Treatment of a multiple sclerosis animal model by a novel nanodrop formulation of a natural antioxidant

Abstract

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is associated with demyelination, neurodegeneration, and sensitivity to oxidative stress. In this work, we administered a nanodroplet formulation of pomegranate seed oil (PSO), denominated Nano-PSO, to mice induced for experimental autoimmune encephalomyelitis (EAE), an established model of MS. PSO comprises high levels of punicic acid, a unique polyunsaturated fatty acid considered as one of the strongest natural antioxidants. We show here that while EAE-induced mice treated with natural PSO presented some reduction in disease burden, this beneficial effect increased significantly when EAE mice were treated with Nano-PSO of specific size nanodroplets at much lower concentrations of the oil. Pathological examinations revealed that Nano-PSO administration dramatically reduced demyelination and oxidation of lipids in the brains of the affected animals, which are hallmarks of this severe neurological disease. We propose that novel formulations of natural antioxidants such as Nano-PSO may be considered for the treatment of patients suffering from demyelinating diseases. On the mechanistic side, our results demonstrate that lipid oxidation may be a seminal feature in both demyelination and neurodegeneration.

Keywords: EAE; PSO; nanodrops; neurodegeneration; oxidative stress.

Figures

Figure 1

Droplet size analysis. Notes: (A) DLS results for Nano-PSO and (B) cryo-TEM image of Nano-PSO. Abbreviations: DLS, dynamic light scattering; PSO, pomegranate seed oil; cryo-TEM, cryogenic transmission electron microscope.

Figure 2

Nano-PSO as an α-EAE agent. Notes: Mice were induced for EAE and treated from day 1 of the induction either with PSO or with Nano-PSO. (A) Designated EAE-induced groups were fed either with normal mouse chow (untreated group; n=10) or with chow enriched with PSO at the concentration in which 3 g (daily intake) comprises the levels designated in the figure insert: 100 (n=10) or 300 μL (n=8) PSO. P<0.05 for all PSO-treated groups versus the untreated group. (B) Designated EAE-induced groups were either left untreated or treated (by gavage) with 150 μL solution comprising 0.2, 0.8 (n=6), or 10 μL (n=7) PSO in the form of Nano-PSO. P<0.05 for 0.8 and 10 μL PSO-treated group versus the untreated group. Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune encephalomyelitis.

Figure 3

Individual α-EAE activity of Nano-PSO ingredients. Notes: Mice were induced for EAE and treated from day 1 of induction (by gavage) with the reagents described in the insert of the figure (n=7 for each of the groups). Nano-PSO was administrated at a dose of 2 μL PSO per 150 μL solution. Mice were scored daily for EAE signs for 2 additional weeks. P<0.05 for the results in the Nano-PSO group versus all others. Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune encephalomyelitis.

Figure 4

Nano-PSO in the prevention and treatment of EAE. Notes: Mice induced for EAE were administered Nano-PSO in two different start points. As shown in the insert, while one group of induced mice was left untreated (n=8), a second group was treated with Nano-PSO from day 1 of the induction (n=6) and a third group from day 7 of the induction (n=7). Mice were scored daily for EAE signs for 2 additional weeks. P<0.05 for both Nano-PSO treatments. Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune encephalomyelitis.

Figure 5

Small Nano-PSO particles are inactive against EAE. Notes: Mice induced for EAE were treated with Nano-PSO in different droplet sizes. As shown in the insert, while one group of induced mice was left untreated (n=8), a second group was treated with 180 nm droplets of Nano-PSO (n=7), and a third group with 30 nm Nano-PSO droplets (n=7). P<0.05 for the untreated group versus the group treated with 180 nm droplets of Nano-PSO. Abbreviations: PSO, pomegranate seed oil; EAE, experimental autoimmune encephalomyelitis.

Figure 6

Pathological markers of EAE in Nano-PSO treated and untreated mice. Notes: Nano-PSO treated and untreated mice were sacrificed 3 weeks after induction of EAE, and their formalin-fixed, paraffin-embedded brain sections as well as those of age-matched naïve mice (C and J) were stained by mAb EO6 (A–D and F), H&E (E and G), and LFB/PAS (brains and spinal cords) (H–J). (D) and (F) represent an enlargement of the squares in (A) and (B); (E) and (G) are serial sections of (D) and (F), respectively. Arrows in (D–G) indicate immune infiltrates. Arrows in (H) represent demyelinated areas. Abbreviations: EAE, experimental autoimmune encephalomyelitis; PSO, pomegranate seed oil; mAb, monoclonal antibody; H&E, hematoxylin and eosin; LFB, Luxol fast blue; PAS, periodic acid Schiff.

Figure 7

TBARS levels in EAE brains. Notes: Samples from naïve, as well as treated and untreated EAE brains were subjected to the TBARS test. Abbreviations: TBARS, thiobarbituric acid reactive substances; EAE, experimental autoimmune encephalomyelitis; MDA, malonaldehyde.