Foren

Stereotactic lesions of the AcbC were made using a standard stereotaxic apparatus (David Kopf Instruments). Rats were first injected with pentobarbital sodium (i.p.; 50 mg/kg body weight, Dainippon Sumitomo Pharma Co., Ltd) dissolved in saline and then maintained on anesthesia with isoflurane (Foren; Abbott Japan Co., Ltd.) for the surgical procedure. Bilateral lesions were made by infusing ibotenic acid (0.6 µl/hemisphere of 5 mg/ml ibotenic acid in PBS; Sigma Aldrich, Japan) through a stainless steel needle (200 µm outer diameter) attached to the stereotaxic device. Coordinates for injection sites were as follows: AP, +1.9 mm rostral to bregma; ML, ±1.7 mm lateral to the midline; V, −6.3 mm ventral to the dura mater. Sham-lesioned rats received injections of the same volume (0.6 µl/hemisphere) of PBS at these coordinates. The needle was left in place for 15–20 min to allow the toxin to diffuse sufficiently within the target sites. Rats were allowed to recover for 7 days following the lesion surgery. For the first 4 days after surgery, rats were given daily i.p. injections of antibiotic solution (0.4 ml/day of 1 mg/ml gentamicin in saline; Schering-Plough).

To prepare mouse models of allodynia, peripheral nerve injury at the distal side of the dorsal root ganglion (DRG) was induced in six wild-type adult mice, as previously described53 (link)54 (link). In brief, after mice were deeply anesthetized with 2–3% isoflurane (Foren; Abbott, Tokyo, Japan), a midline incision was made in the dorsal region of L6 to L4 and the transverse process at L5 was removed using forceps. The exposed L4 spinal nerve root was compressed with forceps and the distal region was immediately transected from the site of compression. The muscle layer and skin were closed with sutures. In the sham-operated group, the L4 spinal nerve root was exposed without any injury. All animals recovered under observation in a heated cage.

Magnetic resonance imaging (MRI) was performed on the mice at 6, 15, and 20 weeks of age using a 7.0-Tesla magnet (BioSpec 70/16; Bruker BioSpin, Ettlingen, Germany) with a cryogenic quadrature RF surface probe (CryoProbe; Bruker BioSpin AG, Fällanden, Switzerland) to improve the sensitivity [15 (link)-18 (link)]. The MRI was performed under general anesthesia induced by the intramuscular injection of ketamine (50 mg/kg; Sankyo, Tokyo, Japan) and xylazine (5 mg/kg; Bayer, Leverkusen, Germany) and maintained by isoflurane (Foren; Abbott, Tokyo, Japan). The animal’s pulse, arterial oxygen saturation, and rectal temperature were monitored during the MRI. The scanning parameters were as follows: Saggital T2-weighted images (RARE, eTE/TR: 37.5/2000 ms), axial T2-weighted images (RARE, eTE/TR: 21.5 ms/1200 ms). To examine the extent of spinal cord compression due to extradural calcified deposits, the transverse areas of the calcification and spinal canal were measured on the axial T2-weighted images of the twy mice, and the canal stenosis ratio was calculated as reported previously [10 (link)].