Specific-pathogen-free male Sprague-Dawley rats (200-250 g) were provided by Shanghai SLAC Laboratory Animal Center, Co. Ltd. (Shanghai, China). The animals were housed in climate-controlled cages at 20°C-25°C and 40%-70% humidity, under a 12 h/12 h light-dark cycle, with food and water ad libitum. The animal protocols followed the guidelines of the International Association of the Study of Pain (in accordance with the UK Animal Act) and had approval from the Ethics Committee of Shaoxing People’s Hospital (Shaoxing Hospital, Zhejiang University School of Medicine) (approval number: 2020-79). The rats underwent adaptive housing for 4 to 5 days before the start of experimental interventions.

A model of streptozotocin (STZ)-induced diabetes was established, based on a previous study [19]. In brief, 45-60 mg/kg of STZ was injected intraperitoneally (i.p.) to induce diabetes. On an Auto Hematology Analyzer (MED-L-900; Youte Medical Instrument Company, Guangzhou, China), the levels of blood glucose were measured after an injection of STZ in blood specimens obtained from the tail vein, to assess the suitability of the model for the study. The model was considered to be suitable with blood glucose levels above 350 mg/dL, after fasting for 3 hours, in rats within 5 weeks post-STZ administration, and used to further examine the effects of inflammatory cytokines in this study.

Rats underwent anesthesia by sodium pentobarbital injection (45 mg/kg, i.p.) and fixation in a stereotaxic apparatus for central cannulation and microinjection [20]. According to previously published reports, the following stereotaxic coordinates were used for the dorsolateral PAG (dl-PAG): 7.6 mm posterior to the bregma, 0.65 mm lateral to the midline, and 4.2 mm ventral to the brain surface. The guide cannula (0.8 mm outer diameter) was implanted and attached to the skull bone with stainless screws and cement (No. 700-00123-00, Screws-M 1.2 L 2.0 mm). A dummy cannula was inserted into and pulled out of the guide cannula twice a day for clogging prevention. After 3-5 days of recovery, drug infusion was initiated in the animals. After this period, cannula connection to an osmotic minipump (Model 2004/2006, Alzet Osmotic Pumps; DURECT Corporation, Cupertino, CA) was carried out to deliver the vehicle, artificial cerebrospinal fluid (aCSF), the mTOR antagonist (rapamycin; Sigma-Aldrich, St. Louis, MO) and a PI3K suppressor (LY294002; Sigma-Aldrich), respectively. Similarly, each inflammatory cytokine receptor antagonist (IL-1Ra, SC144; TNF-α, etanercept, or ETAN) (Tocris Co., Ellisville, MO) was loaded into the pump. These drugs were diluted in aCSF to obtain the target concentration. Rapamycin and LY294002 were administered at a concentration of 300 µM, and proinflammatory cytokine receptor antagonists were utilized at a concentration of 10 µM at 0.25 μL/hour (Alzet Model 1003D/3 day-delivery; DURECT Corporation). The injection catheter was kept in place for at least five minutes for minimizing drug reflux during catheter removal. The rats were euthanized and decapitated for brain tissue dissection under a microscope after the completion of drug administration. Behavioral tests were carried out 1 hour prior to PAG infusion completion. Three tests were performed at 30-minutes intervals.

In accordance with methods provided by Um et al. [21], mechanical paw withdrawal threshold (PWT) was assessed with an electronic von Frey Anesthesiometer (IITC Company Life Science, Woodland Hills, CA) on rat hind paws. For the Von Frey test, individual rats were placed in a dedicated chamber on an elevated mesh floor with halves were painted in white and black, respectively. The apparatus was incorporated with rigid tips (testing pain threshold) with a 0.8 mm end-diameter filament to prevent any misreading. An unbending filament was applied to the rat hind paw vertically, with licking or quick withdrawal of the hindpaw considered a positive response. Six assays were performed within 3 minutes. In order to detect changes in thermal hyperalgesia, a radiant heat plate (IITC 39; IITC Company Life Science) was used for testing rat paw withdrawal latency (PWL). The rats were kept on the plates for 15 minutes for acclimatization, and to reduce their preference before each test. Three continuous stimulations were performed at 10-minutes intervals, and the average value reflected the PWL.

After the completion of all interventions, 2% Evans blue (0.25 μL) was injected via the cannula for site identification. Immediately, the rats were euthanized, and the midbrain was removed for dissection. Then, the specimens were immersed in a paraformaldehyde solution. The proper locations of the injection sites were confirmed by locating the Evans blue dye using histological analysis based on the atlas recommended by a previous study [21].

Tissues extracted from the PAG’s dorsolateral regions under an anatomical microscope were homogenized in the RIPA buffer after all interventions. The homogenates were kept at –80°C. Commercially available enzyme-linked immunosorbent assay kits specific to TNF-α (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China), IL-6 (Jiankang Biological Engineering CO., Ltd., Shanghai, China), and IL-1β (Abcam, Cambridge, UK) were employed to assess cytokine levels in the supernatants as directed by the manufacturers.

Rats were euthanized by i.p. administering sodium pentobarbital (100-120 mg/kg). The dl-PAG tissues were rapidly obtained from the brain and snap frozen in liquid nitrogen for homogenization. Sample processing was then performed as previously described [21]. Because biotin cannot penetrate through the cell membrane, it can exclusively biotinylate cell surface proteins. Unbound biotin was washed. PAG tissue samples then underwent homogenization and centrifugation (12,000 × g, 12 min; 4°C). Protein quantitation in supernatants utilized the bicinchoninic acid method. Protein samples (200 µg) underwent incubation with streptavidin beads (20 µL) for 3 hours at 4°C, followed by washing with the RIPA buffer and centrifugation. The collected beads were boiled in sample buffer (50 µL) for 3 minutes and centrifuged as described above. Equal amounts of total and membrane protein underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis separation and electro-transfer onto polyvinylidene fluoride membranes. After blocking with 5% skim milk in tris buffered saline with Tween 20 for 1 hour at ambient, the membranes underwent successive incubations with rabbit primary antibodies against IL-1R, IL-6R, and TNFR1 (1:500; Neuromics, Edina, MN or Abcam), and horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (1:250). Visualization was performed by enhanced chemiluminescence with the LAS 4000 reagent (GE Healthcare, Chicago, IL). Autoradiography was employed for detection and the NIH Scion image software (NIH, Bethesda, MA) was utilized for quantifying immunoreactive bands by densitometry. In a similar fashion, p-PI3K, p-Akt, and p-mTOR amounts as well as the levels of their non-phosphorylated forms were determined by immunoblot, with primary antibodies from Neuromics or Abcam.

GraphPad Prism 8 (GraphPad Software, San Diego, CA) was employed for data analysis. Data are presented as mean standard error. One- or two-way analysis of variance and post-hoc Bonferroni, Tukey’s, or Dunnett’s tests were performed for comparisons. P < 0.05 indicated statistical significance.

As shown in Fig. 1, a state of stable hyperglycemia was reached 3 weeks after intraperitoneal injection of STZ. This tended to be maintained thereafter, indicating that a successful diabetes model was established. As the blood glucose level increased, the mechanical threshold and latency of pain responses were amplified. STZ evidently decreased PWT and PWL compared with control values in rats 3-5 weeks post-treatment. Therefore, in this study, rats at 5 weeks post-STZ administration were selected as candidates to assess the impacts of inflammatory cytokine signaling.

As depicted in Fig. 2A, B, and C, remarkably increased amounts of phosphorylated p-PI3K, p-Akt, and p-mTOR were recorded after STZ administration in comparison with the control group (n = 8-10). Furthermore, PWT and PWL were significantly decreased in STZ-treated animals (n = 12) in comparison with controls (n = 12). However, no marked differences in total PI3K, Akt, or mTOR protein amounts were observed between the STZ and control groups. Rapamycin (n = 10) and LY294002 (n = 12) were used to block mTOR and PI3K, respectively, in the PAG. In this study, both PWT and PWL were markedly elevated during the 30-minutes test interval (Fig. 2D, E). Importantly, PWT and PWL were elevated in control animals compared with STZ rats receiving the above blockers.

Compared with control values, PAG concentrations of TNF-α, IL-1β, and IL-6 were prominently elevated in STZ rats (Fig. 3A, n = 8). Moreover, the expression levels of the respective cytokine receptors, namely TNFR1, IL-1R, and IL-6R, were also evidently upregulated in the membrane of the PAG tissue (Fig. 3B, C, n = 8-10). However, total PAG protein amounts of TNFR1, IL-1R, and IL-6R were similar in the control and STZ groups. The optical densities of TNFR1, IL-1R, and IL-6R protein bands were 1.08 ± 0.21, 0.98 ± 0.20, and 1.02 ± 0.17, respectively, in control animals, versus 1.01 ± 0.15, 1.11 ± 0.12, and 0.98 ± 0.15, respectively, in the STZ group (n = 8-10 in each group).

In order to further determine the roles of cytokines in pain development and modulation, cytokine receptor blockers (ETAN, IL-1Ra, and SC144) were used to inhibit cytokine receptors in the PAG. The results showed that PWT and PWL in each corresponding antagonist group were remarkably increased during the three tests performed at 30-minutes intervals (Fig. 3D to F, STZ animals administered a given antagonist vs. non-treated STZ animals, n = 8-10).

As shown in Fig. 4, injection of ETAN (TNF-α blocker) and SC144 (IL-1Ra blocker) into the PAG led to reduced p-PI3K and p-mTOR amounts in STZ-treated animals (P < 0.05 vs. STZ animals without receptor inhibitor administration; n = 6-10/group). Interestingly, p-PI3K and p-mTOR amounts were similar in STZ rats receiving inhibitors and control animals.