Suppression of MyD88-dependent signaling alleviates neuropathic pain induced by peripheral nerve injury in the rat

The adult male Sprague-Dawley rats (120–180 g) were used in our study. All animals were housed (4–5 rats per cage) in a standard 12-h light/dark cycle. These experiments were approved by the Institutional Animal Care and Use Committee in Chinese Academy of Medical Sciences, Institute of Basic Medical Sciences. Animals were randomly assigned to treatment or control groups.

In accordance with the study of Bennett and Xie [23], we performed chronic constriction injury (CCI) in the rats anesthetized through intraperitoneal injection of sodium pentobarbital (40 mg/kg) under aseptic condition. After the sciatic nerve of the mid-thigh level on right side was exposed, four snug ligatures of chromic gut suture were loosely tied around the nerve with about 1 mm space between the knots. The sciatic nerves of sham animals were exposed without ligation.

A PE10 catheter (length, 18 cm) was implanted intrathecally according to the protocol as described previously [24, 25]. Briefly, rats were anesthetized with sodium pentobarbital (40 mg/kg) and a 2-cm longitudinal incision was made above vertebrae L5–6.The catheter with guide wire was inserted in the hole that was made by a 23-gauge needle between lumbar vertebrae L5 and 6. The catheter was pushed through the intervertebral space until a sudden movement of tail or the hindlimb was observed and then passed gently 2 cm upward to reach the lumbar enlargement. The tip of the catheter was fixed on the neck area of the rats. After implantation, catheter localization was verified by intrathecal injection of lidocaine and all rats were observed for a minimum of 5 days before being performed with CCI surgery as described above. Rats showing motor weakness or without catheter in spinal space were excluded from the research. An intrathecal injection of 500 μM of MyD88 homodimerization inhibitory peptide (MIP, Novus Biologicals, CO, USA) or control peptide (Novus Biologicals, CO, USA) resolved in PBS was administrated on preoperative and postoperative days. A previous report showed that application of similar dose of MIP did not induce any significant change in locomotor function in the rats [18].

All the behavioral measurements were carried out by an experimenter blinded to schedule during 10 am–3 pm. Mechanical allodynia was assessed by the up-down method using calibrated Electronic von Frey (Electronic von Frey 2393:II TC, USA). Rats were acclimated in suspended cages with wire mesh floor. The probe was applied perpendicularly to the paw with no acceleration at a force and held for approximately 6–8 s. An acute withdrawal of the paw was considered a positive response. Thermal hypersensitivity was tested using plantar test as described previously [24, 25]. The three measurements of mechanic threshold or thermal latency per side were averaged.

The L4–6 spinal dorsal horn and DRGs were harvested from rats that were deeply anesthetized with sodium pentobarbital (40 mg/kg) and snap-frozen in liquid nitrogen. Tissues were homogenized on ice in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1% NP40, 1 mM EDTA, 0.5% Na deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) mixed with proteases inhibitor cocktail and phosphatases inhibitor cocktail (Sigma-Aldrich, MO, USA). After centrifuging, supernatants were collected and denatured with SDS-PAGE loading buffer consisting of 0.25 M Tris-HCl, 52% glycerol, 6% SDS, 5% β-mercaptoethanol, and 0.1% bromophenol blue for 5 min at 95 °C. Equal concentrations of lysates (30 μg) were separated by SDS-polyacrylamide gel electrophoresis gels and transferred to a PVDF membrane (GE Healthcare life science). After blocking with 5% BSA in TBST (Tris-buffered saline with 0.1%Tween 20) for 1 h at room temperature, membranes were incubated in TBST overnight at 4 °C with primary antibodies. The membranes were washed with TBST and then incubated with secondary antibodies diluted with 5% skim milk in TBST for 1 h at room temperature (Table 1 lists the primary and secondary antibodies used for Western blot analysis). Bands were detected with enhanced chemiluminescence reagents eECL Kit (CWBio, Beijing, China) using an ImageQuant LAS 4000 mini Chemiluminescence imaging analysis system (GE Healthcare life science, PA, USA), and the band densities were analyzed using ImageJ software (NIH, USA). For the detecting of total extracellular signal-regulated protein kinase (ERK) or total NF-κB protein, we again blocked the same member that was washed by Western blot antibody Stripping Buffer (CWbio, Beijing, China) with 5% BSA in TBST for 1 h at room temperature after detecting the phosphorylated ERK or phosphorylated NF-κB protein, incubated the member with primary antibodies (anti-total ERK or total NF-κB protein) overnight at 4 °C, and then incubated with secondary antibodies for 1 h at room temperature. Members that were re-incubated by primary and secondary antibodies were detected with enhanced chemiluminescence reagents eECL Kit using ImageQuant LAS4000 mini Chemiluminescence imaging analysis system.

Rats were deeply anesthetized with sodium pentobarbital (40 mg/kg) and then perfused with warm saline followed by fresh 4% paraformaldehyde through the ascending aorta. The L4–6 spinal dorsal horn (SDH) and DRGs were collected, fixed in 4% paraformaldehyde for 4 h, and then dehydrated in 30% sucrose overnight at 4 °C. Tissues were mounted and finally cut to a thickness of 15 μm in a cryostat for immunofluorescent staining. After blocking in 10% normal donkey serum and 0.2% Triton X-100 in PBS for 1 h at room temperature, the tissue sections were incubated overnight at 4 °C in 10% normal donkey serum in PBS containing primary antibodies and then incubated with the proper secondary antibodies or Alexa Fluor 594-conjugated isolectin B4 (IB4) (1:100, Invitrogen/Thermo Fisher Scientific, USA) for 1 h. The slides were then washed in PBS and cover-slipped with VECTASHIELD Mounting Medium with DAPI. Table 2 lists the primary and secondary antibodies used for the immunofluorescence staining analyses. The percentages of positive neurons to total neurons were calculated and statistically analyzed. Images were captured using a confocal laser scanning microscope (FV1000, Olympus) and Olympus FluoView software. At least 12 fields of view (×200) from three sections on each ganglion were examined. DRG neurons were classified according to the cross-sectional areas of soma as small- (area <636 μm²), medium- (area 637–1431 μm²) and large-sized (area >1431 μm²). This classification is based on the criteria as previously described [26].