The Surface Amine Group of Ultrasmall Magnetic Iron Oxide Nanoparticles Produce Analgesia in the Spinal Cord and Decrease Long-Term Potentiation

Abstract

Our previous studies have revealed the ultrasmall superparamagnetic iron oxide in the amine group USPIO-101 has an analgesic effect on inflammatory pain. Here, we further investigated its effect on the spinal cord and brain via electrophysiological and molecular methods. We used a mouse inflammatory pain model, induced by complete Freund's adjuvant (CFA), and measured pain thresholds via von Frey methods. We also investigated the effects of USPIO-101 via an extracellular electrophysiological recording at the spinal dorsal horn synapses and hippocampal Schaffer collateral-CA1 synapses, respectively. The mRNA expression of pro-inflammatory cytokines was detected by quantitative real-time polymerase chain reaction (RT-qPCR). Our results showed intrathecal USPIO-101 produces similar analgesic behavior in mice with chronic inflammatory pain via intrathecal or intraplantar administration. The potentiated low-frequency stimulation-induced spinal cord long-term potentiation (LTP) at the spinal cord superficial dorsal horn synapses could decrease via USPIO-101 in mice with chronic inflammatory pain. However, the mRNA expression of cyclooxygenase-2 was enhanced with lipopolysaccharide (LPS) stimulation in microglial cells, and we also found USPIO-101 at 30 µg/mL could decrease the magnitude of hippocampal LTP. These findings revealed that intrathecal USPIO-101 presented an analgesia effect at the spinal cord level, but had neurotoxicity risk at higher doses.

Keywords: analgesia; inflammatory pain; long-term potentiation; neurotoxicity; pro-inflammatory cytokines; ultrasmall magnetic iron oxide nanoparticles.

Figure 1

The dynamic light scattering (DLS), zeta potential analysis, and Fourier transform infrared spectroscopy (FTIR) spectra of ultrasmall magnetic iron oxide nanoparticles. (A) number distribution of USPIO-101, as measured by DLS. The concentration of USPIO-101 was 1 mg/mL. (B) Zeta potential analysis of USPIO-101. (C) Fourier transform infrared (FTIR) spectra are presented for USPIO-101 (black) and USPIO-102 (red), respectively. The -NH₂ and -CH₂ group signals are expressed in USPIO-101 at the wavelength of 3300~3500 cm⁻¹, 1560~1640 cm⁻¹, or 1405~1465 cm⁻¹ (arrow). The -COOH group signal is expressed in USPIO-102 at the wavelength of 1550~1610 cm⁻¹ (arrow).

Figure 2

USPIO-101 attenuated chronic inflammatory pain via intrathecal or intraplantar injection. Mechanical pain sensitivity was measured using von Frey microfilaments. USPIO-101 attenuated the analgesia behavior in both (A) intrathecal and (B) intraplantar injection (10 mg/mL, 10 μL) after CFA paw injection for four days. The paw withdrawal thresholds were measured every 30 min until there was no difference between the three groups. Data were analyzed by two-way ANOVA and post-hoc with Tukey’s test. *: p < 0.05 vs. PBS sham group.

Figure 3

The effect of USPIO-101 on spinal cord LTP in CFA paw-injected mice. (A) Time courses of the slope of fEPSPs recorded before and after low-frequency stimulation (LFS, arrow) in spinal cord slices from CFA- or saline-treated mice. (C) The bar graph represents the magnitudes of potentiation, which averaged 20 fEPSPs recorded 30~40 min after LFS. **: p < 0.01 (unpaired t-tests) vs. CFA-treated ipsilateral group. (D) Time courses of the slope of fEPSPs recorded before and after LFS (arrow) in spinal cord slices from CFA-treated mice with USPIO-101 (10 or 30 µg/mL), respectively. USPIO-101 (10 or 30 µg/mL) applied for 7.5 min before LFS stimulation, respectively. (F) The bar graph represents the magnitudes of potentiation, which averaged 20 fEPSPs recorded 30~40 min after LFS at a concentration of 10 or 30 µg/mL. * or **: p < 0.05 or p < 0.01 vs. control group (one-way ANOVA). (B,E) Twenty recorded fEPSPs at time points a and b were averaged in each group. The slope of each fEPSP was expressed as % of the baseline fEPSP slope, which was the average of 20 fEPSPs at the beginning of 10 min recording. n indicates the number of slices recorded.

Figure 4

The effect of USPIO-101 on basal transmission (A) and spinal cord LTP (B,C) at the spinal cord superficial dorsal horn synapses, respectively. (A) After baseline recording for 10 min, USPIO-101 (10 µg/mL) was applied for another 15 min, then washed for 15 min. No difference was observed in the basal transmission after application of USPIO-101 to the spinal cord superficial dorsal horn synapses. (C) Time courses of the slope of fEPSPs recorded before and after LFS (arrow) in spinal cord slices. Drugs: USPIO-101 (10 µg/mL) was applied for 7.5 min before LFS stimulation. (E) The bar graph represents the magnitudes of potentiation, which averaged 20 fEPSPs recorded 30~40 min after LFS. *: p < 0.05 vs. control group (one-way ANOVA). (B,D) The traces shown in the graph are averaged 20 recordings of fEPSPs at times a and b in each group. The expression and analysis of the baseline fEPSP slope are the same as Figure 2. n indicates the number of slices recorded.

Figure 5

Intracellular reactive oxygen species level after treatment with USPIO-101 in SH-SY5Y or SM826 cells. (A,B) Hydrogen peroxide (H₂O₂) as positive control and USPIO-101 (10 or 30 μg/mL) were used in SH-SY5Y cells for 30 min (A, H₂O₂ 1 mM) and 24 h (B, H₂O₂ 0.2 mM), respectively. (C) Lipopolysaccharide (LPS) 1 μg/mL as positive control and USPIO-101 (10 or 30 μg/mL) were used in SM826 cells for 24 h, respectively. RFU means the relative fluorescence unit in 3 × 10⁴ cells/well in 96-well plates. Data shown are the mean ± SEM of three independent experiments performed in triplicate. * or ***: p < 0.05 or p < 0.001 vs. control group. ###: p < 0.001 vs. the H₂O₂ group (one-way ANOVA).

Figure 6

USPIO-101 potentiates the LPS-induced NF-kB and COX-2 mRNA expression in SM826 microglial cells. (A) The time scale for treating USPIO-101 and LPS stimulation. (B–D) The levels of mRNAs encoding the pro-inflammatory cytokines TNF-α (B), NF-κB (C), and COX-2 (D) were analyzed by qRT-PCR and normalized by the expression of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) at three time points after LPS stimulation: 30, 60, or 120 min. Data are presented as mean ± SEM (n = 4). * or ** or ***: p < 0.05, or p < 0.01, or p < 0.001 vs. control; # or ## or ###: p < 0.05, or p < 0.01, or p < 0.001 vs. LPS.

Figure 7

The effect of USPIO-101 on hippocampal LTP. (A) Time courses of the slope of fEPSPs recorded before and after theta-burst stimulation (TBS, arrow) in hippocampal slices. Drugs: USPIO-101 (10 or 30 µg/mL) was applied for 7.5 min before TBS stimulation. (B) The traces shown in the graph are the average of 20 recorded fEPSPs at times a and b in each group. (C) The bar graph represents the magnitudes of potentiation, with the average of 20 fEPSPs recorded 30~40 min after TBS at a concentration of 10 or 30 µg/mL. *: p < 0.05 vs. control group. #: p < 0.05 vs. USPIO-101 group (one-way ANOVA). n indicates the number of slices recorded. The expression and analysis of the baseline fEPSP slope are the same as Figure 3.