Behavioral examinations

Cold allodynia was measured using the acetone drop method at the hind paw as described by Choi et al. [11], with small modification. The rat was placed on top of a wire mesh grid, and access to the hind paws was allowed. Acetone (0.1 ml) was applied to the plantar surface of the left hind paw by using a pipette with polyethylene tube and the duration of paw withdrawal was recorded in seconds using a stopwatch. The minimum and maximum values were 0.1 s and 20 s, respectively. This was conducted three times, with an interval of 5 min between applications, and the total individual withdrawal durations were then calculated.

Mechanical hyperalgesia of the paw was evaluated using the pin-prick test, as previously described by Erichsen and Blackburn-Munro [12]. The surface of the injured hind paw was touched with the point of a bent gauge needle (at a 90° angle to the syringe) at an intensity sufficient to produce a reflex withdrawal response. Paw withdrawal latency was then recorded in seconds using a stopwatch with a minimum value of 0.1 s.

The thermal nociceptive threshold, employed as an index of thermal hyperalgesia, was assessed by the hot plate method, using a plate maintained at a temperature of 52.5 ± 1.0°C. The rat was placed on the hot plate, and the withdrawal latency with respect to licking of the hind paw was recorded in seconds. A cut-off time of 15 s was used [13].

The ‘paint brush’ behavioral test is well-established for investigating dynamic responses to a mechanical stimulus. Normal rats never withdraw from a smooth paint brush, so a response to this stimulus is described as allodynia. The rat was placed in a cylinder with a wire mesh floor and a smooth paint brush was used to rub the plantar surface of the hind paw from the heel to the toes. This stimulus was applied five times, with intervals of 5 s, and the number of withdrawals was noted (between 0 and 5). The same procedure was repeated twice, with a gap of 5 min, and the number of withdrawals across the three tests was summed to obtain a single cumulative score of mechanical dynamic allodynia. The minimum and maximum values were 0 and 15, respectively [14].

For assessment of mechanical state allodynia, the rat was placed in an individual plastic cage with a wire mesh bottom. After 20 min, the mechanical threshold was measured by applying a series of eight calibrated von Frey filaments (0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, and 15.0 g; Stoelting Co., USA) to the mid-plantar surface of the hind paw, ipsilateral to the nerve injury. This was conducted until a positive sign of pain behavior was elicited, at which point it was then held for 6 s. A brisk withdrawal or paw flinching was considered a positive response, in which case the next lower force filament was then applied. In the absence of such a response, the next greater force filament was then applied. In the absence of a response at 15.0 g of pressure, this cut-off value was assigned to the animal. The mechanical stimulus producing a 50% likelihood of withdrawal was determined using the up-down method [15].

Drugs were injected in 10 µl volumes by a direct lumbar puncture method between the L5 and L6 vertebrae [16]. Briefly, rats were anesthetized with sevoflurane in oxygen delivered via a nose cone. The lumbar region was shaved and prepared with Betadine solution, and the intervertebral spaces were widened by placing the animal on a 50 ml syringe body. Then, animals were administered the test drug at the L5-6 interspace using a 0.5-inch 30-gauge needle (Becton-Dickinson, USA) connected to a Hamilton syringe (Microliter™ #702, Hamilton Co., USA). The Hamilton syringe was filled with 10–20 µl of the test drug. The needle plunger was then slowly pushed over a 30 s period, delivering a volume of 10 µl, and the needle was immediately removed. The exact subarachnoid positioning of the tip of the needle was monitored using a tail- or paw-flick test [16]. Then, the animals recovered in their home cage before behavioral testing was conducted. All pain treatment groups and drugs were tested in a randomized order. The experimenters were blind to the drug treatment groups.

On the 28th day, after behavioral examination (cold allodynia, mechanical hyperalgesia, thermal hyperalgesia, mechanical dynamic allodynia, and mechanical static allodynia), the antinociceptive and anti-inflammatory effects of intrathecal R-PIA were estimated as follows. A pretreatment dose of saline (10 μl) or DPCPX (10 μg/10 μl) was intrathecally administered 15 min before intrathecal administration of R-PIA (0.3, 1.0, and 3.0 μg/10 μl). Measurements of cold and mechanical static allodynia were performed 15, 30, 45, 60, 90, 120, and 180 min after intrathecal R-PIA administration. Thirty minutes after the final measurements of cold and mechanical static allodynia (i.e., at 210 min), the animals were sacrificed. The sciatic nerve was then obtained, and biochemical estimations were conducted (Fig. 1). The doses of R-PIA and DPCPX administered in this study were chosen based on previous in vivo study involving rat models of neuropathic pain [17].