Spinal nerve ligation and assessment of mechanical allodynia

Forty-six adult male Sprague-Dawley rats weighing 250–300 g (Orient Bio, Kyunggi-Do, Korea) were used. The rats were housed in separate cages and acclimatized for at least 7 days in a controlled-temperature room before use. The room had artificial light with a 12/12 h day/night cycle, and food pellets and water were provided ad libitum. We used the rat model of chronic spinal nerve ligation-induced peripheral neuropathy described by Kim and Chung [13]. The surgical procedure was performed aseptically under inhaled isoflurane anesthesia (3% for induction and 2% for maintenance) with 100% oxygen. The lower back was shaved and a left paravertebral incision (L3–S2) was made. After resection of the left L6 transverse process, the left L5–L6 spinal nerve was ligated tightly with 6–0 black silk under a microscope. The skin wound was closed with 5–0 black silk, and bacteriostatic powder was applied topically to the wound site. The sham operation was identical, with the exception that L5–L6 spinal nerve ligation was not performed. Development of the neuropathic pain was confirmed by a withdrawal of the right hindpaw in response to a tactile stimulus using von Frey filaments (Stoelting, Wood Dale, IL, USA) at 6 days after the nerve injury and followed up at 3 and 6 weeks, as previously described [14]. Briefly, the rats were placed in a Plexiglas box with a stainless steel mesh floor and allowed to acclimatize for at least 30 min before performing the von Frey test. Eight filaments (0.41, 0.70, 1.20, 2.00, 3.63, 5.50. 8.50 and 15 g) were consecutively applied to lift the plantar surface of the right hindpaw. Withdrawal of the right hindpaw was considered a positive result, and the absence of withdrawal within 5 s was considered a negative result. Development of hyperalgesia was determined by the comparison with the baseline values, obtained from the right hindpaw during the presurgical period (P < 0.05) (Fig. 1). After confirming the development of neuropathic pain, the rats were randomly allocated into the 1-, 3-, or 6-week group, or the sham-operated group, and half of the spinal cord ipsilateral to the nerve injury was harvested. The 1-week timepoint was included to detect early changes in the spinal cord in acute phase because T cell infiltration begins 3 days after nerve injury. The 3-week timepoint was included as this is the time of peak T cell infiltration [7]. The 6-week timepoint represents the chronic phase of the disease.

The results of von Frey mechanical allodynia test at 6, 21 and 42 days. * P < 0.05 versus sham-operated group