Eugenia Murawska-Ciałowicz

Zbigniew Jethon

Jan Magdalan

Lidia Januszewska

Marzena Podhorska- Okoł Ów

Marcin Zawadzki

Tomasz Sozański

Piotr Dzie Rgiel

Marzena Podhorska-Okołów

Piotr Dzięgiel

Effects of melatonin on lipid peroxidation and antioxidative enzyme activities in the liver, kidneys and brain of rats administered with benzo(a)pyrene

Keywords: Melatonin, Benzo(a)pyrene, Lipid peroxidation, Antioxidative enzymes, Superoxide dismutase, Glutathione reductase, Catalase

Benzo(a)pyrene [B(a)P] is a widespread pollutant with a mutagenic, carcinogenic and strong prooxidative properties. The present study evaluated the melatonin effects on lipid peroxidation products levels and on activity of antioxidative enzymes in the course of B(a)P intoxication. Control rats were treated with 0.9% NaCl; another group was given 10 mg melatonin/kg b.w.; a third group was injected twice a week with B(a)P at the dose of 10 mg/kg b.w.; the fourth group received both B(a)P and melatonin at the dose as above. The experiment continued for 3 months. In homogenates of brain, liver and kidneys lipid peroxidation was appraised by evaluation of malonyldialdehyde and 4-hydroxyalkenal (MDA+4HDA) levels. Activities of glutathione peroxidase (GPx), superoxide dysmutase (SOD) and catalase (CAT) and concentration of reduced glutathione (GSH) were also estimated. In animals receiving both B(a)P and melatonin lower levels of MDA+4HDA were observed in all organs as compared to the group treated with B(a)P only. Following administration of B(a)P, GSH level decreased in brain and kidney. Melatonin in combination with B(a)P induced rises in the GSH level in liver and brain, as compared to the receiving B(a)P alone. The activity of SOD increased in the rats treated with melatonin alone but the highest activity was observed in rats treated with B(a)P plus melatonin. CAT activity in the melatonin-treated group increased in brain and liver. Similarly to SOD, activity of the enzyme significantly increased in the group treated in combination with B(a)P and melatonin, as compared to the remaining groups in all tested tissues. The results suggest that melatonin protects cells from the damaging action of B(a)P. According to our knowledge, there are no studies describing the effects of melatonin on lipid peroxidation markers and antioxidative enzymes during intoxication of B(a)P in the brain, liver and kidneys. The results of present

processes of oxidation of organic matter (e.g., carbon and petrol) and in culinary processes such as roasting, curing and frying. The compounds enter human body from various sources, due to industrial emission, from a polluted environment and from contaminated food [START_REF] Srivastava | Mechanism of differential efficacy of garlic organosulfides in preventing benzo(a)pyrene-induced cancer in mice[END_REF][START_REF] Roos | Induction of cytochrome P 450 1A1 in multiple organs of minipigs after oral exposure to soils contaminated with polycyclic aromatic hydrocarbons (PAH)[END_REF].

Previous studies have shown that B(a)P may induce cancer of lungs, stomach, breast, urinary bladder, prostate and skin [START_REF] Sharma | Differential carcinogenicity of benzo[a]pyrene in male and female CD-1 mouse lung[END_REF][START_REF] Lee | Benzo[a]pyrene diol-epoxide-I-DNA and oxidative DNA adducts associated with gastric adenocarcinoma[END_REF][START_REF] Kumar | Effect of melatonin on two stage skin carcinogenesis in Swiss mice[END_REF].

The mechanism of carcinogenic activity of B(a)P is linked to formation of DNA adducts with metabolites of the compound [START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF][START_REF] Lee | Benzo[a]pyrene diol-epoxide-I-DNA and oxidative DNA adducts associated with gastric adenocarcinoma[END_REF]. Following penetration into the organism, B(a)P undergoes turnover in processes of epoxidation and monoelectron oxidation. Both pathways are catalysed by cytochrome P-450 monoxgenases. The epoxidation yields highly reactive compound, 7,8-diol-9,10-epoxide-2 [BPDE], which manifests the most pronounced carcinogenic activity among all metabolites of B(a)P [START_REF] Sullivan | Free radicals of benzo(a)pyrene and derivatives[END_REF][START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF]. The other pathway of B(a)P turnover involves a monoelectron oxidation process, leading to formation of chionone derivatives, B(a)P-6,12-dione, B(a)P-3,6-dione and B(a)P-1,6-dione [START_REF] Sullivan | Free radicals of benzo(a)pyrene and derivatives[END_REF][START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF]. These derivatives generate oxygen free radicals which cause to oxidative damage of macromolecules (DNA, RNA, protein, lipids and carbohydrates). This results in alterations in cellular structures and, hence, in organic structural and functional disturbances [START_REF] Sharma | Differential carcinogenicity of benzo[a]pyrene in male and female CD-1 mouse lung[END_REF][START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF][START_REF] Lee | Oxidation of erythrocyte protein and lipid, and hemolysis in rabbit red blood cells treated with benzo[a]pyrene or adriamycin[END_REF].

Melatonin is present both in the plant world and in animals and it is a highly effective antioxidant. In its reaction with free radicals it donates electrons [START_REF] Reiter | Melatonin in relation to the antioxidative defense and immune systems: possible implications for cell and organ transplantation[END_REF][START_REF] Reiter | Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence[END_REF]. Melatonin is a scavenger of both oxygen-and nitrogen-based reactive molecules, including peroxynitrite anion (ONOO -) and its decomposition products, including hydroxyl radical (OH • ), nitrogen dioxide (NO 2 ), and carbonate radical (CO3 •-) [START_REF] Korkmaz | Melatonin: an established antioxidant worthy of use in clinical trials[END_REF].

Melatonin reacts rapidly with highly toxic OH • resulting in several hydroxylated products formation. One of them, cyclic -3-hydroxymelatonin may serve as a biomarker of the amounts of OH • scavenged [START_REF] Tan | Melatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation[END_REF][START_REF] Reiter | Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence[END_REF]. Apart from OH • , melatonin directly inactivates NO 2 , CO3 •-, hydrogen peroxide (H 2 O 2 ) and singlet oxygen ( 1 O 2 ) [START_REF] Gilad | Melatonin is a scavenger of peroxynitrite[END_REF][START_REF] Zang | Scavenging of reactive oxygen species by melatonin[END_REF][START_REF] Reiter | Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence[END_REF][START_REF] Almeida | Oxidation of melatonin by singlet molecular oxygen (O2 (1deltag)) produces N1-acetyl-N2-formyl-5methoxykynurenine[END_REF][START_REF] Fischer | Melatonin suppresses reactive oxygen species induced by UV irradiation in leucocytes[END_REF][START_REF] Chuang | Effect of melatonin on temporal changes of reactive oxygen species and glutathione after MPP(+) treatment in human astrocytoma U373MG cells[END_REF]Chen, 2004, Korkmaz et al., 2009). The products of melatonin oxidation: N 1acetyl-N 2 -formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) have been also described as potent scavengers [START_REF] Carampin | Some biochemical properties of melatonin and the characterization of a relevant metabolite arising from its interaction with H 2 O 2[END_REF][START_REF] Tan | One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species?[END_REF][START_REF] Manda | AFMK, a melatonin metabolite, attenuates X-ray-induced oxidative damage to DNA, proteins and lipids in mice[END_REF]. Interaction of melatonin with the lipid radical (LOO • ) awaits clarification [START_REF] Reiter | Melatonin in relation to the antioxidative defense and immune systems: possible implications for cell and organ transplantation[END_REF][START_REF] Reiter | Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence[END_REF]. Besides its ability to direct scavenge radicals and radical products, melatonin also augments the activities of antioxidative enzymes, reductase [START_REF] Longoni | Effects of melatonin on lipid peroxidation induced by oxygen radicals[END_REF][START_REF] Tan | Melatonin directly scavenges hydrogen peroxide: a potentially new metabolic pathway of melatonin biotransformation[END_REF][START_REF] Reiter | Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of the evidence[END_REF][START_REF] Reiter | Melatonin as an antioxidant: physiology versus pharmacology[END_REF][START_REF] Korkmaz | Melatonin: an established antioxidant worthy of use in clinical trials[END_REF]. The findings relating to the stimulation of antioxidative enzymes by melatonin were reviewed by [START_REF] Rodriguez | Regulation of antioxidant enzymes: a significant role for melatonin[END_REF].

Present study demonstrated the protective activity of melatonin in the course of B(a)P intoxication through its effect on the antioxidative defense system.

Material and Methods

Chemicals

B(a)P and melatonin were obtained from Sigma Chemical Co. (St. Louis, USA) and reagent kits from Calbiochem (La Jolla, CA, USA) and OXIS (OXIS International Inc, USA).

Experimental protocol

The studies were performed on 40 male rats of the Buffalo strain, weighing 200 - The animals were placed into four groups, of 10 rats each: the control group received i.p. injections of 0.9% NaCl twice a week (NaCl); melatonin-treated rats, received s.c.

injections of melatonin at the dose of 10mg/kg b.w., three times a week, rats intoxicated with B(a)P received an i.p. injection of 10mg B(a)P /kg b.w. twice a week; the final group was intoxicated with B(a)P in combination with melatonin. Melatonin dosage was decided according to previous studies of Dziegiel et al. (2002aDziegiel et al. ( , 2002b) ) and it was administered before each injection of a B(a)P and between them. A dosage of B(a)P was determined according to the study of Konstandi et al. (2007).

B(a)P was dissolved in a sunflower oil [START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF], while melatonin was dissolved in 10% C2H5OH in H2O at the ratio of 1:3. All the injections were administered between hours 13:00 and 14:00. The experiment Experimental protocol was approved by the Local Animal Ethics Committee of Wroclaw Immunology Institute.continued for 3 months.

Forty-eight hours following the last injection of melatonin the animals were sacrificed by administration of bioketan at the dose of 100mg/kg body weight followed by cervical dislocation.

Biochemical analysis

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

8 Brain, liver and kidneys were collected. The isolated organs were cut into smaller fragments, weighed and frozen at the temperature of -86°C. For biochemical tests the samples were thawed, washed with 0.9% NaCl plus EDTA, homogenized in ice-cold 20mM TRIS-HCL buffer, pH 7.4 and centrifuged for 10 min at the temperature of 4°C at 15,000 rpm. The supernatants were decanted and used to estimate lipid peroxidation (malondialdehyde + 4 hydroxynonenal, MDA+4HDA) levels using Lipid Peroxidation Assay Kit (Calbiochem) and of GSH using Bioxytech GSH-400 kit (OXIS). Activities of GPx and SOD were estimated using Bioxytech kit GPx-340 (OXIS) and Bioxytech kit SOD-525 (OXIS) while catalase activity was assayed by the technique of Aebi [START_REF] Aebi | Catalase in vitro[END_REF] against H 2 O 2 . Total protein concentration was estimated by the technique of Lowry [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF], using the Protein Assay Kit (Sigma Diagnostics Co., ST. Louis, USA). All analyses were performed as recommended by manufacturers of the reagent kits.

Statistical analysis

Results were subjected to statistical analysis using the Statistica 5.1 PL software (StatSoft, Cracow, Poland). Significance of differences was tested employing the nonparametric Mann -Whitney U-test at the confidence level of p<0.05.

Results

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 9

In control group levels of lipid peroxidation markers varied with the type of tissue studied. The highest concentration of MDA+4HDA was noted in kidneys. Administration of melatonin induced a decrease of lipid peroxidation markers in homogenates of kidney and liver. In the group receiving B(a)P with melatonin, levels of MDA + 4HDA were significantly lower in all studied tissues, as compared to the group receiving B(a)P alone (Fig. 1).

Initial concentrations of GSH were the highest in liver homogenates. In the group treated with melatonin, GSH concentrations were significantly increased in all studied tissues as compared to the control group, especially in the liver. In the groups intoxicated with B(a)P concentration of GSH was significantly lower in the brain and kidney as compared to the control. In B(a)P plus melatonin group only GSH level in liver homogenates was significantly higher than in the group receiving B(a)P alone. (Fig. 2).

The highest control GPx activities were noted in liver. In the group receiving melatonin, activity of the enzyme was significantly higher than in the control in all studies tissues. Significantly augmented activities of GPx in the group of B(a)P-intoxicated animals as compared to the control group, were demonstrated in homogenates of liver and brain. As compared to the B(a)P group, rats treated with B(a)P plus melatonin demonstrated a significantly higher activity of the enzyme in liver and brain homogenates (Fig. 3).

In the control groups the highest activity of SOD was noted in the liver. Melatonintreated rats demonstrated significantly augmented SOD activity in comparison to the control In rats treated with B(a)P alone activity of SOD was significantly higher in kidney and liver as compared to control and melatonin groups. In rats receiving B(a)P plus melatonin, activity of SOD was significantly augmented as compared to the control and melatonin treated groups in each experimental organ (Fig. 4).

In control the highest CAT activity was observed in liver. As compared to this the group treated with melatonin alone showed significantly higher CAT activity only in the liver.

In rats intoxicated with B(a)P significantly lower CAT activity was observed in comparison to control rats in the kidney, and as compared to the group treated with melatonin alone the values were significantly lower in all organs. In the group treated with B(a)P plus melatonin activity of CAT augmented significantly in all studied tissues, as compared to any other group (Fig. 5).

Discussion

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 11

This study aimed to investigate whether melatonin protects cells from oxidative injury, induced by B(a)P. Benzo(a)pyrene, a five-ring polycyclic aromatic hydrocarbon is a widespread pollutant with a well documented mutagenic and carcinogenic properties. It's action involves generation of free radicals which cause oxidative damage of macromolecules.

Melatonin is a highly effective antioxidant. It was also shown that it inhibits mutagenesis and clastogenic effect of a number of chemical mutagens [START_REF] Anisimov | Melatonin as antioxidant, geroprotector and anticarcinogen[END_REF]. Only a few papers described the effects of melatonin on B(a)P intoxication, showing the possible inhibitory effect of this pineal hormone on B(a)P induced carcinogenesis in rodents. In Vesnushkin's et al. surveys melatonin decreased the incidence of skin tumors and subcutaneous sarcomas after superficial and subcutaneous administration of B(a)P, respectively [START_REF] Vesnushkin | Dose-dependent inhibitory effect of melatonin on carcinogenesis induced by benzo[a]pyrene in mice[END_REF][START_REF] Vesnushkin | Melatonin suppresses benz(a)pyreneinduced carcinogenesis in mice[END_REF]. Moreover, melatonin decreased, enhanced by B(a)P, lipid peroxidation markers both in the serum and tumor tissue. [START_REF] Vesnushkin | Dose-dependent inhibitory effect of melatonin on carcinogenesis induced by benzo[a]pyrene in mice[END_REF]. According to our knowledge, there are no studies describing the effects of melatonin on lipid peroxidation markers and antioxidative enzymes during intoxication of B(a)P in the brain, liver and kidneys. The effects of melatonin on brain were examined since it is clearly dependent on rich oxygen supply and containing vast amounts of lipid. The brain is also characterized by relatively low levels of anti-oxidative enzymes [START_REF] Reiter | Oxidative processes and antioxidative defense mechanisms in the aging brain[END_REF][START_REF] Escames | Melatonin and vitamin E limit nitric oxide-induced lipid peroxidation in rat brain homogenates[END_REF][START_REF] Esparza | Melatonin reduces oxidative stress and increases gene expression in the cerebral cortex and cerebellum of aluminum exposed rats[END_REF]. Moreover, both investigated substances: exogenous melatonin and B(a)P readily cross a blood-brain barrier and reach the brain in a clinical relevant concentrations [START_REF] Menendez-Pelaez | Nuclear localization of melatonin in different mammalian tissues: immunocytochemical and radioimmunoassay evidence[END_REF][START_REF] Moir | Pharmacokinetics of benzo[a]pyrene in the rat[END_REF][START_REF] Saunders | Benzo(a)pyrene-induced acute neurotoxicity in the F-344 rat: role of oxidative stress[END_REF][START_REF] Reiter | Melatonin reduces oxidative/nitrosative stress due to drugs, toxins, metals, and herbicides[END_REF]Zhang et al., 2008). The liver was included because of its multiple detoxication pathway including during B(a)P intoxication. The kidney was studied because its excretes metabolites of B(a)P. Among the selected organs, the lowest level of enzymatic antioxidative protection was manifested by the brain while the highest enzyme

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

12 activities were found in the liver. However, the brain may be physiologically protected by many antioxidants, especially by melatonin, the receptors for which are present in various brain structures [START_REF] Reiter | Oxidative processes and antioxidative defense mechanisms in the aging brain[END_REF][START_REF] Sewerynek | H 2 O 2 -induced lipid peroxidation in rat brain homogenates is greatly reduced by melatonin[END_REF][START_REF] Reiter | Functional aspects of the pineal hormone melatonin in combating cell and tissue damage induced by free radicals[END_REF][START_REF] Uz | The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system[END_REF].

B(a)P crosses a blood-brain barrier and induces acute neurobehavioral toxicity through oxidative stress due to inhibition of the brain antioxidant scavenging system [START_REF] Moir | Pharmacokinetics of benzo[a]pyrene in the rat[END_REF][START_REF] Saunders | Benzo(a)pyrene-induced acute neurotoxicity in the F-344 rat: role of oxidative stress[END_REF]Zhang et al., 2008). Generation of oxygen-based reactive molecules in brain can increase permeability of the blood-brain barrier and modify synaptic transmission (Evans, 1995). B(a)P can also reach the brain directly by passing through the olfactory nerve [START_REF] Persson | Cellular activation and neuronal transport of intranasally instilled benzo(a)pyrene in the olfactory system of rats[END_REF]. In the present study B(a)P has stimulated lipid peroxidation and augmented activities of SOD and GPx in the brain. Administration of melatonin alone or in combination with B(a)P clearly stimulated CAT, SOD and GPx activities. Numerous authors [START_REF] Tan | Melatonin: a potent, endogenous hydroxyl radical scavenger[END_REF][START_REF] Acuna-Castroviejo | Cell protective role of melatonin in the brain[END_REF][START_REF] Escames | Melatonin and vitamin E limit nitric oxide-induced lipid peroxidation in rat brain homogenates[END_REF][START_REF] Bongiovanni | Melatonin decreases the oxidative stress produced by 2,4dichlorophenoxyacetic acid in rat cerebellar granule cells[END_REF][START_REF] Hung | Protective effect of melatonin against hippocampal injury of rats with intermittent hypoxia[END_REF][START_REF] Bharti | Fluoride-induced oxidative stress in rat's brain and its amelioration by buffalo (Bubalus bubalis) pineal proteins and melatonin[END_REF] have shown melatonin to be an effective neuroprotective agent. Its actions involve not only stimulation of antioxidative enzymes but also direct scavenging of oxygen free radicals and prevention of free radical generation [START_REF] Sewerynek | H 2 O 2 -induced lipid peroxidation in rat brain homogenates is greatly reduced by melatonin[END_REF][START_REF] Escames | Melatonin and vitamin E limit nitric oxide-induced lipid peroxidation in rat brain homogenates[END_REF][START_REF] Hung | Protective effect of melatonin against hippocampal injury of rats with intermittent hypoxia[END_REF][START_REF] Reiter | Melatonin reduces oxidative/nitrosative stress due to drugs, toxins, metals, and herbicides[END_REF]. [START_REF] Urata | Melatonin induces gamma-glutamylcysteine synthetase mediated by activation protein-1 in human vascular endothelial cells[END_REF] found that the concentration of GSH increases following administration of melatonin similar to our results.

Among the examined organs, kidneys contained the highest physiological level of lipid peroxidation markers. Thus, they may be assumed to be the least protected from free radicals even if, compared to the brain, they have higher activities of antioxidative enzymes.

Perhaps despite the high physiological activities of the enzymes, they are more sensitive to the becomes mainly trapped in the kidneys, liver and adipose tissue [START_REF] Collins | Risk assessment for benzo(a)pyrene[END_REF]. Urinary excretion includes one forth of the total B(a)P [START_REF] Becher | Determination of exposure to polycyclic aromatic hydrocarbons by analysis of human urine[END_REF]. Moreover, various B(a)P turnover products are eliminated in urine mainly as conjugates with endogenous compounds, i.e., with glutathione [START_REF] Yang | Characterization of neutral metabolites of benzo[a]pyrene in urine from germfree rats[END_REF]. A small percent of unmetabolized B(a)P is also excreted in urine [START_REF] Jongeneelen | Biological monitoring of occupational exposure to polycyclic aromatic hydrocarbons[END_REF]. In the groups receiving melatonin alone or in combination with B(a)P, MDA + 4HDA concentrations in kidney homogenates were significantly lower as compared to the control and B(a)P groups.

Melatonin alone also stimulated renal GPx activity, The activity of SOD was significantly increased following injection of B(a)P. Lower levels of GSH both after administration of B(a)P alone and B(a)P plus melatonin as compared to the control group may have resulted from increased GSH usage by GPx and from it complex formation with B(a)P metabolites [START_REF] Sharma | Differential carcinogenicity of benzo[a]pyrene in male and female CD-1 mouse lung[END_REF]. There was very pronounced reduction in concentration of MDA+4HDA in rats that received melatonin with or without B(a)P to values below those observed in the control rats. This may supports melatonin strong protective actions against lipid peroxidation as observed by others [START_REF] Kim | stress to DNA, protein, and antioxidant enzymes (superoxide dismutase and catalase) in rats treated with benzo(a)pyrene[END_REF]Dziegiel et al., 2002a,b;[START_REF] Dziegiel | Melatonin stimulates the activity of protective antioxidaive enzymes in myocardial cells of rats in the course of doxorubicin intoxication[END_REF][START_REF] Buyukokuroglu | Antioxidative role of melatonin in organophosphate toxicity in rats[END_REF].

Results obtained here indicate that among examined organs the liver is least sensitive to damaging action of oxygen radicals in B(a)P treated rats. Administration of melatonin alone or with B(a)P reduced MDA +4HDA levels compared to the control rats and rats intoxicated with B(a)P. Among the antioxidative enzymes activity of which may be stimulated by melatonin, liver cells are protected by SOD, CAT and GPx and by the augmented levels of GSH [START_REF] Ohta | Therapeutic effect of melatonin on carbon tetrachloride-induced acute liver injury in rats[END_REF][START_REF] Ohta | Effect of melatonin on changes in hepatic antioxidant enzyme activities in rats treated with alpha-naphthylisothiocyanate[END_REF][START_REF] Leaden | The protection of long chain polyunsaturated fatty acids by melatonin during nonenzymatic lipid peroxidation of rat liver microsomes[END_REF]Ohta et al., et al., 2003;[START_REF] Taysi | Melatonin reduces lipid peroxidation and nitric oxide during irradiation-induced oxidative injury in the rat liver[END_REF][START_REF] Mauriz | Melatonin prevents oxidative stress and changes in antioxidant enzyme expression and activity in the liver of aging rats[END_REF]. The least pronounced increase in lipid peroxidation markers in the liver of B(a)P-treated animals clearly proves that the organ is most effectively protected from oxidative stress.

Conclusion

On the basis of present results we conclude that melatonin may protect the brain,

  pyrene [B(a)P] is a polycyclic aromatic hydrocarbon (PAH), formed in

  (GPx), superoxide dysmutase (SOD) and glutathione

250g.

  The animals were kept in conditions consistent with requirements of the local commission for ethical matters of animal experimentation. They were maintained in controlled environmental conditions of ambient temperature (22 ± 2°C) and relative humidity 60%, in a 12:12 light / dark cycle. All animals were fed standard pelleted diet and water ad libitum.

  B(a)P metabolites. Similarly to other polycyclic aromatic hydrocarbons, B(a)P

Fig. 1 .

 1 Fig. 1. Concentration of lipid peroxidation markers (MDA+4HDA) in homogenates of brain, kidney and liver, *p<0.05 as compared with control, # p<0.05 as compared with melatonin, $ p<0.05 as compared with B(a)P in all organs.

Fig. 2 .

 2 Fig. 2. Concentration of reduced glutathione (GSH) in homogenates of brain, kidney and liver, *p<0.05 as compared with control, # p<0.05 as compared with melatonin, $ p<0.05 as

Fig. 3 .

 3 Fig. 3. Activity of glutathione peroxidase (GPx) in homogenates of brain, kidney and liver, *p<0.05 as compared with control, # p<0.05 as compared with melatonin, $ p<0.05 as

Fig. 4 .Fig. 5 .

 45 Fig. 4. Activity of superoxide dismutase (SOD) in homogenates of brain, kidney and liver, *p<0.05 as compared with control, # p<0.05 as compared with melatonin, $ p<0.05 as

study gives a perspective for further studies of its free radical scavenger properties in prevention of oxidative stress dependent diseases, among others cancers caused by carcinogens such as B(a)P.


MSc Marie Meskell

MB Raj Ettarh
email: r.ettarh@ucd.ie

Bch Phd

PhD M B Bch

expression in non-steroidal anti-inflammatory drug-treated mice

Keywords: prostaglandin, kidney, cyclooxygenase, NSAIDs

Immunohistochemical localisation of renal cyclooxygenase-1 expression in non-steroidal anti-inflammatory drug-treated mice

INTRODUCTION

The kidney is an important site for the production of prostaglandins, the biosynthesis of which is mediated by the activity of both isoforms of the arachidonic acid converting enzyme A c c e p t e d m a n u s c r i p t 3 cyclooxygenase (COX-1, COX-2). These bioactive prostanoids are involved in the maintenance of a range of physiological functions in the kidney including regulation of glomerular filtration rate and electrolyte balance, as well as modulation of renal haemodynamics. These functions occur in spatially defined sites in the renal cortex and medulla (such as the juxtaglomerular apparatus, glomerular mesangium, convoluted tubules, collecting duct) (Nasrallah and Herbert, 2005).

Several studies have examined the correlation between PG-mediated renal activity and cyclooxygenase localisation in the kidney. In rats and mice, COX-1 expression has been described in the extraglomerular mesangium, juxtaglomerular apparatus and arteriolar endothelia in the kidney [START_REF] Campean | Key enzymes for renal prostaglandin synthesis: sitespecific expression in rodent kidney (rat, mouse)[END_REF]. Significant expression is also present in cortical collecting ducts and the cortical interstitium but not in the thin limb of the loop of Henle, macula densa and parts of the distal convoluted tubule. Expression of COX-1 has however been reported in glomerular cells and the collecting duct in human kidney [START_REF] Komhoff | Localization of cyclooxygenase-1 and -2 in adult and fetal human kidney: implication for renal function[END_REF]. COX-2 is expressed in the glomeruli in the human kidney [START_REF] Adegboyega | Immunohistochemical expression of cyclooxygenase-2 in normal kidneys[END_REF] and in the thick ascending loop of Henle and macula densa in the rat kidney [START_REF] Harris | Cyclooxygenase-2 in the kidney[END_REF]. The relative importance of both COX isoforms is demonstrated by the finding that kidneys from COX-2 deficient mice fail to develop normally whereas kidneys from COX-1 deficient mice are not associated with any reported abnormalities [START_REF] Dinchuk | Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase 2[END_REF][START_REF] Morham | Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse[END_REF].

COX inhibitors such as nonsteroidal anti-inflammatory drugs (NSAIDs) are widely employed in the treatment and clinical management of a variety of inflammatory, arthritic and neoplastic conditions as well as the provision of analgesia. These inhibitors are classified according to their by unwanted gastrointestinal and renal toxicity [START_REF] Eras | NSAIDs and the kidney revisited: are selective cyclooxygenase-2 inhibitors safe?[END_REF][START_REF] Sigthorsson | COX-1 and 2, intestinal integrity, and pathogenesis of non-steroidal anti-inflammatory drug enteropathy in mice[END_REF] with 1-5% of patients developing renal syndromes that necessitate intervention [START_REF] Whelton | Nephrotoxicity of nonsteroidal anti-inflammatory drugs: physiologic foundations and clinical implications[END_REF]. Decreased renal perfusion and salt excretion as well as renal papillary necrosis are thought to arise from COX-1 inhibition [START_REF] Brater | Reversible acute decrease in renal function by NSAIDs in cirrhosis[END_REF][START_REF] Brater | Effects of non-steroidal anti-inflammatory drugs on renal function: focus on cyclooxygenase-2-selective inhibition[END_REF][START_REF] Brater | Renal effects of COX-2-selective inhibitors[END_REF][START_REF] Whelton | Nephrotoxicity of nonsteroidal anti-inflammatory drugs: physiologic foundations and clinical implications[END_REF]. Is there any correlation between clinical inhibitor therapy and COX-1 enzyme expression in the kidney? To address this question, this study examined the expression of COX-1 in the kidney before and after treatment with two COX inhibitors: indomethacin a non-selective inhibitor and nimesulide a preferentially selective COX-2 inhibitor.

MATERIALS AND METHODS

Animals

Forty-five male CD-1 mice, aged 8 weeks old were used in this study, housed in plastic cages under standard conditions and allowed access to food and water ad libitum. Animals were allocated equally to one of three groups: one group of animals was given indomethacin, a second group received Nimesulide, and the remaining group received vehicle. The vehicle-treated group served as control. There were 15 animals in each group.

Treatments

Each animal received one of the following treatments every 12 hours by intraperitoneal injection:

Indomethacin (Sigma-Aldrich, Dublin, Ireland) 10mg/kg body weight, Nimesulide (Helsinn Birex, Dublin, Ireland) 15mg/kg body weight, or vehicle. All treatments were given for 24 hours.

A c c e p t e d m a n u s c r i p t 5

The doses of inhibitors administered were equipotent [START_REF] Sigthorsson | Comparison of indomethacin and nimesulide, a selective cyclooxygenase -2 inhibitor, on key pathophysiologic steps in the pathogenesis of nonsteroidal anti-inflammatory drug enteropathy in the rat[END_REF][START_REF] Sigthorsson | COX-1 and 2, intestinal integrity, and pathogenesis of non-steroidal anti-inflammatory drug enteropathy in mice[END_REF] and each treatment solution was made by dissolving the drug in 5ml of vehicle. The vehicle solution was made up of 0.9% saline to which a drop Tween 80 dispersing agent was added. Addition of the dispersing agent was necessary because both indomethacin and nimesulide dissolve very slowly in aqueous media.

Tissue retrieval and processing

Each kidney was delivery by laparotomy, cut transversely into several 1-2mm thick specimen slices and immersion fixed in 10% formal saline for 24 hours. Specimens were processed routinely for embedding in paraffin wax. Sections obtained at a thickness of 5ȝm were placed on poly-L-lysine coated glass slides.

Immunohistochemistry

Paraffin sections were dewaxed in xylol, rehydrated through graded alcohols, and antigen retrieval performed by heat-treatment in a microwave oven in 10mM sodium citrate buffer solution pH 6.0 for 10 minutes. Immunostaining was carried out according to the protocol of [START_REF] Coons | The cytology of antibody formation[END_REF]. Sections were treated with 3% hydrogen peroxide in methanol to block endogenous peroxidase activity and pre-incubated with 10% normal goat serum for 1 hour to block extraneous binding sites prior to incubation with primary antibody to COX-1 overnight.

The following antibodies were used: rabbit polyclonal raised against murine COX-1

Ethical Approval

Ethical approval for this study was sought from and granted under institutional ethics regulatory mechanisms and all procedures carried out, under licence, within the provisions of the Cruelty to Animals Act (Ireland) 1876.

RESULTS

Control kidney:

Immunohistochemical analysis revealed expression of COX-1 in both the renal cortex and medulla with significantly more expression evident within the cortex. Immunoreactivity in the superficial cortex was more pronounced than in the rest of the cortex or the medulla, and was localised predominantly to the cell cytoplasmic compartment. However, immunoreactive protein was also present in the nucleus of a minority of cells within the superficial cortex. COX-1 protein was expressed in the glomeruli and parietal cells of the glomerular capsule, epithelial cells of the proximal and distal convoluted tubules, and the collecting ducts (Fig 1a,b).

Cytoplasmic COX-1 protein within the distal tubule was present in the juxtaglomerular region,

Indomethacin-treated kidney:

Immunostaining was present in both the cortical and medullary areas of indomethacin-treated specimens. In contrast to the cytoplasmic localisation of COX-1 immunoreactivity in control kidney, COX-1 staining within superficial cortical cells in indomethacin-treated kidneys was diffuse and confined to the nucleus. COX-1 immunoreactive protein was apparent in the outer parietal layer of the Bowman's capsule and in some cells of the glomerulus (Fig 1c).

Cytoplasmic staining was observed within cells of the proximal and distal convoluted tubules while nuclear immunoreactivity was evident within the cortical collecting ducts. Localization of cytoplasmic COX-1 within the endothelial cells of the renal blood vessels and the medullary collecting ducts was minimal (Fig 1d). There was no staining for COX-1 immunoreactive protein in the cortical or the medullary interstitium.

Nimesulide-treated kidney:

There was significant expression of COX-1 protein in the superficial cortical region adjacent to the renal capsule and evidence of some COX-1 immunoreactivity in the outer parietal epithelial cell layer of Bowman's capsule (Fig 1e). Very minimal, diffuse cytoplasmic staining was evident in the proximal convoluted tubule. There was COX-1 protein expression within cells of the cortical distal convoluted tubules in both the nucleus and cytoplasm, with significant localisation

DISCUSSION

Previous studies have documented the expression of COX-1 in human, rat and murine kidney [START_REF] Campean | Key enzymes for renal prostaglandin synthesis: sitespecific expression in rodent kidney (rat, mouse)[END_REF][START_REF] Khan | Effect of papillotoxic agents on expression of cyclooxygenase isoforms in the rat kidney[END_REF][START_REF] Komhoff | Localization of cyclooxygenase-1 and -2 in adult and fetal human kidney: implication for renal function[END_REF][START_REF] Vitzthum | Gene expression of prostanoid forming enzymes along the rat nephron[END_REF][START_REF] Zidar | Cyclooxygenase in normal human tisues-is COX-1 really a constitutive isoform, and COX-2 an inducible isoform?[END_REF]. Human COX-1 protein expression was reported in the collecting duct cells, endothelial cells of the renal vasculature (moreso in the afferent arteriole at the glomerular entrance), interstitial cells and in the parietal cells of Bowman's capsule [START_REF] Komhoff | Localization of cyclooxygenase-1 and -2 in adult and fetal human kidney: implication for renal function[END_REF].

Expression was also localized to the terminal portion of the distal convoluted tubule. In the rat and mouse, COX-1 expression was observed in the juxtaglomerular apparatus of the distal convoluted tubule, the terminal portion of distal convoluted tubule and the cortical collecting duct [START_REF] Campean | Key enzymes for renal prostaglandin synthesis: sitespecific expression in rodent kidney (rat, mouse)[END_REF]. The mesangial cells within the glomerulus also exhibited strong affinity for the COX-1 antibody. In the murine renal tissue signigicant expression was noted in the cortical interstitium. Findings from our study of COX-1 expression in the kidney of CD-1 mice indicate the presence of COX-1 protein in the juxtaglomerular region of the distal convoluted tubule. Other workers have revealed significant COX-1 expression within the terminal distal convoluted tubule and the connecting tubule [START_REF] Campean | Key enzymes for renal prostaglandin synthesis: sitespecific expression in rodent kidney (rat, mouse)[END_REF][START_REF] Stubbe | Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats[END_REF]. The distal tubule represents an important location for electrolyte absorption. Of interest is the presence of COX-1 protein in the proximal convoluted tubule, a finding that has not previously been reported. COX-1 expression was also evident within the endothelial cells of the renal vasculature and the cortical and medullary collecting ducts which agree with previous reports [START_REF] Vitzthum | Gene expression of prostanoid forming enzymes along the rat nephron[END_REF][START_REF] Zidar | Cyclooxygenase in normal human tisues-is COX-1 really a constitutive isoform, and COX-2 an inducible isoform?[END_REF]. Immunohistochemical analysis revealed COX-

A c c e p t e d m a n u s c r i p t

9 1 staining within the parietal epithelium of the Bowman's capsule. This is in keeping with data from [START_REF] Zidar | Cyclooxygenase in normal human tisues-is COX-1 really a constitutive isoform, and COX-2 an inducible isoform?[END_REF] who localized COX-1 expression in the same area. However this study did not find COX-1 staining within the mesangium of the glomerulus. The cortical and medullary interstitium was negative for COX-1 protein in our study in contrast to [START_REF] Campean | Key enzymes for renal prostaglandin synthesis: sitespecific expression in rodent kidney (rat, mouse)[END_REF] who reported significant murine expression of the protein in the cortical interstitium.

These differences may be due to interspecies variation in renal localization of cyclooxygenase [START_REF] Khan | Effect of papillotoxic agents on expression of cyclooxygenase isoforms in the rat kidney[END_REF].

Cyclooxygenase (COX) exists in two related but unique isoforms in the renal kidney. NSAIDs cause renal toxicity following direct inhibition of COX isoforms. Humans and animals exhibit differences in susceptibility, which may be associated with differences in expression of one or both isoforms in the kidney [START_REF] Radi | Pathophysiology of cyclooxygenase inhibition in animal models[END_REF]. Rats and dogs exhibit greater susceptibility than humans to the development of renal papillary necrosis due to their sluggish blood supply (Khan and Alden, 2002;Khan et al., 2002). Renal damage as a result of NSAID administration is due to ischemic injury through direct inhibition of the vasodilatory effects of renal prostaglandins [START_REF] Silva | Chemical-induced nephropathy: a review of renal tubulointerstitial lesions in humans[END_REF]. Previous studies have looked at COX expression in renal tissue following a single toxic dose (75mg/kg) of indomethacin that caused papillary necrosis [START_REF] Khan | Effect of papillotoxic agents on expression of cyclooxygenase isoforms in the rat kidney[END_REF] accompanied by a slight to moderate decrease in papillary COX expression. This decrease was observed 2 hours after administration of the agent and was also seen in tissue that lacked the light microscopic changes of renal papillary necrosis. The possible mechanism that leads to an initial decrease in COX immunoreactivity in indomethacin-treated animals is not known. In conclusion this study has documented the expression of COX-1 along the nephron and reported the presence of COX-1 in the proximal convoluted tubule, a finding not previously described. This study has also determined that administration of therapeutic doses of indomethacin and nimesulide have different effects on COX-1 expression in the murine kidney and that these effects may be related to drug dose and isoform specificity.

  antagonise one or both isoforms of COX; most commonly prescribed inhibitors inactivate both COX-1 and COX-2. The clinical use of these agents is however limited

  (1:200, Cayman Chemical, Ann Arbor, MI, USA) and biotin-conjugated goat secondary antibody raised against rabbit IgG (Sigma-Aldrich, Dublin, Ireland). Following treatment with secondary biotinconjugated goat anti-rabbit antibody (1:200) for 1 hour, and subsequently with avidin-biotin , Burlingame, CA, USA), antibody binding was visualised with diaminobenzidine (DAB) and sections counterstained with haematoxylin. Slide coverslips were mounted with XAM. Specificity of the antibody was confirmed by omitting the primary antibody (negative control) or by preabsorbing the primary antibody for 60 minutes in a 10-fold molar excess of blocking peptide (Cayman Chemical, Ann Arbor, MI, USA). Intensity of COX immunostaining in the sections was evaluated by light microscopy and assessed by blinded review.

  glomerulus. Interstitial cells in the renal cortex did not show reactivity for COX-1. Within the medulla, pronounced nuclear and cytoplasmic expression of COX-1 was apparent within the endothelial cells of the renal vasculature but the epithelial cells of the medullary collecting ducts and the interstitial cells in the medulla showed poor reactivity for COX-1.

  collecting ducts. Focal cytoplasmic immunostaining was seen in endothelial cells of blood vessels and collecting duct cells in the renal medulla (Fig 1f).

  Dose and duration of administration of NSAIDs are both important in determining effects in many tissues. Although other studies have employed therapeutic doses of NSAIDs over varying durations, effects on COX expression have not been examined(McGarvey et al., 2007a; ., 2007b;[START_REF] Sigthorsson | Comparison of indomethacin and nimesulide, a selective cyclooxygenase -2 inhibitor, on key pathophysiologic steps in the pathogenesis of nonsteroidal anti-inflammatory drug enteropathy in the rat[END_REF] In this study a therapeutic dose (10mg/kg) of indomethacin did not alter COX expression but an equipotent dose of nimesulide (15 mg/kg) caused reduced expression. Taken together with the finding that a toxic dose of indomethacin reduces COX expression prior to renal damage, our results suggest that the effect of NSAIDs on renal COX-1 expression may be dependent on dose and isoform specificity. It would be of interest to determine what effect high/toxic doses or prolonged administration of nimesulide may have on renal tissue. In comparison to the information on the effects of Indomethacin on renal COX-1 expression, there is a lack of information on the effect of selective COX-2 inhibitors such as nimesulide on the expression of COX-1 in renal tissue.

  FIGURE LEGEND

Figure

  Figure


Veronique E Miron

Tanja Kuhlmann

Dr Jack P Antel
email: jack.antel@mcgill.ca

Alexandre Prat

Cells of the oligodendroglial lineage, myelination, and remyelination

Keywords: Myelin, Remyelination, Oligodendrocyte, Oligodendrocyte Progenitor Cell, Multiple Sclerosis, Repair

come

The rapid transduction of electrical impulses, termed action potentials, is required for the efficient function of the vertebrate nervous system. The conduction velocity of these impulses is maximized by the presence of an insulating layer surrounding the axon referred to as the myelin sheath [START_REF] Griffiths | Axonal swellings and degeneration in mice lacking the major proteolipid of myelin[END_REF][START_REF] Lappe-Siefke | Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination[END_REF][START_REF] Yin | Evolution of a neuroprotective function of central nervous system myelin[END_REF]. In the central nervous system (CNS), the oligodendrocyte is the resident cell type responsible for the production of myelin that consists of oligodendroglial plasma membrane loops tightly wound concentrically around the axon. Oligodendrocyte progenitor cells (OPCs) are generated in the ventral neuroepithelium of the neural tube in early embryonic life, more specifically from the motor neuron progenitor (pMN) domain [START_REF] Noll | Oligodendrocyte precursors originate at the ventral ventricular zone dorsal to the ventral midline region in the embryonic rat spinal cord[END_REF][START_REF] Pringle | Dorsal spinal cord neuroepithelium generates astrocytes but not oligodendrocytes[END_REF], and in the dorsal spinal cord and hindbrain / telencephalon of the brain in late embryonic development and early post-natal life [START_REF] Fu | Dual origin of spinal oligodendrocyte progenitors and evidence for the cooperative role of Olig2 and Nkx2.2 in the control of oligodendrocyte differentiation[END_REF][START_REF] Cai | Generation of oligodendrocyte precursor cells from mouse dorsal spinal cord independent of Nkx6 regulation and Shh signaling[END_REF][START_REF] Vallstedt | Multiple dorsoventral origins of oligodendrocyte generation in the spinal cord and hindbrain[END_REF][START_REF] Kessaris | Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage[END_REF]. These proliferative cells migrate into the developing white matter [START_REF] Noble | Development and regeneration in the central nervous system[END_REF][START_REF] Fok-Seang | Distribution and differentiation of A2B5+ glial precursors in the developing rat spinal cord[END_REF][START_REF] Richardson | Isolation of neuronal progenitor cells from the adult human neocortex[END_REF], exit the cell cycle, undergo differentiation into mature oligodendrocytes, and begin to express a subset of myelin-associated proteins [START_REF] Baumann | Biology of oligodendrocyte and myelin in the mammalian central nervous system[END_REF][START_REF] Miller | Regulation of oligodendrocyte development in the vertebrate CNS[END_REF]. Following the extension of flat oligodendroglial membrane sheets, there is recognition and adhesion to axonal targets [START_REF] Duchala | Expression of cell surface markers and myelin proteins in cultured oligodendrocytes from neonatal brain of rat and mouse: a comparative study[END_REF]. Myelin components are then synthesized and transported to the appropriate location within the sheath, axons are wrapped, and the myelin membrane is compacted such that the cytoplasmic myelin leaflets are practically fused and almost devoid of cytoplasm [START_REF] Pedraza | Organizing principles of the axoglial apparatus[END_REF][START_REF] Kursula | Structural properties of proteins specific to the myelin sheath[END_REF][START_REF] Simons | Neuron-glia communication in the control of oligodendrocyte function and myelin biogenesis[END_REF]. In the CNS, one oligodendrocyte can produce up to 40 myelin segments on multiple axons. Consequently, myelinating oligodendrocytes produce as much as 5-50 x 10 3 µm 2 of membrane a day [START_REF] Pfeiffer | The oligodendrocyte and its many cellular processes[END_REF]. Maintenance of this myelin membrane occurs throughout adulthood and comprises of a continual turnover of myelin correlated with a high level of expression of myelin genes long after the completion of the myelination process [START_REF] Lajtha | Turnover of myelin proteins in mouse brain in vivo[END_REF][START_REF] Lebaron | Turnover rate of molecular species of sphingomyelin in rat brain[END_REF].

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT

Signals derived from electrically active neurons (such as growth facots, neurotrophic factors, and electrical activity) regulate cellular events involved in myelination and myelin gene expression in oligodendrocytes [START_REF] Demerens | Induction of myelination in the central nervous system by electrical activity[END_REF][START_REF] Barres | Axonal control of oligodendrocyte development[END_REF][START_REF] Baron | Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains[END_REF][START_REF] Ishibashi | Astrocytes promote myelination in response to electrical impulses[END_REF]. Oligodendrocytes also reciprocate signals for neurons, by producing growth factors and by inducing the organization of the axon into specific sub-domains by controlling the distribution of various axonal proteins [START_REF] Salzer | Polarized domains of myelinated axons[END_REF][START_REF] Butts | Maturation-dependent sensitivity of oligodendrocyte lineage cells to apoptosis: implications for normal development and disease[END_REF]. Gaps of myelin cause the formation of the 'Nodes of Ranvier', which are axonal segments where the sodium channels that regulate electrical impulse conduction are aggregated (Figure 1A). Experimental models of demyelination have indicated that the myelin sheath is crucial in maintaining these nodal domains over the long term [START_REF] Dupree | Oligodendrocytes assist in the maintenance of sodium channel clusters independent of the myelin sheath[END_REF]. Myelin also forms the flanking membrane loops, termed the paranodes, which serve to separate the nodal sodium channels from the potassium channels concentrated in the adjacent juxtaparanodal regions (Figure 1A). Together, the paranodes and juxtaparanodes form the myelin segments called the internodes. The importance of reciprocal communication between axons and oligodendrocytes is apparent in neurological diseases where oligodendrocyte loss and demyelination is associated with a considerable degeneration of axons.

For instance, in multiple sclerosis (MS), oligodendrocyte loss and demyelination are associated with progressive axonal degeneration and neurological decline [START_REF] Trapp | Pathogenesis of tissue injury in MS lesions[END_REF][START_REF] Arnold | Magnetic resonance spectroscopy: imaging axonal damage in MS[END_REF][START_REF] Noseworthy | Multiple sclerosis[END_REF]. Recovery from relapses in MS (remittances) can reflect a number of mechanisms, including the resolution of the exaggerated inflammation response and consequent sparing of neural cells, the functional reorganization of nodal components, and myelin repair (remyelination).

Role of Remyelination in Functional Neural Recovery

Remyelination is the default spontaneous process by which demyelinated axons undergo ensheathment with new myelin sheaths leading to functional recovery [START_REF] Liebetanz | Effects of commissural de-and remyelination on motor skill behaviour in the cuprizone mouse model of multiple sclerosis[END_REF][START_REF] Smith | Histological and ultrastructural analysis of white matter damage after naturally-occurring spinal cord injury[END_REF]. Remyelination of demyelinated lesions has been documented to occur in MS by neuropathologic criteria, where

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 5
completely remyelinated MS lesions are called shadow plaques due to the intermediate levels of myelin staining intensity [START_REF] Prineas | Continual breakdown and regeneration of myelin in progressive multiple sclerosis plaques[END_REF][START_REF] Prineas | Multiple sclerosis: remyelination of nascent lesions[END_REF]. This likely reflects the relative decrease in myelin thickness and internodal length in the new myelin sheaths relative to the original parameters of the sheath [START_REF] Gupta | Chronic nerve compression induces local demyelination and remyelination in a rat model of carpal tunnel syndrome[END_REF]. Remyelination is not only found in inactive lesions but can also be observed in lesions with ongoing demyelinating activity [START_REF] Prineas | Multiple sclerosis: remyelination of nascent lesions[END_REF][START_REF] Gupta | Chronic nerve compression induces local demyelination and remyelination in a rat model of carpal tunnel syndrome[END_REF][START_REF] Raine | Multiple sclerosis: remyelination in acute lesions[END_REF]. Remyelination in MS has also been evidenced by magnetic resonance imaging-based criteria of changes in magnetization transfer ratio within lesions [START_REF] Chen | Magnetization transfer ratio evolution with demyelination and remyelination in multiple sclerosis lesions[END_REF]. The new myelin sheath may either act as a protective physical barrier to damaging inflammatory molecules [START_REF] Redford | A combined inhibitor of matrix metalloproteinase activity and tumour necrosis factor-alpha processing attenuates experimental autoimmune neuritis[END_REF], or may restore trophic support to the axon [START_REF] Byravan | Murine oligodendroglial cells express nerve growth factor[END_REF][START_REF] Strelau | GDNF family members and their receptors: expression and functions in two oligodendroglial cell lines representing distinct stages of oligodendroglial development[END_REF][START_REF] Wilkins | A role for oligodendrocyte-derived IGF-1 in trophic support of cortical neurons[END_REF]. However, some patients demonstrate extensive remyelination and yet clinical deterioration proceeds [START_REF] Patrikios | Remyelination is extensive in a subset of multiple sclerosis patients[END_REF],

suggesting that remyelination alone is not always sufficient in restoring function.

Demyelination in MS has been associated with a disruption of the paranodal region, resulting in altered expression and distribution of proteins such as neurofascin 155, contactin-associated protein (Caspr), and paranodin, as well as ion channels [START_REF] Craner | Molecular changes in neurons in multiple sclerosis: altered axonal expression of Nav1.2 and Nav1.6 sodium channels and Na+/Ca2+ exchanger[END_REF][START_REF] Coman | Nodal, paranodal and juxtaparanodal axonal proteins during demyelination and remyelination in multiple sclerosis[END_REF][START_REF] Howell | Disruption of neurofascin localization reveals early changes preceding demyelination and remyelination in multiple sclerosis[END_REF]. Consequently, nodal sodium channels are then directly adjacent to juxtaparanodal potassium channels, leading to impaired saltatory conduction of action potentials (Figure 1B). The remyelination process is associated with a correct re-distribution of paranodal proteins comparable to the developmental sequence of nodal assembly [START_REF] Dupree | Oligodendrocytes assist in the maintenance of sodium channel clusters independent of the myelin sheath[END_REF] (Figure 1C). Studies have demonstrated that sodium channel re-aggregation at the nodes of Ranvier is an early event in remyelination that occurs prior to new myelin sheath formation [START_REF] Coman | Nodal, paranodal and juxtaparanodal axonal proteins during demyelination and remyelination in multiple sclerosis[END_REF]. However, this aggregation of sodium channels on a demyelinated axon just prior to remyelination may render it more susceptible to injury [START_REF] Smith | Histological and ultrastructural analysis of white matter damage after naturally-occurring spinal cord injury[END_REF].

Role of oligodendrocyte progenitor cells in remyelination

Animal models of toxin-induced demyelination have designated the remyelination process as being mediated by newly infiltrated proliferative OPCs that have differentiated into mature

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6
myelinating phenotypes, rather than previously myelinating post-mitotic mature oligodendrocytes within the lesion. More specifically, retroviral tracing has indicated that proliferative cells eventually give rise to remyelinating oligodendrocytes [START_REF] Gensert | Endogenous progenitors remyelinate demyelinated axons in the adult CNS[END_REF]. OPCs also remyelinate efficiently upon transplantation into focal demyelinated lesions [START_REF] Groves | Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells[END_REF][START_REF] Warrington | Differential myelinogenic capacity of specific developmental stages of the oligodendrocyte lineage upon transplantation into hypomyelinating hosts[END_REF][START_REF] Nunes | Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain[END_REF].

Additionally, lesions in which there is widespread oligodendrocyte death are characterized by a repopulation of OPCs prior to new oligodendrocyte formation [START_REF] Levine | Activation and proliferation of endogenous oligodendrocyte precursor cells during ethidium bromide-induced demyelination[END_REF][START_REF] Sim | The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation[END_REF][START_REF] Watanabe | Differentiation of proliferated NG2-positive glial progenitor cells in a remyelinating lesion[END_REF]. The role of OPCs in protecting axonal integrity has also been highlighted in an animal model of demyelination where depletion of these cells via X-ray irradiation results in increased axonal swellings and decreased axonal numbers in white matter tracts that would normally undergo remyelination [START_REF] Irvine | Remyelination protects axons from demyelinationassociated axon degeneration[END_REF].

The OPCs with remyelinating potential are believed to be randomly distributed within the CNS.

However, areas that are distal from progenitor germinal zones, such as the optic nerves, are the first to demonstrate impairments in the remyelination process [START_REF] Ziv | Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury[END_REF]. OPCs have been termed as stem cells of sort due to their multi-potentiality, self-renewal potential, and rapid proliferative response to injury. OPCs are highly proliferative, motile, bipolar cells expressing high levels of the GT3 ganglioside A2B5, PDGF receptor alpha (PDGFαR), and the CSPG NG2 [START_REF] Pfeiffer | The oligodendrocyte and its many cellular processes[END_REF]. The expression of these molecules and hence OPC specification are regulated by the basic helix-loophelix (bHLH) transcription factors Olig1, Olig2, Mammalian achate schute Homolog 1 (Mash1), and the zinc finger transcription factor Myelin transcription factor 1 (MyT1) [START_REF] Sim | The age-related decrease in CNS remyelination efficiency is attributable to an impairment of both oligodendrocyte progenitor recruitment and differentiation[END_REF][START_REF] Fancy | Increased expression of Nkx2.2 and Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS[END_REF][START_REF] Watanabe | Transient upregulation of Nkx2.2 expression in oligodendrocyte lineage cells during remyelination[END_REF]. OPCs are also very responsive to agents that promote their differentiation along the oligodendroglial lineage, such as insulin-like growth factor (IGF)-1, ciliary neurotrophic factor (CNTF), and thyroid hormone (T3) [START_REF] Tosic | Triiodothyronine has diverse and multiple stimulating effects on expression of the major myelin protein genes[END_REF][START_REF] Jones | Triiodothyronine is a survival factor for developing oligodendrocytes[END_REF]. Differentiation requires the function of Olig1, Olig2, Nkx2.2, Nkx2.6, MyT1, and sex determining region Y box (Sox)-10 [START_REF] Nunes | Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain[END_REF][START_REF] Fancy | Increased expression of Nkx2.2 and Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS[END_REF][START_REF] Watanabe | Transient upregulation of Nkx2.2 expression in oligodendrocyte lineage cells during remyelination[END_REF][START_REF] Nicolay | Transcriptional control of oligodendrogenesis[END_REF], likely due to interaction of these transcription factors with the promoters of myelin genes [START_REF] Qi | Control of oligodendrocyte differentiation by the Nkx2.2 homeodomain transcription factor[END_REF][START_REF] Stolt | Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10[END_REF][START_REF] Sohn | Identification of Sox17 as a transcription factor that regulates oligodendrocyte development[END_REF] (Figure 1D).

A C C E P T E D

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The directed migration of these cells to presumptive white matter has been suggested to be mediated by the chemo-attractant PDGF and semaphorin 3F, the chemo-repellents netrin-1, semaphorin 3A, and the ephrins, as well as the stop-signals CXCL1 and tenascin C [START_REF] Noble | Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell[END_REF][START_REF] Tsai | The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cord by arresting their migration[END_REF][START_REF] Jarjour | Netrin-1 is a chemorepellent for oligodendrocyte precursor cells in the embryonic spinal cord[END_REF][START_REF] Cohen | Exploring oligodendrocyte guidance: 'to boldly go where no cell has gone before[END_REF][START_REF] De | The molecular orchestra of the migration of oligodendrocyte precursors during development[END_REF].

OPCs can be identified in the adult human brain, consisting of 5-8% of total glial cells [START_REF] Jones | Triiodothyronine is a survival factor for developing oligodendrocytes[END_REF].

Interestingly, adult OPCs demonstrate slower motility, a prolonged cell cycle, and poorer survival relative to developmental OPCs [START_REF] Wren | In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells[END_REF][START_REF] Tang | Long-term culture of purified postnatal oligodendrocyte precursor cells. Evidence for an intrinsic maturation program that plays out over months[END_REF][START_REF] Ruffini | Distinctive properties of human adult brain-derived myelin progenitor cells[END_REF]. The restriction of these cells to the oligodendroglial lineage in vivo is unlikely: these cells were originally termed O-2A cells due to their potential to generate both oligodendrocytes and astrocytes in vitro. In addition, they can develop into neurons within neurogenic domains of the CNS (i.e. hippocampus) and have the capacity to generate Schwann cells following immune-mediated white matter injury [START_REF] Magnus | Evidence that nucleocytoplasmic Olig2 translocation mediates brain-injury-induced differentiation of glial precursors to astrocytes[END_REF]. The generation of OPCs from differing developmental pathways likely contributes to the heterogeneous population of OPCs observed in development [START_REF] Magnus | Evidence that nucleocytoplasmic Olig2 translocation mediates brain-injury-induced differentiation of glial precursors to astrocytes[END_REF][START_REF] Spassky | Single or multiple oligodendroglial lineages: a controversy[END_REF][START_REF] Richardson | Isolation of neuronal progenitor cells from the adult human neocortex[END_REF]; whether this heterogeneity exists in adulthood is unknown. Subsets of precursors expressing varying levels and combinations of markers do exist in adult white matter [START_REF] Kitada | Transcription factor co-expression patterns indicate heterogeneity of oligodendroglial subpopulations in adult spinal cord[END_REF]. Precursors with differential responses to growth factors can also be isolated from the adult rat forebrain [START_REF] Mason | A2B5+ and O4+ Cycling progenitors in the adult forebrain white matter respond differentially to PDGF-AA, FGF-2, and IGF-1[END_REF]. These precursor subsets may differentially contribute to remyelination and may be under the control of distinct regulatory mechanisms. OPCs can mature into pre-oligodendrocytes, expressing an unidentified sulfatide recognized by the O4 antibody [START_REF] Bansal | Proligodendroblast antigen (POA), a developmental antigen expressed by A007/O4-positive oligodendrocyte progenitors prior to the appearance of sulfatide and galactocerebroside[END_REF] . Although still proliferative, these cells are nonmotile likely due to the adoption of a multi-processed complex morphology. Once these cells have then progressed to differentiation into immature oligodendrocytes, they have undergone cell cycle-arrest. They can still be identified by the O4 antibody, yet expression of OPC markers such as A2B5 and PDGFαR is repressed (Figure 1D). Arborizations are elaborated and maturityassociated markers are acquired (galactocerebroside (GalC)) without the formation of myelin 1D). The subsequent final stage of differentiation into mature oligodendrocytes comprises of the acquisition of myelin proteins and axonal contact (Figure 1D). Interestingly, factors that promote survival and differentiation, such as CNTF, are not necessarily sufficient in causing myelin formation to occur in vivo [START_REF] Talbott | CNTF promotes the survival and differentiation of adult spinal cord-derived oligodendrocyte precursor cells in vitro but fails to promote remyelination in vivo[END_REF].

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 8 (Figure
Whether mature oligodendrocytes can themselves contribute to remyelination has been a topic of great debate. This has been negated by the findings that these cells are post-mitotic, nonmigratory, and unable to remyelinate upon transplantation into lesions [START_REF] Targett | Failure to achieve remyelination of demyelinated rat axons following transplantation of glial cells obtained from the adult human brain[END_REF][START_REF] Zhao | Differences in the early inflammatory responses to toxin-induced demyelination are associated with the age-related decline in CNS remyelination[END_REF]. Mature oligodendrocytes do re-extend processes following sub-lethal injury; however these processes are intercalated between axons and do not ensheath them [START_REF] Keirstead | Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord[END_REF].

Requirements for remyelination: inflammation-dependent OPC activation

The 'activation' of OPCs is thought to be required for remyelination to occur. This involves the adoption of a hypertrophic morphology, increased expression of genes associated with oligodendroglial differentiation (such as the transcription factors Olig2 and NK2 transcription factor related locus 2 (Nkx2.2)), and responsiveness to mitogens and chemoattractants that are released from glia in the injured tissue [START_REF] Fancy | Increased expression of Nkx2.2 and Olig2 identifies reactive oligodendrocyte progenitor cells responding to demyelination in the adult CNS[END_REF][START_REF] Franklin | The biology of CNS remyelination: the key to therapeutic advances[END_REF]. This activation process may be required to prevent OPC differentiation in the normal intact white matter [START_REF] Zawadzka | Myelin regeneration in demyelinating disorders: new developments in biology and clinical pathology[END_REF]. This activation response is proportional to the inflammatory reaction that succeeds demyelination and is required for successful remyelination to occur in animal model systems [START_REF] Chari | Corticosteroids delay remyelination of experimental demyelination in the rodent central nervous system[END_REF][START_REF] Glezer | Innate immunity triggers oligodendrocyte progenitor reactivity and confines damages to brain injuries[END_REF]. Myelin debris has been shown to be inhibitory to OPC differentiation and is phagocytosed by reactive macrophages that have entered the lesion area [START_REF] Kotter | Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation[END_REF]. Furthermore, chemokines and cytokines such as tumor necrosis

Glial Scar in lesion

The glial scar formed within the lesion, comprised of hypertrophic reactive astrocytes and meningeal fibroblasts, likely serves to form a barrier between inflamed damaged tissue and normal brain but is also thought to secrete factors that may inhibit OPC migration into the scar and subsequent differentiation. For example, some plaques with a reactive astrocytic core have high levels of CXCL-1 and ephrins at their perimeter that may impede OPC migration into the lesion [130,131]. Also, the presence of myelin-associated debris in the glial scar, including myelin associated glycoprotein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and NogoA, can prevent axonal regeneration and inhibit OPC differentiation [134]. OPCs grown on myelin substrates in vitro show impairments in maturation [132]. Additionally, the injection of myelin debris into a focal demyelinated lesion prevents the remyelination that normally succeeds [START_REF] Kotter | Myelin impairs CNS remyelination by inhibiting oligodendrocyte precursor cell differentiation[END_REF]. The scar is also hypothesized to have upregulated levels of other molecules inhibitory to axonal regeneration and may render the axon unsuitable for remyelination, such as chondroitin sulfate proteoglycans (CSPGs) and semaphorins [133]. The presence of astrocytes within the lesion has been previously hypothesized to be sufficient to dampen the rate of remyelination via inhibition of OPC process extension and migration [START_REF] Fok-Seang | Cytokine-induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth[END_REF]. Nonetheless, transplanted OPCs are able to repopulate astrocytic lesions and endogenous remyelination does not occur in the adult when astrocytes are depleted [134]. LIF released from astrocytes can indeed promote myelin formation [START_REF] Ishibashi | Astrocytes promote myelination in response to electrical impulses[END_REF]. Some have argued that the glial scar is not a cause of failed remyelination but rather a consequence of it [START_REF] Franklin | The biology of CNS remyelination: the key to therapeutic advances[END_REF].

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Temporal discordance of remyelination with inflammation

The slow rate of OPC repopulation of demyelinated lesions observed in animal systems has lead to the hypothesis that remyelination may be unsuccessful in MS due to a temporal discordance between OPC migration into the lesion and the inflammation required for OPC activation that is generated from myelin breakdown [START_REF] Franklin | The biology of CNS remyelination: the key to therapeutic advances[END_REF]. Delaying OPC infiltration into lesions by their transplantation at a site distal from the demyelinated area results in impaired remyelination [122]. However, increasing progenitor recruitment to demyelinated lesions in aged animals via PDGF upregulation in astrocytes is not sufficient in rescuing the failed remyelination process [142]. This suggests that deficits in OPC migration cannot explain the deficiency in remyelination with aging.

Therapeutic strategies to promote remyelination

Current studies using experimental animal models of demyelination are aimed at discovering therapeutic strategies to overcome these obstacles and to replace myelin under circumstances in which endogenous remyelination is unsuccessful. Remyelination efficiency may be improved either by supplementing the CNS with an exogenous source of OPCs or promoting the endogenous repair process.

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  in maintaining electrical impulse conduction in the central nervous system. The oligodendrocyte is the cell type responsible for myelin production within this compartment. The mutual supply of trophic support between oligodendrocytes and the underlying axons may indicate why demyelinated axons undergo degeneration more readily; the latter contributes to the neural decline in multiple sclerosis (MS). Myelin repair, termed remyelination, occurs in acute inflammatory lesions in MS and is associated with functional recovery and clinical remittances. Animal models have demonstrated that remyelination is mediated by oligodendrocyte progenitor cells (OPCs) which have responded to chemotactic cues, migrated into the lesion, proliferated, differentiated into mature oligodendrocytes, and ensheathed demyelinated axons. The limited remyelination observed in more chronic MS lesions may reflect intrinsic properties of neural cells or extrinsic deterrents. Therapeutic strategies currently under development include transplantation of exogenous OPCs and promoting remyelination by endogenous OPCs. All currently approved MS therapies are aimed at dampening the immune response and are not directly targeting neural processes.

11 [ 12 5. 6 . 1

 111261 factor (TNF) α, interleukin (IL)-1β, IL-11, and Chemokine C-X-C motif ligand (CXCL)-2 can promote OPC proliferation, differentiation, and remyelination[START_REF] Arnett | TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination[END_REF][START_REF] Vela | Interleukin-1 regulates proliferation and differentiation of oligodendrocyte progenitor cells[END_REF][START_REF] Maysami | Modulation of rat oligodendrocyte precursor cells by the chemokine CXCL12[END_REF][START_REF] Zhang | Interleukin-11 potentiates oligodendrocyte survival and maturation, and myelin formation[END_REF]. Inflammatory cells and reactive astrocytes, which can both be found within MS lesions, can release growth factors thatA C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT9 can affect the remyelination process[START_REF] Ikeda | Acute up-regulation of brain-derived neurotrophic factor expression resulting from experimentally induced injury in the rat spinal cord[END_REF][START_REF] Besser | Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors[END_REF][START_REF] Kerschensteiner | Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation?[END_REF][START_REF] Stadelmann | BDNF and gp145trkB in multiple sclerosis brain lesions: neuroprotective interactions between immune and neuronal cells?[END_REF]. Accordingly, the migration and differentiation of transplanted progenitors in the commonly used inflammatory model of MS, experimental autoimmune encephalomyelitis (EAE) corresponds to the peak of inflammation[START_REF] Ben-Hur | Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells[END_REF]. The proinflammatory cytokines IL-1β and TNFα synergistically enhance OPC migration to FGF[START_REF] Fok-Seang | Cytokine-induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth[END_REF].5.5 Animal models to study remyelinationVarious animal model systems have been developed to study different aspects of remyelination.5.4.1 Lysolecithin and Ethidium Bromide focal injectionsA common model used to study the remyelination process in the absence of concomitant demyelination and exaggerated inflammation involves focal injections of toxins such an ethidium bromide or lysolecithin either into the spinal cord or white matter tracts of the brain.This induces a reproducible demyelination in predefined areas of the central nervous system (CNS) by inducing cell death of oligodendrocytes (using ethidium bromide) and myelin membrane solubilization (using lysolecithin) at the site of injection[START_REF] Blakemore | Remyelination in the spinal cord of the cat following intraspinal injections of lysolecithin[END_REF][START_REF] Riet-Correa | Ethidium bromide-induced demyelination of the sciatic nerve of adult Wistar rats[END_REF][100]. These models are attractive in their succinct temporal separation between de-and remyelination processes and the spontaneous OPC-mediated remyelination that occurs within 4 weeks post-injection. There are, however, confounding issues of toxin-induced axonal damage, BBB breakdown, and moderate traumatic injury with consequent immune cell infiltration at the injection site [101-103]. 5.4.2 Oral Cuprizone administration Another commonly used model involves oral administration of the toxin cuprizone (oxalic acid bis (cyclohexylidene hydrazide)) that induces oligodendrocyte cell death, myelin membrane vacuolization, and demyelination in specific white matter tracts of the brain (i.e. cerebellar A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 10 peduncles, corpus callosum, and anterior commissure) [104]. Although the mechanism of demyelination of cuprizone is unknown, its copper chelation functions have implicated the deregulation of copper-dependent mitochondrial functions and resultant impairment in energy production in oligodendrocytes [105]. Interestingly, some areas of white matter are spared from cuprizone toxicity (i.e. spinal cord, optic nerve) suggesting regional differences in oligodendroglial / myelin energy requirements. Following cessation of cuprizone treatment, demyelinated lesions demonstrate robust spontaneous remyelination [106]. OPC proliferation and recruitment to lesion areas is initiated during the period of cuprizone-induced demyelination, causing an overlap in demyelination and repair processes [107]. This model is characterized by a significant lesional infiltration and reactivity of microglia and astrocytes that likely contribute to the secretion of remyelination-promoting factors; there is no evidence of T lymphocyte involvement or of BBB breakdown [108]. 5.4.3 Inflammatory models As previously mentioned, the inflammatory process is critical in inducing OPC activation and subsequent remyelination. Several models of inflammation-induced demyelination show spontaneous remyelination even in the presence of an exaggerated immune response. For instance, the EAE model in which animals are immunized against the myelin-oligodendrocyte glycoprotein (MOG) antigen is characterized by a relapse-remitting phenotype, however remyelination is not very extensive perhaps due to the dense infiltration of macrophages and microglia in the lesion over prolonged periods of time. The only indication of impressive levels of remyelination in EAE is observed in focal models of cortical demyelination involving immunization with subclinical doses of MOG and injection of pro-inflammatory mediators A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 109]. Another model, Theiler's murine encephalomyelitis (TME), consists of injection of a single-stranded ribonucleic acid (RNA) picovirus into the CNS to induce immune-mediated oligodendroglial cell death, axonal damage, and demyelination in the gray and white matter of the CNS [110]. This model can be characterized by varying levels of remyelination which renders it appropriate for testing of agents that can potentially inhibit or enhance the myelin repair process [111]. Both these models have been used as standards for the development and testing of therapeutic strategies for autoimmune diseases. 5.6 Postulates for failure of remyelination in MS Although a limited number of MS patients have extensive spontaneous remyelination in up to 96% of lesions [112], most patients eventually demonstrate impaired or insufficient remyelination as the disease progresses [112, 113]. OPCs are increased in numbers in actively demyelinating MS plaques, yet are depleted over time [114-117]. Failure in remyelination would imply that chronically demyelinated axons are rendered more susceptible to degeneration and immune-attack. Indeed, chronically demyelinated plaques are comprised of severely injured axons that are progressively cleared over time [118, 119]. Axons that have been chronically demyelinated for long periods of time, however, can still be remyelinated under acute inflammation [120]. There are a multitude of hypotheses as to why remyelination fails in MS, which may reflect either changes in environmental inputs or intrinsic pathways regulating OPC function [121, 122]. A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Immune-mediated injury to OPCs and new myelin sheath A postulate that supports that former hypothesis proposes that the new myelin sheath and OPCs are subject to immune-mediated injury to the same degree as the initial insult to the oligodendrocytes and myelin [122]. Post-mortem analysis of MS plaques indicates that remyelination may be interrupted by inflammation [123]. In acute demyelinating animal models with a single insult and no continuing injury to myelin, the ensuing remyelinating is complete [124]. Antibodies recognizing progenitor cell-specific antigen (AN2) have been identified in the CSF of relapse-remitting MS patients and could induce OPC lysis [125]. 5.6.2 Exhaustion of OPC source Another proposal is that repeated demyelinating insults, as observed in the relapse-remitting form of MS, causes an exhaustion of the OPC source. Accordingly, recurrent demyelinating insults to the corpus callosum with oral administration of the oligodendrocyte toxin, cuprizone, resulted in accumulative failure in remyelination and progressive diminishment in repopulation of mature oligodendrocytes [126]. However, successive demyelination of the brain stem with ethidium-bromide does not affect the efficiency of remyelination nor progenitor numbers [127].This suggests that remyelination failure may be regionally defined due to exhaustion of distinct progenitor pools. Proliferating OPCs are rare in MS lesions suggesting that depletion of progenitors is conceivable following repeated episodes or extended periods of demyelination and recurrent episodes of remyelination[117, 127, 128, 129].

14 5. 6 . 4

 1464 Upregulation of developmental regulators of myelination An additional hypothesis for the failure in remyelination in MS proposes that re-expression of developmental regulators of myelination, such as Leucine rich repeat and Ig domain-containing (LINGO)-1 and polysialic acid-neural cell adhesion molecule (PSA-NCAM), may be reexpressed on stressed axons within the lesion and repel OPCs [135]. This is supported by the finding that non-differentiated and non-myelinating OPCs are closely associated to the axon in some MS lesions [136]. In addition, reactivation of the Wnt signaling pathway in MS lesions suggests that disregulation of this pathway may contribute to inefficient myelin repair; indeed, disregulation of this pathway in an experimental model of demyelination interfered with remyelination [137]. 5.6.5 Aging-related deficits in inflammation and OPC differentiation The remyelination process has also been shown to decline with age in experimental animal models; this is relevant in the context of MS that runs over several decades of life [138, 139]. One explanation could relate to prolonged exposure to inflammatory mediators that induce an accumulation of neural damage over time [108]. This age-related failure in remyelination has also been attributed to delayed clearance of myelin debris by phagocytic macrophages, consequently impairing OPC differentiation [124]. This is supported by studies of MS lesions in which early lesions and periplaque white matter (PPWM) have an increased number of OPCs (Olig2+) co-expressing a mature oligodendrocyte marker NogoA, thereby suggesting a differentiation block as a potential cause for remyelination failure [117]. The aforementioned importance of inflammation in inducing the activation of OPCs and resultant remyelination would also suggest that OPCs in un-remyelinated MS lesions may still be functional yet are not A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT15 immersed in the appropriate inflammatory environment that would induce differentiation [136, 140]. Furthermore, studies have demonstrated that remyelination efficiency is regulated by agedependent epigenetic control of gene expression, and disregulation of this process may contribute to the decline in remyelination with age [141].

16 5. 7 . 1 Figure 1 .

 16711 Figure Legend:Figure 1. Myelin characteristics, Demyelination, and Remyelination.

Fig. 1

 1 Fig. 1

Acknowledgements:

Parts of this chapter were previously published in Veronique E. Miron's doctoral thesis at

McGill University (Montreal, Quebec).

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