Blocking mammalian target of rapamycin (mTOR) improves neuropathic pain evoked by spinal cord injury

Animals

All animal protocols were approved by the Animal Care and Use Committee of our Medical Research Institute, and were carried out in accordance with the guidelines of the International Association for the Study of Pain. Male Wistar rats weighing 200-250 g were obtained from the Center for Experimental Animal Sciences. The rats were housed in individual cages with free access to food and water and were kept in a temperature-controlled room (25°C) on a 12/12 h light/dark cycle.

A model of spinal cord injury

There are a number of animal models generally used to study the mechanisms of spinal cord injury. For example, SCI is induced by epidural balloon inflation and by application of an impactor on the spinal cord of the rat [14-16]. In the current study, all the rats were anesthetized by sodium pentobarbital (60 mg/kg, intraperitoneally) and a laminectomy was then performed to expose spinal segment T10. The Infinite Horizon impactor (Precision Systems and Instrumentation, Lexington, KY, USA, 0.0015 N, 1 s dwell time) was used to produce a contusive spinal injury [14]. Following the injury, the musculature and skin were sutured. The animals were allowed to recover from the surgery. A subcutaneous injection of 0.3 mL of enrofloxacin (25 mg/mL twice daily) was given for 7 days and bladders were manually expressed twice daily. Behavioral test to examine mechanical allodynia and thermal hyperalgesia was performed 4 weeks following SCI.

Intrathecal catheter for administration of drugs

The rats were anesthetized by sodium pentobarbital (60 mg/kg, intraperitoneally) in order to implant an intrathecal catheter for administration of drugs 3 days prior to each experiment. Briefly, one end of polyethylene-10 tubing was inserted intrathecally through an incision in the cisternal membrane and advanced 7-9 cm caudal until the tip of the catheter was positioned at the lumbar spinal level (L5 to L6). The other end of the intrathecal tubing was sutured to the musculature and skin at the incision site and externalized to the back of the rat. In each experiment, a Hamilton microsyringe (250 μL) was connected to the intrathecal tubing and used to deliver 100 μl of dimethyl sulfoxide (DMSO) as vehicle control, antagonists to mTOR (rapamycin, 10 μg, obtained from Selleck Chem, Houston, TX, USA) and inhibitor of PI3K (LY294002, 10 μM, obtained from Sigma-Aldrich, St. Louis, MO, USA).

In a subset of studies, in order to examine the effects of mTOR and PI3K on the levels of substance P and CGRP and the effects of blocking PI3K on mTOR expression, rapamycin (10 μg) and LY294002 (10 μM) were intrathecally given using an infusion pump in control rats and rats following SCI, respectively. The pump was set to constantly deliver vehicle or the drugs over a period of 3 h. At the end of infusion, the superficial dorsal horn tissues were obtained under an anatomical microscope for Western Blot and ELISA experiments.

Behavioral test

To quantify the mechanical sensitivity of the hind paw, rats were placed in individual plastic boxes and allowed to acclimate for > 30 min. Mechanical paw withdrawal threshold (PWT) of a rat hind paw in response to the stimulation of von Frey filaments was determined. A series of calibrated von Frey filaments (ranging from 0.5 to 18.0 g) were applied perpendicularly to the plantar surface of the hind paw with a sufficient force to bend the filaments for 60 s or until paw withdrew. In the presence of a response, the filament of next lower force was applied. In the absence of a response, the filament of next greater force was applied. To avoid injury during tests, the cutoff strength of the von Frey filament was 18 g. The tactile stimulus producing a 50% likelihood of withdrawal was determined using the “up-down” method [17]. Each trial was repeated two times at approximately 2 min intervals. The mean value was used as the force producing a withdrawal response [18].

To determine thermal hyperalgesia, rat paw withdrawal latency (PWL) to a radiant heat was measured as described previously [19]. Rats were placed individually in plastic cages on an elevated glass platform and allowed for 30 min acclimation. Each hind paw received three stimuli with a 10 min interval, and the mean of the three withdrawal latencies was defined as PWL. The heat was maintained at a constant intensity. To prevent tissue damage, the cut-off latency was set at 20 s. All the behavioral tests were performed in a blind style.

Western blot analysis

The rats were euthanized and the superficial dorsal horn (lumbar 4-6) tissues were dissected under an anatomical microscope. The tissues were then homogenized in ice-cold radioimmunoprecipitation assay buffer containing 25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) with protease inhibitor cocktail kit (Sigma-Aldrich, St. Louis, MO). Then the tissues were centrifuged. The supernatant was then diluted to the same volume and applied to SDS-PAGE. Membranes were incubated with the rabbit anti-p-mTOR/p-S6K1/p-4EBP1antibodies; rabbit anti-mTOR /S6K1/4EBP1 antibodies; rabbit anti-p-PI3K p85/ anti- PI3K p85, respectively. All these primary antibodies were purchased from the Abcam Company (Cambridge, UK); and goat antirabbit secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Immunoreactive proteins were detected by enhanced chemiluminescence. The membrane was also processed to detect β-actin for equal loading. The optical density of protein bands was first analyzed using the NIH Scion image Software ImageJ (http://rsb. info.nih.gov/ij/), and values for densities of immunoreactive bands/β-actin band densities from the same lane were determined. Each of the values was then normalized to a control sample.

ELISA measurements

To examine the levels of substance P and CGRP in the superficial dorsal horn of the spinal cord (L4-L6), enzyme-linked immunosorbent assay (ELISA) methods were employed. Substance P was measured using substance P ELISA kit following the manufacturer’s instructions (Abcam Company, Cambridge, UK). Briefly, the diluted tissue supernatant (100 μl) was placed in a 96-well goat anti-mouse IgGcoated plate and incubated for 2 hours. After incubation, the plate was washed using the provided washing buffer, and the color was developed by adding p-nitrophenyl phosphate (pNPP, 200 μl) substrate after 45 min and determined by an ELISA plate reader. The amount of substance P was calculated by using a substance P standard curve. In a similar way, the CGRP content of the samples (100 μl supernatant) was determined using a commercial CGRP ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, the diluted samples were placed in a 96-well plate incubated with pre-coated anti-rat IgG antibody overnight, washed and developed, and quantified [20].

Statistical analysis

All data were analyzed using a one-way analysis of variance. Values were presented as means ± standard deviation (SD). For all analyses, differences were considered significant at level of α < 0.05. All statistical analyses were performed by using SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL, USA).

Effects of mTOR and PI3K on SCIinduced hyperalgesia

After mechanical and thermal hyperalgesia were well established in rats, rapamycin and LY294002 were intrathecally injected. Mechanical and thermal sensitivities were examined 0, 1, 2, 3, 4, 5, 6, 7 and 8 h after injection of the drugs. Figure 1 (left panel) showed that rapamycin (number of rats =15) significantly attenuated SCI-induced mechanical and thermal hyperalgesia (all P values < 0.05 vs. vehicle control, number of rats = 10). The inhibitory effects of rapamycin on mechanical and thermal hyperalgesia began from about 60 min after its administration, peaked at 2-3 h and lasted for 8 h (PWT) and 6 h (PWL). Similarly, Figure 1 (right panel) demonstrated that LY294002 had significant attenuating effects on SCI-induced mechanical and thermal hyperalgesia in a time manner (all P values < 0.05, LY294002 vs. vehicle, number of rats = 12 for each group). At this dose, the effects of LY294002 appeared at about 60 min, peaked at 2-3 h and lasted for about 8 h (PWT) and ~6 h (PWL) after injection.

Figure 1

Effects of blocking mTOR and PI3K pathways on SCI-evoked mechanical and thermal hyperalgesia. Intrathecal administration of rapamycin (left panel) and LY294002 (right panel) increased PWT and PWL in SCI rats. *all P values < 0.05, indicated vs. vehicle control. Number of rats = 10 in vehicle controls; and number of rats = 15 in group of rapamycin; number of rats = 12 in each of vehicle control LY294002 groups.

Expression of mTOR signaling pathways engaged in SCI-induced hyperalgesia

Figure 2A showed the protein expression of p-mTOR, p-S6K1 and p-4E-BP1 as well as mTOR, S6K1 and 4E-BP1 in control rats and SCI rats. SCI significantly increased the protein levels of p-mTOR and mTOR-mediated p-S6K1 and p-4E-BP1 in the superficial dorsal horn as compared with control rats (all P values < 0.05, SCI rats vs. control rats, number of rats = 6-10 in each group). Note that total protein of mTOR, S6K1 and 4E-BP1 levels was not significantly increased in SCI rats. We also examined the effects of blocking PI3K on expression of p-mTOR and p-S6K1. Figure 2B demonstrated that the protein expression of p-mTOR and p-S6K1 was significantly increased in SCI rats (number of rats = 6-10) as compared with control rats (number of rats = 6-8). When LY294002 was infused into the spinal cord of SCI rats via a pump, the amplified activities of p-mTOR and p-S6K1 evoked by SCI were significantly attenuated (number of rats = 6-10 in each group).

Figure 2

Expression of mTOR pathways in the superficial dorsal horn of the spinal cord of control rats and SCI rats. A. Typical bands and averaged data showing that p-mTOR, p-S6K1 and p-4E-BP1 in the dorsal horn of the spinal cord were upregulated in SCI rats. *all P values <0.05 vs. control rats (number of rats = 6-10 in control and SCI). There were insignificant differences in total protein expression of mTOR, S6K1 and 4E-BP1 in control rats and SCI rats (number of rats = 6-10). B. Typical bands and averaged data showing expression of p-mTOR/mTOR and p-S6K1/S6K1 were amplified in the dorsal horn of SCI rats with vehicle treatment (number of rats = 6-10) as compared with control rats (number of rats = 6-8). Blocking PI3K pathways by LY294002 attenuated increases in p-mTOR and p-S6K1 in SCI rats. *all P values <0.05 vs. control animals and SCI animals infused with LY294002 (number of rats = 6-10 in each group).

The levels of substance P and CGRP

In additional experiments, we examined the effects of SCI on the levels of substance P and CGRP in the superficial dorsal horn of the spinal cord. Figure 3 showed that substance P and CGRP were significantly increased in SCI rats (number of rats = 15) as compared with control rats (number of rats = 15). Furthermore, blocking individual mTOR and PI3K signaling pathways by intrathecal injection of rapamycin and LY294002 (number of rats = 10 in each group) significantly attenuated amplifications in substance P and CGRP evoked by SCI. Note that a greater inhibitory effect on substance P and CGRP was observed by LY294002 compared with rapamycin.

Figure 3

Effects of blocking mTOR and PI3K pathways on the levels of substance P and CGRP in the superficial dorsal horn of the spinal cord. SCI significantly increased substance P and CGRP as compared with controls and blocking mTOR and PI3K by rapamycin and LY294002 significantly attenuated the enhancement in substance P and CGRP evoked by SCI. Note that a greater inhibitory effect on substance P and CGRP was observed by LY294002. *P = 0.0081 (for substance P) and P = 0.0093 (for CGRP), indicated SCI rats (number of rats = 15) vs. control rats (number of rats =15) and SCI rats with rapamycin (n=10) and LY294002 (number of rats =10) injection. #P = 0.017 (for substance) and P = 0.012 (for CGRP), indicated SCI rats with rapamycin injection vs. with LY294002 injection