Manganese Oxide Nanozymes Ameliorate Mechanical Allodynia in a Rat Model of Partial Sciatic Nerve-Transection Induced Neuropathic Pain

Abstract

Background: Reactive oxygen species (ROS) induced oxidative stress is linked to numerous neurological diseases, including neuropathic pain. Natural ROS scavenging enzymes like superoxide dismutase (SOD) and catalase have been found to be efficient in alleviating neuropathic pain. However, their sensitivity towards extreme pH and a short half-life limit their efficacy in vivo. Manganese oxide nanoparticles (MONPs) are recently known to possess ROS scavenging properties. In this study, MONPs were examined for their therapeutic effect on neuropathic pain.

Methods: The MONPs were synthesized by a hydrothermal method. The synthesized MONPs were characterized by UV/Vis, TEM, SEM, FTIR, NTA and XRD. The biocompatibility of the nanoparticles is evaluated in neural cells using LDH assay. MONPs were evaluated for their antioxidant activity by DPPH assay. In addition, in vitro ROS scavenging properties were examined in bone marrow-derived macrophage (BMDM) cells using 2',7'-dichlorofluorescin diacetate (DCFDA) assay. To evaluate the in vivo efficacy of nanoparticles, neuropathic pain was induced in Wistar rats by partial sciatic nerve transection (PSNT). On post-transection days 14 to 18, rats were intrathecally injected with MONPs and paw withdrawal threshold was measured. The spinal cords were collected and processed for Western blotting and histological analysis.

Results: The synthesized MONPs were biocompatible and showed effective antioxidant activity against DPPH free radical scavenging. Further, the nanoparticles scavenged ROS efficiently in vitro in BMDM and their intrathecal administration significantly reduced mechanical allodynia as well as the expression of cyclooxygenase-2 (COX-2), an important mediator of chronic and inflammatory pain in the spinal dorsal horns of PSNT rats.

Conclusion: As ROS play a significant role in neuropathic pain, we expect that MONPs could be a promising tool for the treatment of various inflammatory diseases and might also serve as a potential nanocarrier for the delivery of analgesics.

Keywords: MONPs; allodynia; reactive oxygen species and neuropathic pain.

Figure 1

(A) Transmission electron microscopy micrograph of MONPs. (B) Scanning electron micrograph of MONPs. (C) X-ray powder diffraction patterns of MONPs and (D) Fourier-transform infrared spectra of MONPs. Abbreviation: MONPs, manganese oxide nanoparticles.

Figure 2

Effect of MONPs on LDH leakage in Cns1 neuronal cells. Cytotoxicity was determined by LDH release after 24 h of exposure to MONPs (10–100 μg/mL). Data are mean ± SD. Results were calculated as percentage of the positive control (Triton X-100-lysed Cns1) (n = 3). Abbreviations: MONPs, manganese oxide nanoparticles; LDH, lactate dehydrogenase.

Figure 3

Absorption spectra of DPPH and oxidation of DPPH radicals. (A) As DPPH radicals are scavenged by the MONPs, there is a decrease in absorbance at 517nm which is monitored by ultraviolet-visible spectroscopy (B) DPPH free radical scavenging percentage at different concentrations of MONPs. Abbreviations: MONPs, manganese oxide nanoparticles; DPPH, 2,2-diphenyl-2-picrylhydrazyl hydrate.

Figure 4

MONPs reduce ROS production induced by TLR1/2 agonist PAM3CSK4. The bone-derived macrophages are either untreated (Blank) or treated with MONPs (20 μg) or stimulated with triacylated bacterial lipopeptide PAM3CSK4 (5 μg/mL) or pre-incubated MONPs for 30 mins followed by the addition of PAM3CSK4 (5 μg/mL). The cells were further incubated for 12 hrs and readings were noted at FL-1 channel using a flow cytometer after the addition of DCFHDA. ROS generation was measured in triplicate. The data shown are the mean ± SD values of 3 individual experiments. **p < 0.01 versus untreated control, ***p < 0.001 versus untreated control. Abbreviations: MONPs, manganese oxide nanoparticles; PAM3CSK4, PAM3CSK4, Pam3CysSerLys4; TLR 1/2, Toll-like receptor ½; DCFHDA, 2′,7′-dichlorofluorescin diacetate; ROS, reactive oxygen species.

Figure 5

Experimental scheme for this study. Neuropathic pain was induced by partial sciatic nerve ligation in Wistar rats. MONPs were then administered intrathecally 14 days post nerve transection. Pain tested by using Dynamic Plantar Aesthesiometer was significantly attenuated in rats injected with MONPs compared with the saline-injected group. Scheme created with BioRender imaging software.

Figure 6

Time-course of the change in paw withdrawal threshold to mechanical stimulus in partial sciatic nerve transection (PSNT) rats and the effect of MONPs on mechanical paw withdrawal threshold in Sham and PSNT rats. Differences between paw withdrawal threshold to tactile stimuli in rats with sham surgery (N = 6) and PSNT were assessed preoperatively on the day of surgery (indicated as day –1 on the x-axis) and on day 14 after surgery. After PSNT, significant tactile sensitivity developed on day 14. The effect of intrathecal MONPs (50 μg) NPs on the antihyperalgesic effect was measured for 60 mins after saline or nanoparticle injection from days 14 to 18 after PSNT. p<0.01. Abbreviations: PSNT, partial sciatic nerve transection; MONPs, manganese oxide nanoparticles.

Figure 7

Effect of MONPs intrathecal single injection on COX-2 protein expression in spinal cord of sham surgery and PSNT animals through Western blotting analysis. β-Actin is the loading protein control. Abbreviations: MONPs, manganese oxide nanoparticles; COX-2, cyclooxygenase-2; PSNT, partial sciatic nerve transection.