Iron oxide nanoparticles and magnetic field exposure promote functional recovery by attenuating free radical-induced damage in rats with spinal cord transection

Figures & data

Figure 1 Transmission electron micrographs of naked IONPs (A) and IONPs (arrows) in gel (B). Images of GBM-U87 cells in an untreated culture (C) and after 24 hours of incubation with IONPs 25 μg/mL (D).

Note: Arrowhead indicates intracellular localization of IONPs.

Abbreviation: IONPs, iron oxide nanoparticles.

Figure 2 In vitro cell viability assay and cell counting with varying concentrations of IONPs (10, 25, 50, and 100 μg/mL) and incubation for 24, 48, and 72 hours (A). There was no significant difference in percent viability at any of the time points or concentrations in comparison with controls. The cytotoxicity induced by H₂O₂ 10 mM was attenuated by 25 μg/mL IONPs (B). Live cell counting with the trypan blue exclusion method using different concentrations of IONPs (C), showing no significant difference in percentage of viable cells observed at any of the time points or concentrations in comparison with the control. Cytotoxicity induced by H₂O₂ 10 mM was attenuated by 25 μg/mL IONPs (D).

Notes: Statistical significance (P < 0.05) is shown by ^aControl versus IONP, H₂O₂, IONP + H₂O₂; ^bH₂O₂ versus NP + H₂O₂.

Abbreviations: IONPs, iron oxide nanoparticles; H₂O₂, hydrogen peroxide.

Table 1 Effect of iron oxide nanoparticle implantation and/or exposure to a magnetic field on general body condition

Variables	Groups	Preinjury	Weeks post SCI					P-value (one-way ANOVA)
		Basal	1	2	3	4	5
General body condition
Mean weekly Fl (g)	SCI (n = 10)	30.4 ± 0.84	6.3 ± 1.83	12.1 ± 2.08	16.2 ± 1.61	19.6 ± 2.11	21.6 ± 1.57	0.0001
	NP (n = 7)	30.43 ± 2.14	6.29 ± 1.70	12.57 ± 1.81	15.57 + 1.71	18.43 ± 2.3	21.57 + 1.61	0.0001
	MF(n = 8)	30 ± 1.07	10.63 ± 3.16a,b	14.5 ± 2.39	15.63 ± 3.33	20.88 ± 2.47	22.63 ± 1.40	0.0001
	NP + MF(n = 14)	29.86 ± 0.95	13.22 ± 2.08a,c	16.71 ± 2.7a,c	20.29 ± 1.9a,b,c	22.8 ± 1.4a,c	25.14 ± 0.8a,b,c	0.0001
	P-value (repeated-measures ANOVA)	0.670	0.0001	0.0001	0.0001	0.0013	0.0001
Mean weekly Wl (mL)	SCI (n = 10)	39 ± 4.59	12.8 ±2.66	17.9 ±2.38	21.6 ±2.37	27.8 ± 3.05	29 ± 2.45	0.0001
	NP (n = 7)	37.15 ± 2.67	10.57 ± 2.22	16.57 ± 1.81	21.57 + 2.15	25.29 ± 1.88	28 ± 1.83	0.0001
	MF(n = 8)	39.38 ± 4.17	15.63 ± 3.54a	20.75 ± 4.26a	24 ± 2.77	28.37 ± 4.44	30.13 ± 1.52	0.0001
	NP + MF(n = 14)	38.57 ± 3.06	18.72 ± 2.61a,c	24.57 ± 1.7a,b,c	28.14 ± 12.11a,b,c	30.57 ± 2.10c	32.93 ± 2.26a,b,c	0.0001
	P-value (repeated-measures ANOVA)	0.671	0.0001	0.0001	0.0001	0.0039	0.0001
Absolute change in BW from baseline (g)	SCI (n = 10)	0	−34.5 ± 7.98	−50.0 ± 12.9	−53.0 ± 12.95	−49.0 ± 16.5	−45.0 ± 17.95	0.0001
	NP (n = 7)	0	−23.0 ± 8.0	−39.3 ± 8.9	−45.0 ± 9.6	−40.7 ± 9.6	−35.0 ± 7.1	0.0001
	MF(n = 8)	0	−30.0 ± 6.5	−38.1 ± 6.5a	−38.2 ± 6.5a	−36.5 ± 7.4	−30.6 ± 8.6	0.007
	NP + MF(n = 14)	0	−22.1 ±5.1a	−26.4 ± 5.7a,b,c	−26.9 ± 6.1a,b,c	−21.1 ± 6.0a,b,c	−13.2 ± 7.0a,b,c	0.0001
	P-value (repeated-measures ANOVA)	1	0.0001	0.0001	0.0001	0.0001	0.0001

Notes: Statistical significance (P < 0.05) is shown by;

a SCI versus MF, NP, and NP + MF groups;

b NP versus MF and NP + MF groups;

c MF versus NP + MF group.

Abbreviations: ANOVA, analysis of variance; IONPs, iron oxide nanoparticles; Fl, food intake; Wl, water intake; BW, body weight; SCI, spinal cord injury group; MF, SCI + magnetic field group; NP, SCI + IONP implantation group; NP + MF, SCI + IONP implantation + magnetic field group.

Figure 3 Effect of IONP implantation and MF exposure on mean (± SD) BBB score (A) and mean (± SD) time of stay on the moving Rotarod (B). Significant recovery was observed in both these tests after IONP implantation and MF exposure starting from week 1 which continued throughout the observation period. Mean (± SD) hind paw withdrawal latency (C) and urinary bladder function (D) were also significantly better in the NP + MF group.

Notes: Statistical significance (P < 0.05) is shown by ^aSCI versus MF, NP, NP + MF groups; ^bNP versus MF/NP + MF groups; ^cMF versus NP + MF group.

Abbreviations: BBB, Basso, Beattie, and Bresnahan locomotor rating; IONPs, iron oxide nanoparticles; SD, standard deviation; SCI, spinal cord injury group; MF, SCI + magnetic field group; NP, SCI + IONP implantation group; NP + MF, SCI + IONP implantation + magnetic field group.

Figure 4 Histological analysis of lesion site. Representative images of stained sections from each group showing extent of the lesion (A) and presence of viable motor neurons (arrowhead) (B) in the NP + MF group. Masson’s trichrome staining for collagenous connective tissue (C). Dense collagenous staining was observed in the SCI and NP groups (D), indicating scar formation; this was significantly less in the MF group and was absent in the NP + MF group.

Notes: Scale bar (A and C) = 1000 μm and (B and D) = 100 μm.

Abbreviations: IONPs, iron oxide nanoparticles; SCI, spinal cord injury group; MF, SCI + magnetic field group; NP, SCI + IONP implantation group; NP + MF, SCI + IONP implantation + magnetic field group.

Figure 5 Mean (± SD) total tissue damage volume was significantly less in all groups in comparison with the SCI group, but least in the NP + MF group.

Notes: Statistical significance (P < 0.05) is shown by ^aSCI versus MF, NP, and NP + MF groups; ^bNP versus MF and NP + MF groups; ^cMF versus NP + MF group.

Abbreviations: IONPs, iron oxide nanoparticles; SCI, spinal cord injury group; SD, standard deviation; MF, SCI + magnetic field group; NP, SCI + IONP implantation group; NP + MF, SCI + IONP implantation + magnetic field group.

Figure 6 Prussian blue staining for iron (A), showing that IONPs diffused out of the gel and concentrated in tissues near the site of the lesion. This aggregation of IONPs was high in the presence of MF in the NP + MF group. The arrowhead indicates the location of IONPs inside the motor neurons and axons (B). Transmission electron microscope images of tissues showed cytoplasmic localization of IONPs (arrows) (C).

Notes: Scale bar (A) = 100 μm, (B) = 10 μm, and (C) = 500 nm.

Abbreviations: IONPs, iron oxide nanoparticles; SCI, spinal cord injury group; MF, SCI + magnetic field group; NP, SCI + IONP implantation group; NP + MF, SCI + IONP implantation + magnetic field group.

Figure 7 Proposed mechanism for free radical scavenging by IONPs (A) and interaction of radical pairs with MF (B).

Notes: The presence of mixed valence states for Fe²⁺ and Fe³⁺ in the Fe₃O₄ acts as an antioxidant, enabling the IONPs to scavenge free radicals from the cell. Hydroxyl radical (°OH) produced from H₂O₂ reacts with an electron donor (Fe²⁺) and results in generation of a pair of radicals (Fe³⁺ + ⁻OH + °OH) by electron transfer from Fe²⁺ to °OH. An external MF affects interconversion between singlet and triplet states of the radical pair. We believe that low frequency MF leads to spin relaxation and favors generation of a singlet product and prevents generation of oxidative stress by interaction of the triplet product with biomolecules in the cell. Another set of chemical reactions between the ions in the cell and Fe₃O₄ then appear to be involved in reversing the oxidation state from Fe³⁺ to Fe²⁺. We believe that this is indicative of a cyclical regenerative or autocatalytic reaction on the part of IONPs.

Abbreviations: IONPs, iron oxide nanoparticles; Fe²⁺, ferrous ion; Fe³⁺, ferric ion; MF, magnetic field; H₂O₂, hydrogen peroxide; ⁻OH, hydroxyl ion; °OH, hydroxyl radical; Fe₃O₄, iron (II,III) oxide.