The Melatonin Type 2 Receptor Agonist IIK7 Attenuates and Reverses Morphine Tolerance in Neuropathic Pain Rats Through the Suppression of Neuroinflammation in the Spinal Cord

Abstract

Background: Morphine analgesic tolerance (MAT) limits the clinical application of morphine in the management of chronic pain. IIK7 is a melatonin type 2 (MT2) receptor agonist known to have antioxidant properties. Oxidative stress is recognized as a critical factor in MAT. This study sought to assess the impact of IIK7 on the progression of MAT and its potential to reverse pre-existing MAT.

Methods: Wistar rats underwent partial sciatic nerve transection (PSNT) surgery to induce neuropathic pain (NP). Seven days post nerve transection, we implanted an intrathecal (i.t.) catheter and linked it to an osmotic pump. Rats were randomly divided into the following groups: sham-operated/vehicle, PSNT/vehicle, PSNT/IIK7 50 ng/h, PSNT/MOR 15 g/h, and PSNT/MOR 15 g + IIK7 50 ng/h. We implanted two i.t. catheters for drug administration and the evaluation of the efficacy of IIK7 in reversing pre-established MAT. We linked one to an osmotic pump for MOR or saline continuous i.t. infusion. On the 7th day, the osmotic pump was disconnected, and 50 μg of IIK7 or the vehicle was administered through the second catheter. After 3 h, 15 μg of MOR or saline was administered, and the animal behavior tests were performed. We measured the levels of mRNA for Nrf2 and HO-1, pro-inflammatory cytokines (PICs), and the microglial and astrocyte activation in the spinal cord.

Results: The co-administration of IIK7 with MOR delayed MAT development in PSNT rats by restoring Nrf2 and HO-1 while also inhibiting the microglial-cell and astrocyte activation, alongside the suppression of PICs. Additionally, a single injection of high-dose 50 μg IIK7 was efficient in restoring MOR's antinociception in MOR-tolerant rats.

Conclusions: Our results indicate that the co-infusion of ultra-low-dose IIK7 can delay MAT development and a high dose can reverse pre-existing MAT.

Keywords: IIK7; MOR tolerance; MT2 receptor agonist; neuroinflammation; neuropathic pain.

Figure 1

Impact of ultra-low dose IIK7 co-infusion on MOR tolerance development.

Figure 2

IIK7 co-infusion alleviates MOR-induced antinociceptive tolerance against acute pain. The baseline tail-flick response was assessed on Day −1 (one day prior to PSNT) and on Day 7 post PSNT. Following the baseline tail-flick measurements on Day 7, osmotic pumps were implanted, and the tail-flick response was assessed on Days 8, 10, 12, and 14 through constant i.t. delivery of the following: vehicle (1 µL/h), IIK7 (50 ng/h), MOR (15 μg/h), and a combination of MOR (15 μg/h) and IIK7 (50 ng/h) in both the sham and PSNT groups. Statistical significance levels are indicated as follows: ** p < 0.01, and *** p < 0.001, in comparison to the PSNT + Vehicle group. Statistical significance is indicated by ## p < 0.01, and ### p < 0.001 when comparing PSNT + MOR to PSNT + MOR + IIK7 (n = 6).

Figure 3

IIK7 co-infusion alleviates MOR-induced analgesic tolerance in neuropathic−pain rats. For the behavior tests, the baseline (a) mechanical paw withdrawal threshold and (b) thermal withdrawal latency were measured on Day −1 (1 day before performing PSNT) and Day 7 after PSNT. Following the baseline measurement on Day 7, osmotic pumps were installed, and mechanical paw withdrawal threshold and thermal paw withdrawal were evaluated on Days 8, 10, 12, and 14 through constant intrathecal infusion of the vehicle (1 µL/h), IIK7 (50 ng/h), MOR (15 μg/h), and MOR (15 μg/h) + IIK7 (50 ng/h) in the sham and PSNT groups. ** p < 0.01, and *** p < 0.001 compared to PSNT + Vehicle group. Statistical significance is indicated by ## p < 0.01, and ### p < 0.001 when comparing PSNT + MOR to PSNT + MOR + IIK7 (n = 6).

Figure 4

Relative mRNA expression levels of the neuroprotective proteins (a) Nrf2 and (b) HO-1 were assessed using RT-qPCR. The asterisk indicates a statistically significant difference among the following comparisons: sham + Vehicle versus PSNT + Vehicle, PSNT + Vehicle versus PSNT + IIK7, and PSNT + Vehicle versus PSNT + MOR. The symbol # denotes a statistically significant difference between PSNT + MOR and PSNT + MOR + IIK7. Significance levels are indicated as follows: * p < 0.05; ## p < 0.01; ###/*** p < 0.001. Six animals were included in each group (n = 6).

Figure 5

Impact of vehicle, PSNT, IIK7, or MOR or MOR + IIK7 infusion on pro-inflammatory cytokine concentrations in the spinal cord. PSNT surgery and MOR infusion significantly elevated all three PIC levels—TNF-α (a), IL-1β (b), and IL-6 (c)—in the spinal cord samples. The asterisks indicate statistically significant differences between the following comparisons: sham + Vehicle versus PSNT + Vehicle, PSNT + Vehicle versus PSNT + IIK7, and PSNT + Vehicle versus PSNT + MOR. # indicates a statistically significant difference between PSNT + MOR and PSNT + MOR + IIK7. Statistical significance levels are indicated as follows: */# p < 0.05; **/## p < 0.01; *** p < 0.001. Sample size consisted of 6 animals per group.

Figure 6

Impact of a high-dose IIK7 single injection on rats with pre-established MOR tolerance.

Figure 7

In rats tolerant to MOR, IIK7 restores the antinociceptive action of MOR. On Day 7, following the i.t. infusion of either MOR or saline, the antinociceptive effects were assessed after the disconnection of the i.t. pump. The rats were given i.t. injections of either 50 μg of IIK7 or vehicle 3 h after the infusion was stopped. Thirty minutes later, they were given a 15 μg MOR challenge, and for 120 min, tail-flick latency was measured every 30 min. For at least six rats, all data are displayed as means ± SDs. The asterisks indicate statistically significant differences among the groups Sal/Veh/Sal compared to MOR/Veh/MOR, MOR/Veh/MOR compared to Sal/Veh/MOR, and MOR/Veh/MOR compared to MOR, IIK7/Saline. # indicates statistically significant difference between MOR/Veh/MOR and MOR/IIK7/Mor: **/## p < 0.01; *** p < 0.001. Six animals were included in each group (n = 6).

Figure 8

In PSNT rats tolerant to MOR, IIK7 effectively restores the antinociceptive action of MOR. On Day 7, following the conclusion of the i.t. infusion of either MOR or saline, the antinociceptive impact of the drug was assessed. The rats were given i.t. injections of either 50 μg of IIK7 or vehicle three hours after the infusion was stopped. Thirty minutes later, they were given a 15 μg MOR challenge, and for 120 min, (a) Mechanical paw withdrawal threshold and (b) Thermal paw withdrawal latency were measured every 30 min. For at least six rats, all data are displayed as means ± SDs. The asterisks indicate statistically significant differences among the groups Sal/Veh/Sal compared to MOR/Veh/MOR, MOR/Veh/MOR compared to Sal/Veh/MOR, and MOR/Veh/MOR compared to MOR, IIK7/Saline. # indicates a statistically significant difference between MOR/Veh/MOR and MOR/IIK7/MOR with the following p-values: * p < 0.05; **/## p < 0.01; *** p < 0.001. (n = 6 animals per group).

Figure 9

(A) Co-infusion of IIK-7 reduces microglial cell activation induced by morphine. Spinal cord samples were treated with IBA-1 and the nucleus was stained with DAPI (blue) seven days after the infusion of different combinations. Fluorescence microscopy was then used to acquire and blend the images. The shown sections are from rats that were infused with (a) sham/vehicle, (b) PSNT/vehicle, (c) PSNT/IIK-7, (d) PSNT/MOR, and (e) PSNT MOR + IIK-7 treatments. The dot-like red triangular structures represent resting microglia and chained morphology outlined in red circles indicates activated microglial cells. Red arrows indicate pericellular edema. (B) Impact of IIK-7 co-infusion on morphine-induced microglial cell activation. Active microglial cells were observed in the spinal cords of MOR-infused rats seven days post infusion compared with sham animals. IIK-7 markedly reduced microglial cell activation induced by morphine. Asterisks indicate statistically significant differences among the following comparisons: sham vs. PSNT + saline, PSNT + saline vs. PSNT+MOR, and PSNT MOR vs. PSNT + MOR + IIK-7. * p < 0.05; ** p < 0.01; *** p < 0.001 (n = 6).

Figure 10

(A) In morphine-tolerant rat spinal cords, IIK-7 co-infusion decreases morphine-induced astroglial cell activation. Spinal cord slices were treated with GFAP and the nuclei were stained with DAPI stain (blue) seven days after the infusion of different combinations. Fluorescence microscopy was then used to acquire and blend the images. The shown sections are from rats that were infused with (a) sham/vehicle, (b) PSNT/vehicle, (c) PSNT/IIK-7, (d) PSNT/MOR, and (e) PSNT MOR + IIK-7. The stellate shapes of astrocytes with several intricate processes, outlined in red, were evident in GFAP-positive cells. The effects of 1× and 2× reduced astrogliosis. Arrows indicate pericellular edema. (B) The quantitative analysis of GFAP-positive cells. A marked positivity for IL-1β in the spinal cords of MOR-infused rats was observed 7 days post infusion compared with sham animals. The IIK-7 significantly attenuated morphine-induced astroglial cell activation. Asterisks indicate statistically significant differences among the following comparisons: sham vs. PSNT + Vehicle, PSNT + Vehicle vs. PSNT + MOR, and PSNT MOR vs. PSNT + MOR + IIK-7. * p < 0.05; ** p < 0.01; *** p < 0.001 (n = 6).