Animals
Male Sprague–Dawley rats (180 g to 200 g) were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences. The animals were allowed to adapt to the laboratory for minimus of 2 h prior to testing, and used only once. All animal procedures and experimental protocols in this study were approved by the “Institutional Animal Care and Use Committee (IACUC)” of Chinese Academy of Medical Sciences (“Tab of Animal Experimental Ethical Inspection” number: Acuc-A02–2015–005; IACUC Chairman: Minli Li). All protocols were also consistent with the NIH Guide for Care and Use of Laboratory Animals (NIH Publication No. 80–23).
Intrathecal catheter implant surgery
Each rat was anesthetized with 50 mg/kg sodium pentobarbital intraperitoneally (i.p.) and an intrathecal catheter was implanted as described by Yaksh and Rudyb [12]. Briefly, a polyethylene tubing (PE-10) filled with sterile normal saline was inserted through a small incision on the atlantooccipital membrane and extended caudally to the lumbar enlargement of the spinal cord. Studies involving rats with chronic intrathecal catheters were conducted after implantation. Rats were monitored daily for signs of neural dysfunction and were removed from the study if neurological dysfunction was noted. Overall, no animals were excluded in the diabetic model experiments.
Induction and assessment of diabetes in rats
Experimental diabetes was induced by a single i.p. injection of STZ (Sigma-Aldrich Co., St. Louis, MO, USA). The STZ solution was prepared freshly by dissolving normal saline and was injected intraperitoneally at a dose of 65 mg/kg on the seventh day after surgery as previously described [13]. Diabetes induction was assessed by weekly measurement of the tail vein blood glucose level by using a blood glucose meter (HEA-214, OMRON, USA). Body weight was also monitored. Only rats with blood glucose concentration exceeding 240 mg/dL were considered diabetic and used for the study.
The effect of KN93 on diabetic models
The rats were randomly divided into seven groups (n = 6 per group): Group Naive; Group Normal Saline (NS), which received only the NS; Group STZ, which received STZ; Group STZ + Sham, which sham operation after administration of STZ; Group STZ + DMSO, which received DMSO intrathecally after the administration of STZ; Group STZ + KN93 (50 μg), which received KN93 (50 μg) intrathecally after the administration of STZ; Group STZ + KN93 (100 μg), which received KN93 (100 μg) intrathecally after the administration of STZ.
KN93 obtained from Calbiochem was dissolved in sterile DMSO 20 μl and injected intrathecally on 15, 17, 19 days after the STZ administration respectively. The dose of KN93 was chosen based on previous publications [14].
Behavioral tests
Paw mechanical withdrawal threshold (MWT) was used to measure mechanical allodynia and was assessed by testing the left hind paw withdrawal response to Electronic Von Frey filaments (Stoelting Co., USA). Brisk withdrawal or paw flinching was considered a positive response. Each rat was tested five times per stimulus strength.
Paw thermal withdrawal latency (TWL) was used to measure thermal hyperalgesia and performed by using a heat pain stimulator (PL-200, Taimen Biotech Company, Chengdu). The measurement was repeated five times for each rat (interval ≥ 5 min), and the mean was calculated as PWL for this measurement.
Western blot analysis
Total protein was extracted from the L4 and L5 DRGs using methods described earlier [15]. The primary antibodies against pCaMKIV, CaMKIV, HMGB1 (1:500; Santa Cruz, USA) were used in Western blot (WB). To verify equal loading of protein, the blots were reprobed with primary monoclonal antibody against β-actin (ProteinTech Company, USA).
Immunohistochemistry
Immunofluorescent labeling of the following markers was performed on different group rat lumbar DRG cryosections using the methods as previously described [16]. The primary antibodies in the immunohistochemistry: rabbit polyclonal and mouse monoclonal antibodies against pCaMKIV, HMGB1, isolectin B4 (IB4), protein gene product 9.5 (PGP9.5), calcitonin gene-related peptide (CGRP), 1:200, Santa Cruz. The secondary antibodies used: Alexa Fluor 555-conjugated donkey-anti-mouse, 1:500; Alexa Fluor 488-conjugated donkey-anti-rabbit, 1:500, Invitrogen.
The cells were visualized using a laser confocal microscopic imaging system (Nikon A1; Nikon Co.Ltd., Tokyo, Japan) with a water immersion 10 objective lens (Plan Apo 60x1.20 PFS WI) and a 1 st dichroic mirror (405/488/561/640). The green signal was excited by 488 nm light from an Ar laser and red signal was excited by 561 nm light from a DPSS laser. The images were 400 times magnification and the scale bar in Fig. 4 was 20 μm.
Transfection of siRNA
RAW 264.7 cells (2*104) were plated in 0.5 ml of growth medium (without antibiotics) in each well of a 24-well plate. The sequence of negative control siRNA (N.C. siRNA) and CaMKIV siRNA obtained from Zhang X et al. [11]. The protocol is also comes from Zhang X [11].
Statistical analysis
Data were presented as means ± standard deviation. A behavioral test was evaluated by two-way repeated measures analysis of variance (ANOVA). The expression of pCaMKIV and HMGB1 were analyzed by one-way or two-way ANOVA with Student Newman Keuls post hoc analysis. P-values of <0.05 were considered statistically significant.
