Skip to main content
NCBI home page
Cellular and Molecular Neurobiology logoLink to Cellular and Molecular Neurobiology
. 2015 Oct 14;36(6):943–954. doi: 10.1007/s10571-015-0279-2

Ozone Therapy in Ethidium Bromide-Induced Demyelination in Rats: Possible Protective Effect

Neveen A Salem 1,✉, Naglaa Assaf 2, Manal F Ismail 3, Yasser A Khadrawy 4, Mohga Samy 5
PMCID: PMC11482425  PMID: 26467344

Abstract

Multiple sclerosis, an autoimmune inflammatory disease of the central nervous system, is characterized by excessive demyelination. The study aimed to investigate the possible protective effect of ozone (O3) therapy in ethidium bromide (EB)-induced demyelination in rats either alone or in combination with corticosteroids in order to decrease the dose of steroid therapy. Rats were divided into Group (1) normal control rats received saline, Group (2) Sham-operated rats received saline, Group (3) Sham-operated rats received vehicle (oxygen), Group (4) EB-treated rats received EB, Group (5) EB-treated rats received O3, Group (6) EB-treated rats received methylprednisolone (MP), and Group (7) EB-treated rats received half the dose of MP concomitant with O3. EB-treated rats showed a significant increase in the number of footfalls in the grid walk test, decreased brain GSH, and paraoxonase-1 enzyme activity, whereas brain MDA, TNF-α, IL-1β, INF-γ, Cox-2 immunoreactivity, and p53 protein levels were increased. A significant decline in brain serotonin, dopamine, norepinephrine, and MBP immunoreactivity was also reported. Significant improvement of the above-mentioned parameters was demonstrated with the administration of either MP or O3, whereas best amelioration was achieved by combining half the dose of MP with ozone.

Keywords: Ethidium bromide, Ozone, Methylprednisolone, Multiple sclerosis, Oxidative stress, Cox-2, P53, Rat

Introduction

MS is a complex disease with several pathophysiological processes: inflammation, demyelination, oxidative stress, axonal damage, and repair mechanisms that participate in this disorder (Hafler 2004). Since reactive oxygen species (ROS) play a pivotal role in the initial phase as well as the chronic stage of MS, antioxidant therapy might be an attractive approach to limit disease progression. Notably, there are a number of disadvantages to the use of exogenous antioxidants for MS treatment, as most antioxidant compounds do not efficiently cross the blood–brain barrier (BBB) and have a narrow therapeutic window (Carlson and Rose 2006).

Steroids are postulated to positively impact MS at the molecular level by many mechanisms including reduction in the production of adhesion molecules, pro-inflammatory cytokines, and circulating CD4 T-lymphocytes and B lymphocytes (Anderson and Goodkin 1998). A number of studies support the concept that short course of high single dose of methylprednisolone (MP) is considered to be the drug of choice to accelerate recovery from acute attacks of MS relapses for many years. The most commonly used dosage regimen is 500–1000 mg of IV MP as a single dose per day for 3–5 days (Zorzon et al. 2005). Steroid therapy also presents the risk of numerous adverse events although such outcomes are more typical in long-term steroid therapy (Anderson and Goodkin 1998).

Ozone (O3), a natural highly reactive gaseous molecule, is formed when oxygen is exposed to an electric discharge or/and UV radiation. It can act as a disinfectant, an oxygen donor, an immunomodulator, a paradoxical inducer of antioxidant enzymes, a metabolic enhancer, an inducer of endothelial nitric oxide synthase, and possibly an activator of stem cells with consequent neovascularization and tissue reconstruction (Barber et al. 1999). Ozone–oxygen mixture showed beneficial effects in cerebrovascular ischemia, humoral immunity deficiency, heart ischemia, and autoimmune diseases such as MS and rheumatoid arthritis (Bocci et al. 2011). The total ozone dose is equivalent to the gas volume (O3 + O2 present as carrier) in (ml) multiplied by ozone concentration (µg/ml). Exposure of human blood to a mixture of oxygen and ozone is not toxic for blood, provided exposure times and concentrations are appropriate. Actually the optimal concentration ranges between 70 and 90 μg/ml. However, the concentration of 70 μg/ml was found to be the safest (Travagli et al. 2010). Ozone has been administered via several routes, including local irrigation, intra-arterial, intramuscular, subcutaneous, and intra-articular and as enemas (Romero et al. 1993). Repeated rectal administrations of ozone have shown to induce a sort of cross-tolerance to free radicals released after hepatic and renal ischemic/reperfusion (I/R) (León et al. 1998).

The current study aimed to focus attention on the possible protective effect of ozone–oxygen therapy in ethidium bromide (EB)-induced demyelination in rats either alone or in combination with corticosteroids where the combination may allow decreasing the dose of steroid therapy.

Materials and Methods

Materials

Animals

Male adult Wistar albino rats weighing 180 ± 10 g were obtained from the Animal House Colony, National Research Center, Cairo, Egypt. They were housed in stainless steel wire-meshed cages under environmentally controlled conditions. The ambient temperature was 25 ± 2 °C and the light/dark cycle was 12/12 h. The animals had free access to water and standard rodent chow diet. All animals received human care in compliance with guidelines of the Ethical Committee of National Research Centre, Egypt Centre and followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.

Chemicals

Ozone was generated by an ozone generator system (EXT120-T). It is a medical oxygen-fed ozone generator for ultra pure medical applications (Longevity resources Inc., Canada). Ozone obtained from medical grade oxygen was used immediately and it represented only about 3 % of the gas (O2 + O3) mixture. It produces 1–120 µg/ml ozone concentrations. The O3 concentration is measured by using an UV spectrophotometer at 254 nm as recommended by the Standardization Committee of the International Ozone Association. The ozone dose is the product of the O3 concentration (expressed as mg/l) by the gas (O2 + O3) volume (Devesa et al. 1993). Ozone was administered by rectal insufflations (RI). Rats received five applications per week, one per day. The application started with a dose of ozone of 0.5 mg/(kg day) (25 µg/ml) in the first week and increased to 1 mg/(kg day) (50 µg/ml) in the second week. The volume of ozone administered was 4–5 ml/rat according to the animal weight. Unless otherwise stated, all chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA).

Experimental Demyelination with EB

Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). After shaving the hair from the fronto-occipital area, antisepsis was performed with 2 % iodine solution. A hole of 0.5 cm was made using orthodontic roof motor and number 2 drill to the right of the bregma until the dura mater was exposed. With the use of a Hamilton syringe fitted with a 30-gauge needle, EB solution (3 µl of 0.1 %) was injected in the cisterna pontis (basal), an enlargement of the subarachnoid space on the ventral surface of the pons. The dura mater left open and the skin together with remainder of the subcutaneous tissue was sutured with a nylon thread 4.0. Two groups of rats were subjected to the same surgical procedure but injected with sterile saline (0.9 %) and served as sham-operated groups.

Experimental Design

On the 7th day of the surgical procedure, rats were divided into the following groups (n = 8): Group (1) Naïve rats received oral saline and served as normal control, Group (2) Sham-operated rats were injected with saline intracranially, Group (3) Sham-operated rats received vehicle (oxygen), Group (4) EB-treated rats were injected intracranially with 3 µl of 0.1 % EB, Group (5) EB-treated rats received O3 [0.5 mg/(kg day), RI] for 5 days in the first week increased to 1 mg/(kg day) for 5 days in 2nd week, Group (6) EB-treated rats received 30 mg/kg MP sodium succinate intraperitoneally (Solu-Medrol® Upjohn Co, Kalamazoo, MI), and Group (7) EB-treated rats received half the dose of MP concomitant with O3 [0.5 mg/(kg day)] for 5 days then increased to 1 mg/(kg day) for 5 days in 2nd week.

At the end of the experimental period, deficits in descending fine motor control was examined by the grid walk test which assesses the ability of all rats to navigate across a 1-m-long runway with irregularly assigned gaps (0.5–5.0 cm) between round metal bars, as described previously by Metz et al. (2000). Crossing this runway required that animals accurately place their limbs on the bars. In the test, every animal crossed the grid at least three times. The numbers of footfalls (errors) for hindlimbs were counted in each crossing, and the mean was calculated. On the next day, all rats were decapitated in deep ether anesthesia. Whole brains were isolated and divided into two halves. The first portion was formalin-fixed and paraffin-embedded for subsequent immunohistochemical assessment of Cox-2 and MBP. The second portion was washed with ice-cold saline, weighed and homogenized with 0.1 M phosphate buffer saline at pH 7.4, to give a final concentration of 10 % w/v and centrifuged at 3000×g for 15 min at 4 °C. The resulting supernatant was used for subsequent biochemical assessments.

Methods

Oxidative Stress Markers

Lipid peroxidation was estimated by measuring thiobarbituric reactive substances (TBARS) in brain samples according to the method of Placer et al. (1966) and the results were expressed as nanomole MDA per g wt tissue. Brain glutathione was evaluated by the method of Ellman (1959) and expressed as µg/(g wt) tissue. The determination of paraoxonase 1(PON1) activity was carried out in accordance with the method of Gatica et al. (2006). This assay involves the hydrolysis of phenylacetate (substrate) by paraoxonase1/arylesterase activity releasing phenol. The phenol formed after the addition of a 40-fold diluted homogenate sample was spectrophotometrically measured at 217 nm. Blanks were included to correct the spontaneous hydrolysis of phenylacetate. The activity of PON1 was expressed in K unit/(g wt) tissue. One unit was defined as the enzyme quantity that disintegrates 1 nmol phenylacetate per minute.

Brain Cytokine Levels

Brain TNF-α, IL-1β, and interferon-γ (IFN-γ) were determined by enzyme-linked immuno-sorbent assay following the methods of Kitaura et al. (2004), Tamaoki et al. (1999), and Sommandas et al. (2005), respectively using ELISA kits (Invitrogen Corporation Camarillo, CA, USA) and microtiter plate reader (Fisher Biotech, Germany).

Brain Monoamines Neurotransmitters

Determination of brain serotonin (5-hydroxytryptamine, 5-HT), dopamine, and norepinephrine (NE) was carried out using high-performance liquid chromatography (HPLC) system, Agilent technologies 1100 series equipped with a quaternary pump (Quat pump, G131A model). Separation was achieved on ODS-reversed phase column (C18, 25 × 0.46 cm i.d. 5 µm). The mobile phase consisted of potassium phosphate buffer/methanol 97/3 (v/v) and was delivered at a flow rate 1 ml/min. UV detection was performed at 270 nm, and the injection volume was 20 µl. The concentration of the neurotransmitters was determined by external standard method using peak areas. A linear standard curve was constructed where samples concentration was obtained directly from the curve.

Immunohistochemical Assessment of Cox-2 and Myelin Basic Protein (MBP)

Brain sections were cut into 4 µm then fixed in a 65 °C oven for 1 h. Triology (Cell Marque, CA-USA. cat# 920 p-06) is a product that combines the three pretreatment steps: deparaffinization, rehydration, and antigen unmasking. Slides were placed in a coplin jar filled with 200 ml of triology working solution and the jar is placed in the autoclave at 120 °C for 15 min after which pressure is released and the coplin jar is removed to allow slides to cool for 30 min. Sections were then washed and immersed in Tris-buffered saline (TBS). Quenching endogenous peroxidase activity was performed by immersing slides in 3 % hydrogen peroxide for 10 min. Power Stain TM 1.0 Poly HRP DAB Kit Cat# 54-0017 (Genemed Biotechnologies, CA-USA) was used to visualize any antigen–antibody reaction in the tissues. Two to three drops of the rabbit polyclonal primary antibody (Cox-2, Thermoscientific, CA-USA) and MBP (Sternberger Monoclonals Inc., Berkeley, CA, USA) were applied, and the slides were incubated in the humidity chamber overnight at 4 °C. Henceforward, poly horseradish peroxidase (HRP) enzyme conjugate was applied to each slide for 20 min. Diaminobenzidine (DAB) chromogen was prepared and 2–3 drops were applied on each slide for 2 min. DAB was rinsed, after which counterstaining with Mayer Hematoxylin and cover slipping were performed as the final steps before slides were examined under light microscope.

For each positive section, six microscopic fields showing the highest immunopositivity were captured at a magnification ×200 using a digital video camera mounted on a light microscope (CX21, OLYMPUS, JAPAN). Images were then transferred to the computer system for analysis using image analysis software (Image J, 1.46a, NIH, USA). Brown stain of any intensity was considered positive, whereas the background blue stain was considered negative. Optical density was then calculated automatically representing the immunostaining intensity of Cox-2 immunopositive cells.

Western Blot Analysis

Brain tissue was homogenized with lysis buffer [20 mM Tris/HCl, pH 8, 150 mM NaCl, 2 % sodium deoxycholate, 2 mM phenylmethylsulphonylfluoride (PMSF), 1 mM phenanthroline, 1 mM p chloromercuribenzoic acid, and 1 mM iodoacetamide] and centrifuged at 45,000 rpm for 60 min at 4 °C. Approximately, 15 μg of extract was loaded per lane of a 10 % SDS-PAGE gel, electrophoresed, and transferred to nitrocellulose membrane (Amersham Hybond-C Extra). Immunoblots were probed with mouse anti-P53 monoclonal antibody (Thermo Scientific DO-7 Mouse IgG2-b raised against recombinant wild-type P53 protein expressed in E. coli). The blots were incubated with the secondary antibody (Sigma Goat, anti-mouse IgG-SAB3701029). Visualization of the secondary antibody was done using 3,3′-diaminobenzidine (DAB) liquid substrate system (Sigma Cat. No. D7304), and the blots were photographed using Olympus C-4000 Zoom Digital Camera (Olympus Optical Co., LTD).

Results

Grid Walk Test to Assess the Motor Behavior

EB-treated rats showed a significant increase in the number of footfalls in the grid walk test compared to sham-operated rats (12.87 ± 0.8 vs. 0.28 ± 0.05) (p < 0.05). Comparisons between groups showed significant improvement with the administration of ozone (9.3 ± 0.6), MP (5.5 ± 0.3), where the best amelioration was achieved by combining half the dose of MP with ozone (3.2 ± 0.12) as compared to EB-treated rats (p < 0.05) (Fig. 1).

Fig. 1.

Fig. 1

Open in a new tab

Grid walk test. The average number of footfall errors is depicted on the ordinate, different treatments on the abscissa. EB ethidium bromide, MP methylprednisolone, O 3 ozone. Data are expressed as mean ± S.E. Groups with different letters are significantly different at p < 0.05, n = 6

Oxidative Stress Markers and Cytokines Levels

Brain PON1 activity and GSH were significantly lowered in EB-treated rats in comparison to sham-operated animals. Experimental demyelination showed a significant elevation in brain MDA, TNF-α, IL-1β, and INF-γ when compared to sham-operated group. Ozone treatment revealed an enhancement in PON1 activity and GSH, whereas MDA was significantly decreased in comparison to EB-treated rats. Treatment with MP showed a significant increase in PON1 activity and GSH while MDA, TNF-α, IL-1β, and INF-γ were significantly lowered compared to demyelinated group. Combination of half dose of MP with ozone succeeded in nearly normalizing the above-mentioned parameters (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

Brain oxidative stress markers and cytokines levels in the different experimental groups. EB ethidium bromide, O 3 ozone, MP methylprednisolone, PON1 paraoxonase 1, MDA malondialdehyde, GSH glutathione, TNF-α tumor necrosis factor-α, IL-1β interleukin-1β, IFN-γ interferon-γ. Data are expressed as mean ± S.E. Groups with different letters are statistically different (p < 0.05), n = 6

Brain Neurotransmitters, Inflammatory and Apoptotic Markers

As shown in (Fig. 3), EB-treated rats showed a significant decline in 5-HT, dopamine, and NE compared to sham-operated rats. Normalization of the tested neurotransmitters was achieved by treatment with either ozone or MP where a superior effect was observed with MP. Profound enhancement of brain 5-HT, dopamine, and NE was achieved by combining half the dose of MP with ozone. Immunohistochemical assessment of brain Cox-2 (Fig. 4) and Western blotting analysis of p53 (Fig. 5) showed significant increase in demyelinated animals compared to sham-operated ones. No significant change was observed after ozone treatment, however, MP alone and in combination with ozone produced significant decrease in Cox-2 immunoreactivity and p53 protein level in comparison to EB-treated animals.

Fig. 3.

Fig. 3

Open in a new tab

Brain neurotransmitters levels in the different experimental groups. EB ethidium bromide, O 3 ozone, MP methylprednisolone. Data are expressed as mean ± S.E. Groups with different letters are statistically different (p < 0.05), n = 6

Fig. 4.

Fig. 4

Open in a new tab

Immunoreactivity of Cox-2 cells in the hippocampus of rats in the tested groups. A Light photomicrographs demonstrate immunoreactivity in the hippocampus of normal (a), sham-operated (b), sham-operated + vehicle (oxygen) (c), ethidium bromide-treated (d), ethidium bromide + ozone-treated (e), ethidium bromide + methylprednisolone-treated (f), ethidium bromide + ozone + 1/2 dose of methylprednisolone-treated rats (g). B Number of Cox-2 reactive cells in the hippocampus of rats in the tested groups. The means of cells positive to Cox-2 are depicted on the ordinate, different treatments on the abscissa. Groups with different letters are significantly different at p < 0.05, n = 6 (scale bar 20 μm)

Fig. 5.

Fig. 5

Open in a new tab

Western blot analysis of p53 in the different experimental groups. EB ethidium bromide, O 3 ozone, MP methylprednisolone

MBP Immunohistochemistry Evaluation

Immunohistochemical examination of brain MBP (Fig. 6) revealed that the expression of MBP in the hippocampus was significantly suppressed in the EB-treated group as compared to sham-operated animals however, MP alone and in combination with ozone significantly alleviated the EP-induced suppression of MBP expression as compared to EB-treated rats.

Fig. 6.

Fig. 6

Open in a new tab

Photomicrographs of myelin basic protein (MBP)-positive cells in the hippocampus of rats in the tested groups. MBP immunoreactivity in the hippocampus of normal (a), sham operated (b), Sham operated + vehicle(oxygen) (c), ethidium bromide-treated (d), ethidium bromide + ozone-treated (e), ethidium bromide + methylprednisolone-treated (f), ethidium bromide + ozone + 1/2 dose of methyl prednisolone-treated rats (g). b Number of MBP positive cells in the hippocampus of rats in the tested groups. The means of cells positive to MBP are depicted on the ordinate, different treatments on the abscissa. Groups with different letters are significantly different at p < 0.05, n = 6 (scale bar 20 μm)

Discussion

Multiple sclerosis is characterized by demyelination of axons which leads to a deficiency or complete loss in the transmission of nerve impulses (Becq et al. 2005). The present study aimed to investigate the effect of ozone and/or corticosteroid therapy on demyelination pathology following local injection of EB in rats. EB, a chromatin-disrupting agent, is known to induce early astrocyte disappearance and oligodendroglial loss when injected in the CNS leading to blood–brain barrier disruption and primary demyelination, 6 days after the surgical procedure (Bondan et al. 2008). The grid walk paradigm assesses deficits in the descending fine motor control. It requires voluntary fine movement control (Metz et al. 2000). Results of the present study showed a significant difference in the motor behavior (increased number of footfalls) of EB-treated animals in comparison to sham-operated ones, probably due to motor limitation during locomotion and uncertainty to perform the steps needed to complete the grid. Such defects have been correlated to oligodendrocyte loss and, consequently, to the demyelination caused by EB (Bondan et al. 2006; Kerschensteiner et al. 2004).

Lymphocytes and activated macrophages containing phagocytosed myelin were found in the demyelinating lesions from EB-injected rats, in a relationship suggestive of antigenic recognition. Reactive T cells and macrophages in CNS produce cytokines and other mediators like TNF-α, INF-γ, and ROS (H2O2, O2-, NO) that activate caspase protein family. In the brain, the high content of polyunsaturated fatty acids and the low capacity for antioxidant defense and intrinsic risk factors account for the susceptibility to free radical damage (Fragoso et al. 2004). IFN-γ is a pro-inflammatory cytokine made primarily by T-lymphocytes and natural killer cells; under normal circumstances, IFN-γ is usually not detectable in the brain (Bitsch et al. 2000). However, elevated expression of IFN-γ persists in chronic inflammatory diseases such as MS (Popko et al. 1997). Transgenic expression of IFN-γ in the CNS of mice following demyelinating insult, results in oligodendrocyte cell loss and hypomyelination (Balabanov et al. 2007).

Our results revealed that glutathione and PON1 enzyme activity in rat brains were depressed, whereas MDA, IL-1β, TNF-α, and INF-γ were elevated in EB-treated rats as compared to sham-operated ones. These results are in agreement with those obtained in patients with MS, where oxidative stress parameters such as conjugated dienes in cerebrospinal fluid were increased while antioxidant defenses were found to be decreased, suggesting that oxidative stress may be associated with the pathogenesis of demyelinating diseases (Koch et al. 2006). The expression of mRNA for IL-1β, TNF-α, and INF-γ were remarkably upregulated in rat model of MS. Free radicals can activate certain transcription factors, such as nuclear transcription factor-kappa B (NF-κB), which upregulates the expression of genes involved in EAE, such as TNF-α (Glass et al. 2010). PON1 is located in a subfraction of HDL and may function to protect cell membranes against lipid peroxidation. HDL is the only lipoprotein present in the CNS (Rosenblat and Aviram 2009). Ferretti et al. (2005) found low plasma activity of PON1 in patients with MS, suggesting that the exacerbation of oxidative stress decreases the activity of PON1 (Moghtaderi et al. 2011).

Results of the current study revealed a significant decline in 5-HT, dopamine, and NE levels in brains of EB-treated rats compared to sham-operated ones. It has been shown that inflammatory mediators like IL-1 β or TNF-α can influence 5-HT transporter (5-HTT) activity (Mossner and Lesch 1998). Free radicals can convert the parent catechols into their quinone derivatives, which can modify the sulfhydryl group of tyrosine hydroxylase enzyme and result in the formation of cysteinyl-catechols within the enzyme leading to its inhibition (Kuhn et al. 1999). Schott et al. (2003) demonstrated the presence of anti-serotonin antibodies in serum of MS patients leading to inhibition of serotonin binding to human cortical membranes which represents an autoimmune process of the CNS. Polak et al. (2011) demonstrated that there are significant reduction in NE in brains of EAE mice with neuronal damage as indicated by tyrosine hydroxylase positive neuronal cell shrinkage suggesting loss of NE synthesis as a contributing cause. Alternatively, this decrease in NE could be due to increased NE metabolism in the cortex by catechol-O-methyltransferase, whose expression is significantly increased in the brain under inflammatory conditions (Helkamaa et al. 2007).

Arachidonic acid is an n-6 polyunsaturated fatty acid, which is released upon inflammatory stimuli and then converted by cyclooxygenase (Cox)-1 and -2 to prostaglandins (PGs), potent mediators of inflammation. The p53 protein, a transcription factor, exercises a critical regulatory function at the G1-cell cycle checkpoint to allow for the repair of damaged DNA and in apoptosis in response to stress stimuli. Such stimuli include inflammatory cytokines, or DNA-damaging substances some of which are postulated to be involved in MS. Our results revealed a significant increase in Cox-2 immunoreactivity and p53 protein level associated with significant decline in MBP expression in brains of EB-treated rats compared to sham-operated ones. Palumbo et al. (2011) demonstrated that Cox-2 gene expression was consistently upregulated in mice given the neurotoxicant cuprizone to induce brain demyelination. Exposure of human adult oligodendrocytes to the pro-inflammatory cytokine TNF-α was associated with the up-regulation and nuclear translocation of p53 inducing rapid apoptosis in these cells (Ladiwala et al. 1999). EB injection into rats induced hypomyelination in the hippocampus of rats, that was represented by suppressed expression of MBP. MBP is the most abundant protein in the myelin sheath. One possible reason for the reduced MBP staining is a reduction MBP production by the oligodendrocyte or a reduction in the number of oligodendrocytes after EB treatment.

Glucocorticoids (GS) are potent anti-inflammatory drugs and are commonly used in the treatment of autoimmune disorders. Corticosteroids may have promoted spontaneous CNS remyelination by their anti-inflammatory effects; i.e., perturbation of leukocyte trafficking, and inhibition of T-lymphocyte activation, or by interfering with locally secreted cytokines (Gold et al. 2001). Our results revealed that MP administration enhanced rat ability to perform the grid walk test which is reflected on the decrease in the number of footfalls. Hellwig et al. (2004) demonstrated that repeated intrathecal injection of corticosteroids increased the walking distance in MS patients. The bcl-xL gene has several glucocorticoid response elements (GRE) in the promoter region where it is induced by MP in oligodendrocytes. It is believed to exert its anti-apoptotic action by preventing the release of proapoptotic signaling molecules from the inner mitochondrial membrane (Gascoyne et al. 2003). Results of the current study demonstrated that MP was found to significantly decrease the elevated brain levels of TNF-α, IL-1β, and IFN-γ compared to EB-treated animals. MP increased the secretion of Th2 cytokines such as IL-4, IL-10, and IL-13, while it decreased the production of Th1 cytokines such as IFN-γ, TNF-α, and IL-2 in EAE animals and MS patients. MP promotes the differentiation of T cells into the Th2 bias and thereby modulates the susceptibility to EAE and MS (Tischner and Reichardt 2007). Miljković et al. (2009) found that MP inhibited IFN-γ and IL-17 generation by the cells infiltrating the CNS in EAE rats where the number of cells isolated from the spinal cord was significantly decreased. This could be a consequence of the inhibition of encephalitogenic cell infiltration into the CNS which might be due to increased integrity of BBB.

The present study results showed that MP enhanced PON1 activity, brain GSH content, decreased MDA content and raised brain serotonin, NE, and dopamine levels compared to EB-treated rats. These results were in agreement with Mitosek-Szewczyk et al. (2010), Chelmicka-Schorr et al. (1989), and Hall and McGinley (1982).

Moreover, our results revealed decreased p53 and Cox-2 associated with increased MBP expression in MP-treated animals. Treatment with MP decreases the expression of p53 in psoriatic patients (Adişen et al. 2006; Andreas et al. 2009).

Glucocorticoids have multiple side effects and it is a near universal aim of physicians treating inflammatory diseases to reduce glucocorticoid exposure. Ozone has been considered as a pro-drug which, at certain nontoxic doses, can induce a rearrangement of the biochemical pathways with the activation of a second messenger in a cascade with a multisystem action (Rodriguez et al. 2010). Our results showed that ozone exposure induced significant rise in PON-1 activity and GSH, whereas MDA was significantly decreased in rat brain when compared to EB-treated group. However, a non-significant rise in TNF-α, IL-1β, and INF-γ was reported. After administration, ozone is dissolved in biologic fluids and immediately reacts with polyunsaturated fatty acids, antioxidants, reduced glutathione, and albumin. All of these compounds act as electron donors and undergo oxidation, resulting in formation of hydrogen peroxide (H2O2). Hydrogen peroxide is able to act as an ozone messenger for eliciting several therapeutic effects. In contrast to the conventional idea that H2O2 is harmful, it has been widely accepted that it acts as a regulator of signal transduction and is an important mediator of host defense and immune responses (Zamora et al. 2005). Although H2O2 acts immediately and is metabolized, lipid oxidation products distribute throughout the tissues and become late and long-lasting messengers. This process stimulates the innate immune system and supports cellular antioxidant power preparing the host to face physiopathologic conditions mediated by ROS (Bocci 2006). Increased levels of GSH could also result from ozone-induced increases in cellular permeability and release of intracellular GSH as suggested by Jörres et al. (2000). In adult mice exposed prenatally to ozone, Santucci et al. (2006) found increased expression of brain-derived neurotrophic factor (BDNF) suggesting brain cellular adaptive response to ozone. Araneda et al. (2008) found that vascular endothelial growth factor (VEGF)-immunoreactive glial cells are in contact with blood vessel walls and that the blood vessel area was markedly increased during post-ozone recovery indicating revascularization and repair of the BBB. In addition, VEGF exerted neuroprotective effect by stimulating neurogenesis from neural stem cell (Sun et al. 2006).

Current results revealed significant elevation in 5-HT, dopamine, and NE levels after ozone exposure compared to EB-treated group in addition to amelioration in the grid walk test. Soulage et al. (2004) demonstrated that the ozone exposure was found to reduce the rate of dopamine turnover in the striatum. Since ozone has been found to decrease brain catecholamine turnover following long-term exposure, amelioration of the grid walk test may be a direct consequence of the aforementioned effects of ozone on key neurochemicals affecting behavioral response in EB-treated rats. Barragán-Mejía et al. (2002) demonstrated that animals exposed to ozone exhibited a significant cortical 5-HT increase compared with non-exposed animals, suggesting an increased transport of free tryptophan, an immediate precursor of 5-HT, to the brain with a possible enhancement of serotonin synthesis due to the high activity of tryptophan-5 hydroxylase. The present study revealed that ozone exposure resulted in non-significant increase in Cox-2 immunoreactivity and p53 protein level in rat brain as compared to EB-treated group. The study of Schulz et al. (2012) give evidence that intra-abdominal insufflation of ozone induces time-dependently vascular expression of mRNA and protein for Cox-2 in rats. Cox-2 may be pro-inflammatory during the early phase of a carrageenin-induced pleurisy in rats, but may aid resolution at the later phase by generating an alternative set of anti-inflammatory prostaglandins (Gilroy et al. 1999). When there is significant DNA damage in glial cells due to oxidative stress, wild-type p53 protein results in G1 arrest, presumably to allow for the repair of damaged DNA. However, if glial cells are unable to repair damaged DNA, p53 may induce apoptosis. It was reported that p53 accumulation was observed in H2O2-treated human glial cells (Kitamura et al. 1999).

Our results revealed that combination of ozone and half dose of MP succeeded in nearly attaining the ameliorating effect of MP alone. Andreula et al. (2003) gave evidence that the combined intradiscal and periganglionic injection of medical ozone and periganglionic injection of steroids has a cumulative effect that enhances the overall outcome of treatment of lumbar disk herniation. Martrette et al. (2011) demonstrated that ozone-exposed rats had increased levels of plasma corticosterone which could suggest that ozone exposure is a stress in these animals.

In conclusion, ozone therapy approach could be considered as a positive complement to the actual pharmacological therapies addressed to neurodegenerative disorders such as MS, promoting the maintenance of an adequate cellular redox balance together with considerable immune-modulatory effects.

Acknowledgments

The authors would like to thank Dr Abdel Razik (Professor of Pathology, National Research Centre, Egypt) for his kind cooperation in the histopathological examinations involved in this research.

Compliance with Ethical Standards

Conflict of interest

Authors declared no conflict of interest.

References

  1. Adişen E, Gülekon A, Erdem O et al (2006) The effects of calcipotriol and methylprednisolone aseponate on bcl-2, p53 and ki-67 expression in psoriasis. J Eur Acad Dermatol Venereol 20:527–533 [DOI] [PubMed] [Google Scholar]
  2. Anderson PB, Goodkin DE (1998) Glucocorticoid therapy for multiple sclerosis: a critical review. J Neurol Sci 160:16–25 [DOI] [PubMed] [Google Scholar]
  3. Andreas K, Häupl T, Lübke C et al (2009) Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration. Arthritis Res Ther 11:R15. doi:10.1186/ar2605 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Andreula CF, Simonetti L, De Santis F et al (2003) Minimally invasive oxygen–ozone therapy for lumbar disk herniation. Am J Neuroradiol 24:996–1000 [PMC free article] [PubMed] [Google Scholar]
  5. Araneda S, Commin L, Atlagich M et al (2008) VEGF overexpression in the astroglial cells of rat brainstem following ozone exposure. Neurotoxicology 29:920–927 [DOI] [PubMed] [Google Scholar]
  6. Balabanov R, Strand K, Goswami R et al (2007) Interferon-γ-oligodendrocyte interactions in the regulation of experimental autoimmune encephalomyelitis. J Neurosci 27:2013–2024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barber E, Menendez S, Leon OS et al (1999) Prevention of renal injury after induction of ozone tolerance on rats submitted to warm ischaemia. Mediat Inflamm 8:37–41 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barragán-Mejía MG, Castilla-Serna L, Calderón-Guzmán D et al (2002) Effect of nutritional status and ozone exposure on rat brain serotonin. Arch Med Res 33:15–19 [DOI] [PubMed] [Google Scholar]
  9. Becq H, Jorquera I, Ben-Ari Y et al (2005) Differential properties of dentate gyrus and CA1 neural precursors. J Neurobiol 62:243–261 [DOI] [PubMed] [Google Scholar]
  10. Bitsch A, Kuhlmann T, Da Costa C et al (2000) Tumour necrosis factor alpha mRNA expression in early multiple sclerosis lesions: correlation with demyelinating activity and oligodendrocyte pathology. Glia 29:366–375 [DOI] [PubMed] [Google Scholar]
  11. Bocci VA (2006) Scientific and medical aspects of ozone therapy. State of the art. Arch Med Res 37:425–435 [DOI] [PubMed] [Google Scholar]
  12. Bocci V, Zanardi I, Travagli V (2011) Has oxygen-ozone therapy a future in medicine? J Exp Integr Med 1:5–11. doi:10.5455/jeim.161210.ir.002 [Google Scholar]
  13. Bondan EF, Lallo MA, Orsini H et al (2006) Evaluation of locomotor activity after a local induction of toxic demyelination in the brainstem of Wistar rats. Arq Neuropsiquiatr 64:496–503 [DOI] [PubMed] [Google Scholar]
  14. Bondan EF, Lallo MA, Graça DL (2008) Ultrastructural study of the effects of cyclosporine in the brainstem of Wistar rats submitted to the ethidium bromide demyelinating model. Arq Neuropsiquatr 66:378–384 [DOI] [PubMed] [Google Scholar]
  15. Carlson NG, Rose JW (2006) Antioxidants in multiple sclerosis: do they have a role in therapy? CNS Drugs 20:433–441 [DOI] [PubMed] [Google Scholar]
  16. Chelmicka-Schorr E, Kwasniewski M, Thomas B et al (1989) The β-adrenergic agonist isoproterenol suppresses experimental allergic encephalomyelitis in Lewis rats. J Neuroimmunol 25:203–207 [DOI] [PubMed] [Google Scholar]
  17. Devesa E, Menéndez S, Rodrõguez MM et al (1993) Ozone therapy in ischemic cerebro-vascular disease. In: Proceedings 11th ozone world congress (USA) ozone in medicine, pp M-4-10–M-4-18
  18. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82:70–77 [DOI] [PubMed] [Google Scholar]
  19. Ferretti G, Bacchetti T, Principi F et al (2005) Increased levels of lipid hydroperoxides in plasma of patients with multiple sclerosis: a relationship with paraoxonase activity. Mult Scler 11:677–682 [DOI] [PubMed] [Google Scholar]
  20. Fragoso G, Martınez-Bermudez AK, Liu HN et al (2004) Developmental differences in HO-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3. J Neurochem 90:392–404 [DOI] [PubMed] [Google Scholar]
  21. Gascoyne DM, Kypta RM, Vivanco MM (2003) Glucocorticoids inhibit apoptosis during fibrosarcoma development by transcriptionally activating Bcl-xL. J Biol Chem 278:18022–18029 [DOI] [PubMed] [Google Scholar]
  22. Gatica LV, Vega VA, Zirulnik F et al (2006) Alterations in the lipid metabolism of rat aorta: effects of vitamin A deficiency. J Vasc Res 43:602–610 [DOI] [PubMed] [Google Scholar]
  23. Gilroy DW, Colville-Nash PR, Willis D et al (1999) Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med 5:698–701 [DOI] [PubMed] [Google Scholar]
  24. Glass C, Saijo K, Winner B et al (2010) Mechanisms underlying inflammation in neurodegeneration. Cell 140:918–934 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gold R, Buttgereit F, Toyka KV (2001) Mechanism of action of glucocorticosteroid hormones: possible implications for therapy of neuroimmunological disorders. J Neuroimmunol 117:1–8 [DOI] [PubMed] [Google Scholar]
  26. Hafler DA (2004) Multiple sclerosis. J Clin Invest 113:788–794 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hall ED, McGinley PA (1982) Effects of a single intravenous glucocorticoids dose on biogenic amine levels in cat lumbar spinal cord. J Neurochem 38:1787–1790 [DOI] [PubMed] [Google Scholar]
  28. Helkamaa T, Reenilä I, Tuominen RK et al (2007) Increased catechol-O-methyltransferase activity and protein expression in OX-42-positive cells in the substantia nigra after lipopolysaccharide microinfusion. Neurochem Int 51:412–423 [DOI] [PubMed] [Google Scholar]
  29. Hellwig K, Stein FJ, Przuntek H et al (2004) Efficacy of repeated intrathecal triamcinolone acetonide application in progressive multiple sclerosis patients with spinal symptoms. BMC Neurol 7(4):18 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jörres RA, Holz O, Zachgo W et al (2000) The effect of repeated ozone exposures on inflammatory markers in bronchoalveolar lavage fluid and mucosal biopsies. Am J Respir Crit Care Med 161:1855–1861 [DOI] [PubMed] [Google Scholar]
  31. Kerschensteiner M, Stadelmann C, Buddeberg BS et al (2004) Targeting experimental autoimmune encephalomyelitis lesions to a predetermined axonal tract system allows for refined behavioral testing in an animal model of multiple sclerosis. Am J Pathol 164:1455–1469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kitamura Y, Ota T, Matsuoka Y et al (1999) Hydrogen peroxide-induced apoptosis mediated by p53 protein in glial cells. Glia 25:154–164 [PubMed] [Google Scholar]
  33. Kitaura H, Sands MS, Aya K et al (2004) Marrow stromal cells and osteoclast precursors differentially contribute to TNF-α-induced osteoclastogenesis in vivo. J Immunol 173:4838–4846 [DOI] [PubMed] [Google Scholar]
  34. Koch M, Ramsaransing GS, Arutjunyan AV et al (2006) Oxidative stress in serum and peripheral blood leukocytes in patients with different disease courses of multiple sclerosis. J Neurol 253:483–487 [DOI] [PubMed] [Google Scholar]
  35. Kuhn DM, Arthur RE Jr, Thomas DM et al (1999) Tyrosine hydroxylase is inactivated by catechol-quinones and converted to a redox-cycling quinoprotein. Possible relevance to Parkinson’s Disease. J Neurochem 73:1309–1317 [DOI] [PubMed] [Google Scholar]
  36. Ladiwala U, Li H, Antel JP et al (1999) p53 induction by tumor necrosis factor-α and involvement of p53 in cell death of human oligodendrocytes. J Neurochem 73:605–611 [DOI] [PubMed] [Google Scholar]
  37. León OS, Menéndez S, Merino N et al (1998) Ozone oxidative preconditioning: protection against cellular damage by free radicals. Mediat Inflamm 7:289–294 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Martrette JM, Thornton SN, Trabalon M (2011) Prolonged ozone exposure effects behaviour, hormones and respiratory muscles in young female rats. Physiol Behav 103:302–307 [DOI] [PubMed] [Google Scholar]
  39. Metz GA, Merkler D, Dietz V et al (2000) Efficient testing of motor function in spinal cord injured rats. Brain Res 883:165–177 [DOI] [PubMed] [Google Scholar]
  40. Miljković Z, Momcilović M, Miljković D et al (2009) Methylprednisolone inhibits IFN-gamma and IL-17 expression and production by cells infiltrating central nervous system in experimental autoimmune encephalomyelitis. J Neuroinflammation 6:37. doi:10.1186/1742-2094-6-37 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mitosek-Szewczyk K, Gordon-Krajcer W, Walendzik P et al (2010) Free radical peroxidation products in cerebrospinal fluid and serum of patients with multiple sclerosis after glucocorticoid therapy. Folia Neuropathol 48:116–122 [PubMed] [Google Scholar]
  42. Moghtaderi A, Hashemi M, Sharafaddinzadeh N et al (2011) Lack of association between paraoxonase 1 Q192R polymorphism and multiple sclerosis in relapse phase: a case–control study. Clin Biochem 44:795–798 [DOI] [PubMed] [Google Scholar]
  43. Mossner R (1998) Lesch KP Role of serotonin in the immune system and in neuroimmune interactions. Brain Behav Immun 12:249–271 [DOI] [PubMed] [Google Scholar]
  44. Palumbo S, Toscano CD, Parente L et al (2011) Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination. Prostaglandins Leukot Essent Fatty Acids 85:29–35 [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Placer ZA, Cushman LL, Johnson BC (1966) Estimation of lipid peroxidation (malondialdehyde) in biochemical systems. Anal Biochem 16:359–365 [DOI] [PubMed] [Google Scholar]
  46. Polak PE, Kalinin S, Feinstein DL (2011) Locus coeruleus damage and noradrenaline reductions in multiple sclerosis and experimental autoimmune encephalomyelitis. Brain 134:665–677 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Popko B, Corbin JG, Baerwald KD et al (1997) The effects of interferon-γ on the central nervous system. Mol Neurobiol 14:19–35 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rodriguez ZZ, Guanche D, Alvarez RG et al (2010) Preconditioning with ozone/oxygen mixture induces reversion of some indicators of oxidative stress and prevents organic damage in rats with fecal peritonitis. Inflamm Res 58:371–375 [DOI] [PubMed] [Google Scholar]
  49. Romero VA, Menéndez CS, Gómez MM et al (1993) Ozone therapy in the advanced stages of arteriosclerosis obliterans. Angiologia 45:146–148 [PubMed] [Google Scholar]
  50. Rosenblat M, Aviram M (2009) Paraoxonases role in the prevention of cardiovascular diseases. Biofactors 35:98–104 [DOI] [PubMed] [Google Scholar]
  51. Santucci D, Sorace A, Francia N et al (2006) Prolonged prenatal exposure to low level ozone affects aggressive behaviour as well as NGF and BDNF levels in the central nervous system of CD-1 mice. Behav Brain Res 166:124–130 [DOI] [PubMed] [Google Scholar]
  52. Schott K, Schaefer JE, Richartz E et al (2003) Autoantibodies to serotonin in serum of patients with psychiatric disorders. Psychiatry Res 121:51–57 [DOI] [PubMed] [Google Scholar]
  53. Schulz S, Ninke S, Watzer B et al (2012) Ozone induces synthesis of systemic prostacyclin by cyclooxygenase-2 dependent mechanism in vivo. Biochem Pharmacol 83:506–513 [DOI] [PubMed] [Google Scholar]
  54. Sommandas V, Rutledge EA, Van Yserloo B et al (2005) Aberrancies in the differentiation and maturation of dendritic cells from bone-marrow precursors are linked to various genes on chromosome 4 and other chromosomes of the BB-DP rat. J Autoimmun 25:1–12 [DOI] [PubMed] [Google Scholar]
  55. Soulage C, Perrin D, Cottet-Emard JM et al (2004) Central and peripheral changes in catecholamine biosynthesis and turnover in rats after a short period of ozone exposure. Neurochem Int 45:979–986 [DOI] [PubMed] [Google Scholar]
  56. Sun Y, Jin K, Childs JT et al (2006) Vascular endothelial growth factor-B (VEGFB) stimulates neurogenesis: evidence from knockout mice and growth factor administration. Dev Biol 289:329–335 [DOI] [PubMed] [Google Scholar]
  57. Tamaoki J, Kondo M, Kohri K et al (1999) Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages. J Immunol 163:2909–2915 [PubMed] [Google Scholar]
  58. Tischner D, Reichardt HM (2007) Glucocorticoids in the control of neuroinflammation. Mol Cell Endocrinol 275:62–70 [DOI] [PubMed] [Google Scholar]
  59. Travagli V, Zanardi I, Bernini P et al (2010) Effects of ozone blood treatment on the metabolite profile of human blood. Int J Toxicol 29:165–174 [DOI] [PubMed] [Google Scholar]
  60. Zamora ZB, Borrego A, Lopez OY et al (2005) Effects of ozone oxidative preconditioning on TNF-alpha release and antioxidant prooxidant intracellular balance in mice during endotoxic shock. Mediat Inflamm 2005:16–22 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zorzon M, Zivadinov R, Locatelli L et al (2005) Long-term effects of intravenous high dose methylprednisolone pulses on bone mineral density in patients with multiple sclerosis. Eur J Neurol 12:550–556 [DOI] [PubMed] [Google Scholar]

Articles from Cellular and Molecular Neurobiology are provided here courtesy of Springer

RESOURCES