2.1. Animal preparation
Male Sprague-Dawley rats (n=24, 250-300 g, Koatech, Pyeongtaek, Korea) were used in this experiment. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei University Health System (protocol number 2016–0061). All experiments were performed in accordance with the IACUC guidelines and regulations. Animals were housed in plastic cages with soft bedding on a 12-hour light/dark cycle (light cycle: 08:00 ~ 20:00), at a constant temperature (22 ± 2℃) and humidity (50 ± 10%). Experimental procedure according to the time sequence is summarized in Fig. 1. Rats were anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (50 mg/kg). Deep anesthesia was verified by loss of nociception in response to a tail pinch stimulation. The surgical procedure for nerve injury was performed following the methods described in our previous report [22, 23]. The left sciatic nerve was exposed, and three major divisions were clearly separated. Then, the tibial and sural nerves were tightly ligated and transected. Complete hemostasis was confirmed, and the wound was closed with muscle and skin sutures.
Changes in the mechanical paw withdrawal threshold were measured before nerve injury (pre) and at 1, 4, and 7 days after nerve injury. Rats were habituated for 10 min to the testing cages, which consisted of metal mesh floors under plastic domes. Mechanical allodynia was measured by assessing thresholds for hind paw withdrawal upon stimulation with an electrical von Frey (Ugo Basile, Varese, Italy). Mechanical forces were recorded with each withdrawal. Responses were measured seven times, and the means were calculated after the maximum and minimum values were excluded (supplementary fig.1).
2.2 Agent administration
On postoperative day 7, all rats were anesthetized with urethane (0.5 g/kg, i.p.) and α-chloralose (25 mg/kg). In addition, atropine (20 µg/kg, i.p.) was injected to avoid excessive mucus secretion in the trachea. The depth of anesthesia was determined by a lack of flexor muscle response to hind-paw pinching. Then, the rats were mounted on the surgical stage. Skin incision was performed on the right ventral aspect of the neck, and the common carotid artery and external carotid artery (ECA) were gently exposed. Polyethylene tubing (PE-10) was cannulated into the ECA.
Following catheter insertion, a 20% D-mannitol solution (35±2℃, 5 ml/kg, Dai Han Pharm, Seoul, Korea) was infused to open the blood–brain barrier. To investigate pain-dependent signal changes in the whole brain following mTOR inhibition, nerve-injured rats were divided into three groups (Control, n=8; Torin1, n=8 and XL388, n=8). Then, the vehicle (0.06 % DMSO in saline, 1 ml), Torin1 (400 nM in vehicle, 1 ml), and XL388 (500 nM in vehicle, 1 ml) were infused in each group of rats. Following the application, 20 mM of manganese chloride (MnCl2–4H2O, Sigma, St. Louis, MO, USA) was injected for MR imaging. All infusions were injected via the ECA (150 µl/min) using a syringe pump (22 Infusion Syringe Pump, Harvard Apparatus, Holliston, MA, USA). For noxious stimulation, electric stimulations (3 mA, 2 Hz, 1 ms width; A385, WPI, Sarasota, FL, USA) were applied to the left hind paw during manganese chloride injection.
2.3. MR measurements
MR imaging was performed using a 9.4-T horizontal Biospec small bore scanner (Bruker BioSpin, Ettlingen, Germany) with an 86-mm volume coil to transmit the radio frequency (RF) and a four-channel array coil to receive the signal. After tuning and shimming, T2 multi-slice spin echo sequence images were acquired for positioning. T1W images were taken at the same positions. High-resolution anatomical images were obtained with a rapid acquisition with relaxation enhancement (RARE) protocol using the following parameters: Effective TE = 28 ms, TR = 4750 ms, FOV = 32 x 32 mm, matrix = 192 x 192, and RARE factor = 8. Fifteen slices of T1W images were acquired using the RARE protocol with shortened TR. The imaging parameters were RARE factor = 2, TE/TR = 8/412 ms, FOV = 32 x 32 mm, matrix = 192 x 192, and slice thickness = 1 mm. Respiration rates of animals were monitored during the experiments. Functional analysis was performed using ParaVision (Version 5.0, Bruker BioSpin). The whole brain was outlined on axial slices, and the mean signal was calculated. To quantify data from the series of T1W images and measure the Mn2+ enhancement in separated brain regions, ROIs were set to the entire brain regions that were covered in coronal images. ROIs, the insular cortex (IC), primary somatosensory cortex of the hind limb (S1HL), motor cortex 1/2 (M1/2), anterior cingulate cortex (ACC), and visual cortex 1/2 (V1/2) were manually drawn on each separate image. Signal magnitudes from each brain region were normalized to signal intensity in the temporalis muscle. Image analysis was performed using ParaVision (Bruker Biospin), MRVision (MRVision Co., Winchester, MA, US), and ImageJ software (National Institutes of Health, Bethesda, MD, US). Data are presented as mean ± standard error of the mean (SEM). After MRI scans were taken, the rats were humanely killed with an overdose of urethane (1.5 mg/kg).
2.4. Statistical analysis
To quantify the data from T1W images and the measurement of Mn2+ enhancement in the brain, each ROI was set to the brain atlas covered in coronal images. Relative signal intensities were calculated and compared within groups. Data are presented as mean ± SEM. One-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc pairwise comparisons were used to identify the mean difference of the ROI contrast. All statistical analyses were performed with Prism 5 software (Version 8.03; GraphPad software Inc, San Diego, CA, US). P < 0.05 indicated a statistically significant difference.