Male Sprague–Dawley rats (3–4 weeks of age) were housed in individual cages. The rats had free access to food and water. Room temperature was controlled at 23 ± 2°C during a 12 h light/dark cycles. All animal experimental protocols were approved by the Animal Research Ethics Committee of Hubei University of Science and Technology (2016-03-005). To evaluate the effect of intrathecal treatment with metformin on sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats, animals were randomly divided into three groups (n = 18, 6 rats in each group). In control group (CON), rats were injected corresponding vehicle intraperitoneally and intrathecally, respectively. In paclitaxel-treated group (PAC), rats received intraperitoneal injection (i.p.) of paclitaxel (2 mg/kg per injection at day 1, 3, 5, and 7) and intrathecal injections (i.t.) of 50 μl vehicle. In paclitaxel-metformin (i.t.)-treated group (MET), metformin (100 ug in 50 ul, at day 8, 9, and 10) was intrathecally treated in paclitaxel-treated rats. To further investigate the effect of systemic treatment with metformin on sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats, rats were randomly divided into three groups (n = 18, 6 rats in each group). In control group (CON), rats were injected corresponding vehicle intraperitoneally. In paclitaxel-treated group (PAC), rats received intraperitoneal injection (i.p.) of paclitaxel (2 mg/kg per injection at day 1, 3, 5, and 7) and 50 μl vehicle (i.p. at day 8, 9, and 10). In paclitaxel-metformin (i.p.)-treated group (MET), metformin (200 mg/kg, at day 8, 9, and 10) was intraperitoneally treated in paclitaxel-treated rats. At day 11 or 12, spinal cord slices were prepared to record sEPSCs in all rats. To study the acute effect of metformin on sEPSCs, spinal slices (at day 11 or 12) from paclitaxel-treated rats were incubated by oxygenated ACSF containing metformin (2 mM) or vehicle for 1 h, and then sEPSCs were recorded.

Intrathecal catheters were implanted on rats 15 days before paclitaxel treatment. Under deep anesthesia with a mixture of ketamine (75 mg/kg) and medetomidine (0.5 mg/kg), rats were inserted a polyethylene-10 catheter into their subarachnoid space through L5–L6 intervertebral space, and the tip of the catheter was located at the L5 spinal segmental level. To establish a mechanical allodynia model, rats received intraperitoneal injection (i.p.) of paclitaxel (Sigma-Aldrich, 2 mg/kg) at day 1, 3, 5, and 7, with a cumulative dose of 8 mg/kg. Paclitaxel was dissolved in a 1/1 Cremophor EL (Sigma-Aldrich)/ethanol solution then further prepared with 0.9% saline for injection. Rats in the control group received the vehicle (Cremophor EL/ethanol, 1:1) on the same four alternate days. Metformin (Sigma-Aldrich) was treated at day 8, 9, and 10. Metformin was dissolved in artificial cerebrospinal fluid and delivered through the intrathecal catheter. Metformin was also dissolved in 0.9% saline and intraperitoneally injected. After paclitaxel and metformin were applied, rats were sacrificed to prepare spinal cord slices for electrophysiological recordings at day 11 or 12.

The rats were anesthetized with 1.5 mg/kg urethane (10%, i.p.). A laminectomy was performed, and the lumbar segment was quickly removed and immersed in oxygenated (95% O₂ and 5% CO₂) cold ACSF. The ACSF contained 95.0 mM NaCl, 1.8 mM KCl, 0.5 mM CaCl₂, 7.0 mM MgSO₄, 1.2 mM KH₂PO₄, 26.0 mM NaHCO₃, 15.0 mM Glucose and 50.0 mM Sucrose. Its pH was adjusted to 7.3–7.4 with NaOH and osmolarity to 310–320 mOsm/L with sucrose. The pial-arachnoid membrane was removed from the section. The L5 spinal segment was fixed on the cutting bracket with cyanoacrylate glue, then, 350 μm thick spinal cord slices were cut with a vibratome (Leica VT1200S Microsystem). Slices were incubated in ACSF oxygenated at 35°C for at least 1 h and then transferred to the recording chamber.

The slice was placed into a recording chamber and continuously perfused with recording solution at a speed of 3–4 ml/min. The recording solution contained 127.0 mM NaCl, 1.8 mM KCl, 2.4 mM CaCl₂, 1.3 mM MgSO₄, 1.2 mM KH₂PO₄, 15.0 mM glucose, and 26.0 mM NaHCO₃, oxygenated with 95% O₂, and 5% CO₂, at a pH of 7.3–7.4, and an osmolarity of 300–310 mOsm/L. Recordings of sEPSCs were performed using whole-cell voltage-clamp. All recordings were conducted in lamina II neurons at the L5 spinal level. Lamina II outer zone was identified by its distinctive translucent appearance and neurons were visualized using a water-immersion objective on an upright infrared Olympus microscope (BX51WI, Japan) with differential interference contrast/infrared illumination. sEPSCs were recorded using an EPC-10 amplifier and PULSE program (HEKA Electronics, Lambrecht, Germany). The recording pipette had a resistance of 8–10 MΩ when filled with the internal solution containing: 140.0 mM K-gluconate, 3.0 mM KCl, 4.0 mM NaCl, 0.2 mM EGTA, 10 mM HEPE and 2.0 mM Mg-ATP. Only a seal resistance of ≥ 2 GΩ and an access resistance of 20–35 MΩ were used for electrophysiological recording. The series resistance was optimally compensated by ≥70% and constantly monitored throughout the experiments. In the presence of 10 μM bicuculline and 5 μM strychnine, sEPSCs were recorded from neurons clamped at −70 mV. After a 15 min stabilization period, synaptic events were recorded for 4 min in every slice. A spinal cord slice was obtained from each animal and only a neuron was recorded in a slice.

Male rats were placed in a Plexiglas chamber and adapted to the environment for at least 30 min before behavior experiments. Mechanical allodynia was measured by paw withdrawal threshold (PWT). After rats were coded and pretreated with paclitaxel, different groups of rats then received intrathecal injection of vehicle or different dose (10, 30, and 100 ug in 50 ul, i.t. at day 8, 9, and 10) of metformin, separately. After 30 min metformin injection, the other experimenter tested PWT of the hind plantar using a series of von Frey filaments (Stoelting, Wood Dale, IL, USA). Experimenters who performed behavioral tests were blinded to all treatments.

Spontaneous events were performed and analyzed using Mini Analysis Program version 6.0.3 (Synaptosoft, Decatur, GA, USA). Analysis was performed in Igor pro6.10A software. Data were expressed as mean ± SEM and statistically compared using unpaired t-test or analysis of variance (ANOVA), followed by Bonferroni’s post-hoc test.

To evaluate the effect of metformin on paclitaxel-induced mechanical allodynia, rats were intrathecally injected with different dose of metformin versus vehicle, and then their mechanical thresholds were compared. We observed that rats displayed mechanical allodynia after administration with paclitaxel (4 × 2 mg/kg, cumulative dose 8 mg/kg). Figure 1 shows that the mechanical withdrawal thresholds of rats were decreased 1 day after paclitaxel injection. The decrease was further intensified, which was significantly different from baseline value at day 0. Since day 7, withdrawal threshold of paclitaxel-treated rats remained stable at low levels throughout the experiment. Figure 1 shows intrathecal administration of metformin (10, 30, and 100 ug in 50 ul, i.t. at day 8, 9, and 10) dose-dependently prevented the paclitaxel-induced mechanical allodynia (P < 0.01 and 0.001, Bonferroni’s post-hoc test, compared with vehicle, n = 10 rats). Mechanical allodynia only partially developed in the paclitaxel and 100 ug metformin-treated rats, which was significantly less than that of the paclitaxel–vehicle-treated rats at days 8–15. These results suggested that intrathecal injection of metformin dose-dependently prevents the established mechanical allodynia induced by paclitaxel in rats.

At day 11 or 12, sEPSCs in spinal dorsal horn neurons were recorded. Figure 2A shows the sEPSCs traces from three groups of rats. Cumulative distributions shows that the curve of sEPSC interevent interval was significantly shifted leftward in paclitaxel-treated (PAC) rats (Figure 2B), but no change in the curve of sEPSC amplitude (Figure 2D), compared with those from control (CON) rats. Figures 2C, E shows that sEPSC frequency was significantly increased (1.86 ± 0.26 Hz vs. 0.6 ± 0.07 Hz, P < 0.01, n = 6 neurons from 6 rats) without change in amplitude with paclitaxel, compared with those from control treatment. Metformin reversed this effect (Figures 2B, C). Intrathecal treatment with metformin significantly inhibited the increased frequency of sEPSCs with paclitaxel, as observed in paclitaxel-metformin treated (MET) rats (0.87 ± 0.07 Hz vs. 1.86 ± 0.26 Hz, P < 0.05, n = 6 neurons from 6 rats; Figures 2B, C), but not affected the sEPSC amplitude (Figures 2D, E). The results indicated that intrathecal administration of metformin inhibited the increased frequency of sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats.

Figure 3 shows that incubation of slices with metformin for 1 h had also effects on sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats. Cumulative distributions shows that metformin incubation significantly shifted the curve of sEPSC interevent interval rightward and had no effects on the curve of sEPSC amplitude, indicating that metformin made the intervals between sEPSC events longer, but not significantly changed sEPSC amplitude, compared with vehicle incubation (Figures 3B, D). Figures 3B, D show that metformin incubation also significantly reduced the sEPSC frequency (0.99 ± 0.19 Hz vs. 1.75 ± 0.38 Hz, P < 0.05, n = 6 neurons from 6 rats), rather than sEPSC amplitude, in the slices from paclitaxel-treated rats. These results indicated that metformin had an acute effect on sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats.

Figure 4A shows the sEPSCs traces in spinal dorsal horn neurons from three groups of rats. Cumulative distributions shows that the curve of sEPSC interevent interval was significantly shifted rightward in paclitaxel-metformin (i.p.)-treated (MET) rats, compared with those from paclitaxel-treated (PAC) rats (Figure 4B). However, no change in the curve of sEPSC amplitude was observer between the two groups of rats (Figure 4D). Figures 4C, E show that intraperitoneal injection of metformin also significantly decreased the increased frequency of sEPSCs (1.18 ± 0.37 Hz vs. 1.89 ± 0.42 Hz, P < 0.01, n = 6 neurons from 6 rats), but did not affect the sEPSC amplitude in spinal dorsal horn neurons from paclitaxel-treated rats. The results indicated that systemic treatment with metformin also suppressed the increased frequency of sEPSCs in spinal dorsal horn neurons from paclitaxel-treated rats.