A comparative study of melatonin and immunomodulatory therapy with interferon beta and glatiramer acetate in a mouse model of multiple sclerosis. / Estudio comparativo de melatonina contra los tratamientos inmunomoduladores (interferón beta y acetato de glatirámero) en un modelo murino de esclerosis múltiple.

INTRODUCTION: Multiple sclerosis (MS) is a chronic, demyelinating, autoimmune disease of the central nervous system causing neuroinflammation. Experimental autoimmune encephalitis (EAE) is a model of the disease. MS is classically treated with interferon beta (IFN-ß) and glatiramer acetate (GA). Melatonin (MLT) has been reported to modulate immune system responses. The aim of the present study is to analyse the effects of MLT administration in comparison with the first-line treatments for MS (IFN-ß and GA). METHODS: EAE was induced in male Sprague-Dawley rats; the animals subsequently received either IFN-ß, GA, or MLT. Cerebrospinal fluid (CSF) samples were analysed by multiplex assay to determine the levels of proinflammatory cytokines. The neurological evaluation of EAE was also recorded. RESULTS: All immunised animals developed EAE. We evaluated the first relapse-remission cycle, observing that IFN-ß and GA had better results than MLT in the clinical evaluation. Neither EAE nor any of the treatments administered modified CSF IL-1ß and IL-12p70 concentrations. However, IFN-ß and MLT did decrease CSF TNF-α concentrations. CONCLUSIONS: Further studies are needed to evaluate the molecular mechanisms involved in the behaviour of MLT in EAE, and to quantify other cytokines in different biological media in order for MLT to be considered an anti-inflammatory agent capable of regulating MS.

Effect of melatonin administration on the PER1 and BMAL1 clock genes in patients with Parkinson's disease.

Sleep disorders are a widespread condition in patients with Parkinson's disease (PD), which has been linked to a deregulation of the circadian cycle and therefore of the clock genes. The aim of this study was to evaluate the effect of melatonin (MEL) on the PER1 and BMAL1 clock genes in patients with PD. A double-blind, cross-over, placebo-controlled randomized clinical trial pilot study was conducted in 26 patients with stage 1-3 PD according to the Hoehn & Yahr scale, who received either 25 mg of MEL or a placebo at noon and 30 min before bedtime for three months. The relative expression of the PER1 and BMAL1 genes was measured, as well as the presence of daytime, nocturnal, and global sleepiness, and the progression of PD. The levels of the PER1 and BMAL1 genes at baseline were 0.9 (0.1-3) vs. 0.56 (0.1-2.5), respectively; while after the intervention with MEL or placebo the BMAL1 levels increased to 2.5 (0-3.70) vs. 2.2 (0.10-3.30), respectively (d = 0.387). Fifty percent (50 %) of patients had daytime sleepiness and sixty-five percent (65 %) had abnormal nighttime sleepiness, yet neither group showed changes after the intervention. Patients with PD exhibited an alteration in the levels of the clock genes: MEL increased the levels of BMAL1, but the PER1 levels remained unchanged.

Absence of the tag polymorphism for the risk haplotype HLA-DR2 for multiple sclerosis in Wixárika subjects from Mexico.

The HLA-DRB1*15:01 allele has a demonstrated risk for the development of multiple sclerosis (MS) in most populations around the world. The single nucleotide polymorphism (SNP) rs3129934 is found in linkage disequilibrium with the risk haplotype formed by the HLA-DRB1*15:01 and HLA-DQB1*06:02 alleles, and it is considered a reliable marker of the presence of this haplotype. Native Americans have a null or low prevalence of MS. In this study, we sought to identify the frequency of rs3129934 in the Wixárika ethnic group as well as in Mestizo (mixed race) patients with MS and in controls from western Mexico. Through real-time polymerase chain reaction (PCR) using TaqMan probes, we analyzed the allele and genotype frequencies of rs3129934 in Mestizo individuals with and without MS and in 73 Wixárika subjects from the state of Jalisco, Mexico. The Wixárika subjects were homozygote for the C allele of rs3129934. The allele and genotype frequency in Mestizos with MS was similar to that of other MS populations with Caucasian ancestry. The absence of the T risk allele rs3129934 (associated with the haplotype HLA-DRB1*15:01, HLA-DQ1*06:02) in this sample of Wixárika subjects is consistent with the unreported MS in this Amerindian group, related to absence of such paramount genetic risk factor.

Efficacy of fish oil on serum of TNF α , IL-1 ß , and IL-6 oxidative stress markers in multiple sclerosis treated with interferon beta-1b.

UNLABELLED: Multiple sclerosis (MS) is a chronic inflammatory disease, which leads to focal plaques of demyelination and tissue injury in the central nervous system. Oxidative stress is also thought to promote tissue damage in multiple sclerosis. Current research findings suggest that omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapenta-enoic acid (EPA) and docosahexaenoic acid (DHA) contained in fish oil may have anti-inflammatory, antioxidant, and neuroprotective effects. The aim of the present work was to evaluate the efficacy of fish oil supplementation on serum proinflammatory cytokine levels, oxidative stress markers, and disease progression in MS. 50 patients with relapsing-remitting MS were enrolled. The experimental group received orally 4 g/day of fish oil for 12 months. The primary outcome was serum TNF α levels; secondary outcomes were IL-1 ß 1b, IL-6, nitric oxide catabolites, lipoperoxides, progression on the expanded disability status scale (EDSS), and annualized relapses rate (ARR). Fish oil treatment decreased the serum levels of TNF α , IL-1 ß , IL-6, and nitric oxide metabolites compared with placebo group (P ≤ 0.001). There was no significant difference in serum lipoperoxide levels during the study. No differences in EDSS and ARR were found. CONCLUSION: Fish oil supplementation is highly effective in reducing the levels of cytokines and nitric oxide catabolites in patients with relapsing-remitting MS.

Protective effects of tryptophan on neuro-inflammation in rats after administering lipopolysaccharide.

Tryptophan (TRP), which plays an important role in immune system regulation, protein synthesis, serotonin (5-HT) and melatonin production, is a potent endogenous free radical scavenger and antioxidant. The aim of this work was to determine the efficacy of TRP in neuro-inflammation induced by systemic administration of lipopolysacharide (LPS, 20mg/kg) which promotes the synthesis of free radical (LPO: MDA and 4-HDA), and pro-inflammatory cytokine Interferon-γ (IFN-γ) in different brain regions (cerebral cortex and hippocampus) of rats. Experiments were performed on adult female, pregnant and lactating rats fed with a diet of TRP content (0.5mg/100g protein), cerebral cortex and hippocampus were evaluated for lipid peroxidation (LPO) products, nitrites, nitrates and plasmatic concentration of IFN-γ. LPO levels in LPS+TRP groups were significantly decreased than that obtained in the LPS group. However, there were no observed differences in plasmatic levels of nitrites and nitrates as well as IFN-γ, neither in the cerebral cortex or hippocampus. The TRP has protective effect in the oxidative damage in a model of endotoxic shock in the breading nurslings induced by the systemic administration of LPS, acting as a scavenger of free radicals. So, it can be proposed as an innocuous protector agent in the endotoxic shock process.

Serum oxidative stress is increased in patients with post cholecystectomy bile duct injury.

BACKGROUND: Post-cholecystectomy bile duct injuries are identified by the onset of jaundice as well as elevated bilirubin and alkaline phosphatase levels during the peri-operative period. It is unknown how serum oxidative stress markers are modified in patients with post-cholecystectomy bile duct injuries. OBJECTIVE: To determine serum oxidative stress marker levels (lipid peroxidation by-products, nitrites/nitrates and total antioxidant capacity) in patients with post-cholecystectomy bile duct injuries. PATIENTS AND METHODS: A prospective, transversal and analytical study was designed with two groups. Group 1: 5 healthy volunteer subjects. Group 2: 52 patients with post-cholecystectomy bile duct injuries (43 female and 9 male). An elective bilio-digestive reconstruction was performed at week 8. The serum oxidative stress marker levels were quantified by colorimetric method. RESULTS: Patients with bile duct injuries had a significant increased serum lipid peroxides (malondialdehyde and 4-hydroxy-alkenals) and nitric oxide metabolites (nitrites/nitrates) levels compared to the control group. In contrast, total antioxidant capacity in patients with bile duct injuries remained similar compared to healthy controls. CONCLUSIONS: The results show that oxidative stress is usually associated to bile duct injury.

Apolipoprotein E genotypes in Mexican patients with Parkinson's disease.

BACKGROUND: The association of the apolipoprotein (Apo E) -epsilon4 allele to neurodegenerative diseases such as Parkinson's disease (PD) has been analyzed in several studies. This association has been identified by amyloid deposits and neurofibrillary tangles in the brains of patients with neurodegenerative diseases. METHOD: In this study the possible relationship between Apo E alleles and PD patients was analyzed in 105 patients with PD and 107 healthy controls from a Mexican population. RESULTS: Allele analysis in PD vs. controls was: epsilon2 in 6% and 2.3%, respectively; epsilon3 in 73% and 88.3%; and epsilon4} in 21% and 9.4%. The epsilon3 allele showed a protective risk effect with an Odds ratio (OR) of 0.36 (95%CI 0.20-0.61) and p < 0.05; contrary results were observed for the epsilon4 allele, which showed an increased risk for PD, with an OR of 2.57(95% CI 1.42-4.79) and p < 0.05. Upon multivariate analysis showed PD risk was evident in patients who were carriers of the genotype epsilon3/epsilon4; age group (fifty or more years) and had exposure to pesticides and solvents (p < 0.05). CONCLUSIONS: The epsilon3/epsilon3}; epsilon3/epsilon4 genotypes of the Apo E, were positively associated with sporadic PD.

Scanning electron microscopy of the orbital Harderian gland in the male Atlantic bottlenose dolphin (Tursiops truncatus).

The ultrastructure of the Harderian gland of Atlantic bottlenose dolphin (Tursiops truncatus) was examined by scanning electron microscopy (SEM). We found the following surface features: the typical round appearance of the ascinar glandular unit with a finely granular surface, a thin cortex and immediately below two types of cells: type I cells (characterized by small lipid vacuoles) and type II cells (characterized by large lipid vacuoles). It has been suggested that different cells forms represent a single cell type in varying activity states. Additionally, a coalescent tubular complex, a small balloon-like structures and large globular structures were observed. These structures may be reservoirs of secretion products.

Protective effects of melatonin against caustic esophageal burn injury in rats.

Caustic ingestion is one of the most life-threatening events in the pediatric age group, which requires the immediate management and subsequent treatment of its most significant complication, i.e. alterations in esophageal structure. We investigated whether melatonin could reduce the esophageal burn damage induced by sodium hydroxide. It was assumed that melatonin could be effective because of its function as a direct free radical scavenger, its antioxidative actions and its ability to diminish tissue hydroxyproline (HP) levels. Esophageal burns were induced in male rats by the administration of 10% sodium hydroxide. Lipid peroxidation (LPO) products were then measured at the following times: 0, 1, 6, 24, 48 and 72 hr after treatment. Tissue HP concentrations in the injured area were assessed at 14 days after the administration of sodium hydroxide. The groups received either systemic melatonin or normal saline. There were two, non-ischemic, sham control groups treated with or without melatonin. LPO products, malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA), increased immediately after the administration of sodium hydroxide; this indicates the participation of free radicals in the development of damage. Melatonin diminished the oxidative response and the amount of HP in the late phase of the lesion. Melatonin reduced oxidative damage in the early phase of the esophageal burns induced by sodium hydroxide.

Detection of membrane fluidity in submitochondrial particles of platelets and erythrocyte membranes from Mexican patients with Alzheimer disease by intramolecular excimer formation of 1,3 dipyrenylpropane.

It has been suggested that mitochondrial dysfunction and defects in membrane structure could be implied in AD pathogenesis. The aim of the present work was the study of membrane fluidity in submitochondrial platelet particles and erythrocyte membranes from Mexican patients. Blood samples were obtained from 30 patients with Alzheimer disease and 30 aged-matched control subjects. Membrane fluidity determinations were done using a very low concentration of the fluorescent dipyrenylpropane probe incorporated in both types of membranes. This probe is able to give excimer and monomer fluorescence, therefore it can be used to monitor fluidity changes in biological membranes. The data obtained showed that in submitochondrial particles from AD patients, the excimer to monomer fluorescent intensity ratio was lower (0.231 +/- 0.008) than aged-matched control subjects (0.363 +/- 0.014). Therefore, membrane fluidity was lower in AD samples. On the other hand, we found similar membrane fluidity in erythrocytes from AD patients and aged-matched controls: the fluorescent intensity ratios were 0.312 +/- 0.03 and 0.305 +/- 0.033, respectively. In addition, lipid peroxidation in submitochondrial particles and erythrocyte membranes was higher in AD samples than in aged-matched controls. These data suggest that submitochondrial platelet particles are more sensitive to oxidative stress than erythrocyte membranes.

The orbital Harderian gland of the male atlantic bottlenose dolphin (Tursiops truncatus): a morphological study.

The ultrastructure of the Atlantic Bottlenose dolphin Harderian gland (HG) has been described but some questions remain unanswered. The purpose of this work was to define the gland's structure, ultrastructure and the differences between cells (types I and II) of the male dolphin using optic, fluorescence and electron transmission microscopy. Three different cells were observed under optic and fluorescence microscopic examination, while only two cell types (types I and II) were distinguished by electron transmission microscopy. Type I (oval nuclear envelope) exhibited three different cell populations and type II (indented nuclear envelope) exhibited two different cell populations. Although, we observed both types of vesicles in both types of cells they differed, principally, in quantity. The glands also possessed prominent duct systems, with three orders of complexity. The dolphin orbital HG appears to function as a mixed heterologous gland with two types of cells that exhibit both types of vesicles and other distinguishable differences.

Altered beta-amyloid precursor protein isoforms in Mexican Alzheimer's Disease patients.

OBJECTIVE: To determine the beta-amyloid precursor protein (betaAPP) isoforms ratio as a risk factor for Alzheimer's Disease and to assess its relationship with demographic and genetic variables of the disease. METHODS: Blood samples from 26 patients fulfilling NINCDS-ADRDA diagnostic criteria for AD and 46 healthy control subjects were collected for Western blotting for betaAPP. A ratio of betaAPP isoforms, in optical densities, between the upper band (130 Kd) and the lower bands (106-110 Kd) was obtained. Odds ratios were obtained to determine risk factor of this component. RESULTS: betaAPP ratio on AD subjects was lower than that of control subjects: 0.3662 +/- 0.1891 vs. 0.6769 +/- 0.1021 (mean +/- SD, p<0.05). A low betaAPP ratio (<0.6) showed an OR of 4.63 (95% CI 1.45-15.33). When onset of disease was taken into account, a betaAPP ratio on EOAD subjects of 0.3965 +/- 0.1916 was found vs. 0.3445 +/- 0.1965 on LOAD subjects (p>0.05). CONCLUSIONS: Altered betaAPP isoforms is a high risk factor for Alzheimer's disease, although it has no influence on the time of onset of the disease.

Monosodium glutamate-induced damage in liver and kidney: a morphological and biochemical approach.

It has been demonstrated that high concentrations of monosodium glutamate in the central nervous system induce neuronal necrosis and damage in retina and circumventricular organs. In this model, the monosodium glutamate is used to induce an epileptic state; one that requires highly concentrated doses. The purpose of this study was to evaluate the toxic effects of the monosodium glutamate in liver and kidney after an intra-peritoneal injection. For the experiment, we used 192 Wistar rats to carry out the following assessments: a) the quantification of the enzymes alanine aminotransferase and aspartate aminotransferase, b) the quantification of the lipid peroxidation products and c) the morphological evaluation of the liver and kidney. During the experiment, all of these assessments were carried out at 0, 15, 30 and 45 min after the intra-peritoneal injection. In the rats that received monosodium glutamate, we observed increments in the concentration of alanine aminotransferase and aspartate aminotransferase at 30 and 45 min. Also, an increment of the lipid peroxidation products, in kidney, was exhibited at 15, 30 and 45 min while in liver it was observed at 30 and 45 min. Degenerative changes were observed (edema-degeneration-necrosis) at 15, 30 and 45 min.

Effects of melatonin on the nitric oxide treated retina.

AIMS: Nitric oxide (NO) is a free radical which reportedly causes damage to living cells. This study evaluated the damaging effect of NO and the protection of melatonin on the retina in vivo. METHODS: Female Wistar rats (230-250 g) received two intraperitoneal injections of either melatonin (5 mg/kg) or vehicle alone. After general anaesthesia, the animals received 1 microl intravitreal injections of 0.9% saline and 1 mM sodium nitroprusside (SNP) into the right eye and the left eye, respectively. The animals were divided into two groups and then sacrificed after 24 hours (day 1) and 96 hours (day 4). The mean inner retinal layer thickness (mIRLT), the number of retinas expressing hyperchromatic (HC) nuclei in the inner nuclear layer (INL) and the apoptotic ganglion cell detection were compared. RESULTS: After 1 day, SNP significantly increased the mIRLT by 45% (p = 0.004), initiated more INL nuclear HC expression (p = 0.01) and apoptotic nuclei (p<0.05) compared with the control eyes. Injection of melatonin ameliorated these changes. On day 4, SNP demonstrated similar effects in all parameters on the retina. After the injection of melatonin, both INL HC expression and apoptotic ganglion nuclei in the SNP treated eyes were similar to the controls but the mIRLT was significantly greater than in controls (p = 0.006). CONCLUSION: Uncontrolled NO elevation caused morphological and nuclear changes in the retina. Melatonin significantly suppressed the NO induced increase in mIRLT, INL HC expression, and apoptotic ganglion cells on day 1, but not after day 4. Melatonin may have a protective role in the NO elevated retina.

Scanning electron microscopy of the superficial pineal gland of the 15-day-old rat (Rattus norvegicus).

The presence of a cortex and medulla in the superficial pineal gland has been a controversial point in the morphology of this structure in mammals. The published reports indicate contradictory data especially in rodents. In this study the pineal gland of 15-day-old male rats (Rattus norvegicus) were studied, using scanning electron microscopy, in an attempt to determine whether or not a cortex and medulla are apparent in the pineal gland of young rats. The superficial pineal gland of the 15-day-old rat exhibited both a cortex and a medulla; these areas exhibited different structural organizations. The cortex had a thickness of 40-80 microm and the cells did not show a particular arrangement. The center of the gland was composed of a medulla, which had a width of 1000-1200 microm, and consisted of cells arranged in cords; its morphology was distinctly different from that of the cortex.

Melatonin prevents cytoskeletal alterations and oxidative stress induced by okadaic acid in N1E-115 cells.

Progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, both in neural aging and in neurodegenerative diseases. Cytoskeleton in neurodegenerative diseases is characterized by hyperphosphorylated tau assembled in neurofibrillary tangles. Tau protein promotes microtubule enlargement and its hyperphosphorylation inhibits tubulin assembly. Okadaic acid (OA) causes oxidative stress, tau hyperphosphorylation, and altered cytoskeletal organization similar to those observed in neurons of patients with dementia. Since melatonin acts by both enlarging microtubules and as a free-radical scavenger, in this work we studied the effects of melatonin on altered cytoskeletal organization induced by OA in N1E-115 neuroblastoma cells. Optic microscopy, morphometric analysis, and tubulin immunofluorescence staining of neuroblastoma cells incubated with 50 nM OA showed an intact microtubule network following the neurite profile similar to that observed in the vehicle-incubated cells when melatonin was added to the incubation media 2 h before OA. The melatonin effects on altered cytoskeletal organization induced by OA were dose-dependent and were not abolished by luzindole, the mt(1) melatonin antagonist receptor. Also, increased lipid peroxidation and augmented apoptosis in N1E-115 cells incubated with 50 nM OA were prevented by melatonin. The results support the hypothesis that melatonin can be useful in the treatment of neurodegenerative diseases.

N-acetylserotonin suppresses hepatic microsomal membrane rigidity associated with lipid peroxidation.

N-acetylserotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this work was to test the effect of N-acetylserotonin in stabilizing biological membranes against oxidative stress. Hepatic microsomal membranes from male adult rats were incubated with N-acetylserotonin (0.001-3 mM) before inducing lipid peroxidation using FeCl(3), ADP and NADPH. Control experiments were done by incubating microsomal membranes with N-acetylserotonin in the absence of lipid peroxidation-inducing drugs. Membrane fluidity was assessed by fluorescence spectroscopy and malonaldehyde plus 4-hydroxyalkenals concentrations were measured to estimate the degree of lipid peroxidation. Free radicals induced by the combination of FeCl(3)+ADP+NADPH produced a significant decrease in the microsomal membrane fluidity, which was associated with an increase in the malonaldehyde plus 4-hydroxyalkenals levels. These changes were suppressed in a concentration-dependent manner when N-acetylserotonin was added in the incubation buffer. In the absence of lipid peroxidation, N-acetylserotonin (0.001-3 mM) did not change membrane fluidity nor malonaldehyde plus 4-hydroxyalkenals levels. These results suggest that the protective role of N-acetylserotonin in preserving optimal levels of fluidity of the biological membranes may be related to its ability to reduce lipid peroxidation.

Melatonin, vitamin E, and estrogen reduce damage induced by kainic acid in the hippocampus: potassium-stimulated GABA release.

Melatonin, vitamin E and estrogen have been shown to exert neuroprotective effects against kainic acid (KA)-induced damage in the hippocampus. The aim of the present study was to examine the changes in potassium-evoked gamma-aminobutyric acid (GABA) release in the hippocampus of KA-treated rats and to test the possible protective effects of melatonin, vitamin E or estrogen. Following the treatment of mice with KA, a marked reduction in potassium-evoked [3H]GABA release was observed. Melatonin or estrogen prevented the reduction in potassium-evoked GABA release due to kainate administration. Vitamin E also exhibited some protective effect, but it was less than that provided by melatonin or estrogen. Melatonin, estrogen and, to a lesser extent, vitamin E reduce the physiological toxicity of KA. Since KA is believed to cause neuronal alterations via oxidative processes, it is assumed that the free radical scavenging and oxidative properties of melatonin, estrogen and vitamin E account for the protective effects of these agents.

Different patterns in the histology and autofluorescence of the Harderian glands of the Syrian Hamster, rat, mouse, Mongolian gerbil and guinea pig.

The purpose of this study was to compare the natural fluorescence in the Harderian glands of the Syrian hamster, rat, mouse, Mongolian gerbil and guinea pig (both sexes). For each species, 10 animals (five males and five females) were used. Histological autofluorescence studies were performed using a fluorescence microscope (450-490 nm filter). Two different types of fluorescent cells were observed in both hamster (type AFI high intensity and type AFII, low fluorescence) and rat (type AFI, low fluorescence and type AFII, high fluorescence) Harderian glands. The fluorescence was basally located in all mice cells, whereas it was observed near the epithelial cell nuclei in the Mongolian gerbil (occupying two-thirds and one-third of the cells in males and females, respectively). A high intensity of fluorescence was present throughout the acinar cells in the guinea pig. The patterns of fluorescence identified exhibited a sexual dimorphism in all species studied. These results demonstrate that the Harderian glands of the animal species examined exhibit a variety of histological autofluorescence patterns.

Protective role of melatonin against MPTP-induced mouse brain cell DNA fragmentation and apoptosis in vivo.

OBJECTIVES: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that induces a Parkinsonian-type syndrome in animals which is similar to Parkinson's disease in humans. MPTP toxicity partially depends on the production of free radicals which in turn play a key role in the apoptotic death of neurons. In the present study melatonin, a potent free radical scavenger with antiapoptotic properties, was given to determine whether it would reduce oxidative stress in mice treated with MPTP. MATERIALS AND METHODS: Male mice were given MPTP with or without melatonin and the brain was studied either 6h, 24h, 7 days or 15 days after the last MPTP injection. RESULTS: The results show that melatonin counteracted in vivo MPTP-induced apoptosis in midbrain neurons at 6 and 24 h after MPTP treatment, and partially prevented apoptosis at 7 and 15 days after MPTP administration. MPTP treatment also produced time-dependent cell damage, whereas melatonin reduced the percentage of damaged cells at all time points, the effect being most evident at 15 days after treatment. Moreover, melatonin counteracted MPTP-dependent DNA fragmentation in the midbrain and striatum at 7 and 15 days after drug administration. CONCLUSION: These results support a role for melatonin in protecting neurons against MPTP toxicity in vivo, and suggest that its antiapoptotic action is one of the mechanisms by which melatonin protects neuronal cells from neurotoxic insults.