3 times at intervals of 2 minutes and the average of these values was used to calculate percent of maximum possible effect (%MPE), which was derived as follows: (post drug latency – baseline latency) / (cut off latency-basal latency) × 100.

Data were analysed by GraphPad Prism version 5 (GraphPad Software, San Diego, CA). Values are represented as mean ± standard error of mean. Data was analysed by one-way analysis of variance followed by the Bonferroni multiple comparison test except for behavioral tests of nociception, which were assessed by two-way analysis of variance followed by the Bonferroni multiple comparison test. Values of COX activity could not be analysed as these were based upon the pooling of samples from 4 rats. A P value of less than 0.05 was considered statistically significant.

Compared to basal levels (17.44 nM), total COX activity was consistently increased up to day 3 (Fig. 2). Maximum increase was noted at 8 hours (61.01 nM). Between 2 hours and day 1, COX-2 activity was greater than COX-1 activity. Unexpectedly, under basal conditions, COX-2 activity (8.26 nM) was noted to be only slightly lower than COX-1 activity (9. 18 nM).

LTB4 concentration was relatively low under basal conditions (200.8 ± 51.63 pg/mL) but increased by almost 5 fold at 8 hours (1,043 ± 153.2 pg/mL) (Fig. 3). Thereafter, it remained at a higher level up to day 3.

Hemodynamic parameters like blood pressure and heart rate were not significantly affected by the incision (Fig. 4). Basal values were 118 ± 3.4 mmHg and 83 ± 2.7 mmHg for systolic and diastolic blood pressure, respectively. Two hours after, these were 115 ± 7.6 and 86 ± 5.8 mmHg. The heart rate was 476 ± 46 beats/min and 449 ± 32 beats/min before and 2 hours after incision.

Food (pellets) (basal, 20.1 ± 0.6 g vs. day 1, 12.4 ± 0.8 g) and water (basal, 39.4 ± 0.9 mL vs. day 1, 31.4 ± 0. 9 mL) consumption were reduced 1 day after incision (Fig. 5). Food consumption decreased again on day 3 (16.6 ± 0.8 g).

All the three behavioral parameters demonstrated maximum nociception (highest values for cumulative pain score, %MPE, and lowest values for a 50% withdrawal threshold) immediately after incision (Fig. 6). Among these, treatment with protein C once during the entire experiment, attenuated the cumulative pain score (Fig. 6A). This effect was observable up to day 2. Evoked pain behaviors were unaffected (Fig. 6B, C).