Conjugated Linoleic Acid-Curcumin Attenuates Cognitive Deficits and Oxidative Stress Parameters in the Ethidium Bromide-Induced Model of Demyelination.

Oxidative stress has been shown to play an important role in the pathogenesis of multiple sclerosis (MS). Curcumin (CUR), an antioxidant compound, can be a potent treatment for neurodegenerative diseases, such as MS. CUR has poor bioavailability; therefore, it is used in nanoforms to increase its bioavailability. In the present study, the effects of CUR and conjugated linoleic acid-CUR (Lino-CUR) on spatial memory and oxidative stress in a putative animal model of MS were investigated. Forty-nine adult male Wistar rats (250 ± 50 g) were randomly divided into seven groups (n = 7): control, sham, ethidium bromide (EB), CUR (20 and 40 µg/kg) + EB, and Lino-CUR (20 and 40 µg/kg) + EB groups. Following MS induction, the groups were treated for 5 consecutive days. Finally, spatial memory and levels of oxidative stress parameters were assessed. Treatment with CUR and Lino-CUR at two doses significantly improved spatial memory and reduced oxidative stress parameters in the experimental models of MS. Furthermore, the effects of high dose (40 µg/kg) of Lino-CUR were more remarkable. These findings suggest that the microinjection of CUR in its synthetic form Lino-CUR significantly ameliorated spatial memory, through the reduction of oxidative stress markers in the brain of studied animals as a rat model of MS.

Investigation of in vivo unscheduled DNA synthesis in rabbit corneas following instillation of genotoxic agents.

PURPOSE: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents. MATERIALS AND METHODS: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography. RESULTS: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea. CONCLUSIONS: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

Mitochondrial DNA Manipulations Affect Tau Oligomerization.

BACKGROUND: Mitochondrial dysfunction and tau aggregation occur in Alzheimer's disease (AD), and exposing cells or rodents to mitochondrial toxins alters their tau. OBJECTIVE: To further explore how mitochondria influence tau, we measured tau oligomer levels in human neuronal SH-SY5Y cells with different mitochondrial DNA (mtDNA) manipulations. METHODS: Specifically, we analyzed cells undergoing ethidium bromide-induced acute mtDNA depletion, ρ0 cells with chronic mtDNA depletion, and cytoplasmic hybrid (cybrid) cell lines containing mtDNA from AD subjects. RESULTS: We found cytochrome oxidase activity was particularly sensitive to acute mtDNA depletion, evidence of metabolic re-programming in the ρ0 cells, and a relatively reduced mtDNA content in cybrids generated through AD subject mitochondrial transfer. In each case tau oligomer levels increased, and acutely depleted and AD cybrid cells also showed a monomer to oligomer shift. CONCLUSION: We conclude a cell's mtDNA affects tau oligomerization. Overlapping tau changes across three mtDNA-manipulated models establishes the reproducibility of the phenomenon, and its presence in AD cybrids supports its AD-relevance.

Decomposition of DNA staining agent ethidium bromide by gamma irradiation: Conditions, kinetics, by-products, biological activity, and removal from wastewater.

Ethidium Bromide (Eth-Br) is an intercalating agent commonly used in medical and biological laboratories as a DNA staining dye. Despite its popular use, aqueous solutions containing Eth-Br showed high toxicity, mutagenic capacity, and deactivate DNA transcription. In this study, the removal of Eth-Br from aqueous solutions by gamma irradiation has been fully investigated. Gamma irradiation was capable of achieving a near complete removal of Eth-Br in neutral and non-buffered aqueous solutions at an absorbed dose of 15 kGy. Various experimental conditions were studied and showed that the removal efficiency is not diminished. The addition of hydrogen peroxide (2 %) to the irradiated solutions reduced the D50 and D90 by 50 %. Modeling Eth-Br decomposition showed that the reaction followed pseudo first-order kinetics and reaches at least 90 % removal under all experimental conditions. TOC and HPLC measurements confirmed that Eth-Br is fully mineralized when the absorbed dose reaches 15 kGy. The biological activity of Eth-Br after irradiation treatment was investigated with synthetic DNA and natural DNA. The biological activity of Eth-Br was deactivated at an absorbed dose as low as 5 kGy. Toxicity measurement with E-coli bacteria also confirmed that the absorbed dose of 5 kGy was sufficient to remove Eth-Br toxicity.

The effect of intermittent fasting diet on the hippocampus of adult male mouse after inducing demyelination by ethidium bromide injection / Efecto de la dieta de ayuno intermitente en el hipocampo de ratón macho adulto después de inducir la desmielinización por inyección de bromuro de etidio

Intermittent fasting diet (IF) as a restrictive regimen prevents neural degeneration and stimulates overexpression of various neurotropic factors in the hippocampus of animal models. This study evaluates the potential effect of the IF in the prevention of learning and memory dysfunction and improving the alterations in the number and volume of neurons in an ethidium bromide (EB) induced mouse model of demyelination.Mice were randomly assigned into N group (Normal Diet and normal saline injection), F group (IF and normal saline injection), EBN group (Normal Diet and EB injection), EBF group (IF and EB injection). The hidden platform test was carried out based on path length, escape latency and swim speeds of mice. Stereological studies were determined by the Cavalieri and the Optical Dissector technique. Maintenance of mice on the IF results in significantly decreased the body weight and biochemical parameters, increased total number of neurons and volume of the hippocampus, and improved learning and memory parameters of adult male mice. However, IF in EBF group did not show as excellently as F group. The EBF group displayed significantly spatial memory improvement than that in EBN group. There were no statistically significant differences between EBF and EBN groups in stereological and learning parameters, though the EBF group displayed faster escape latencies, and swam faster and shorter path lengths than the EBN group in these parameters. Therefore as a conclusion, The IF fairly improved some adverse effects of EB in experimental demyelination models.

La dieta de ayuno intermitente (AI) como régimen restrictivo, previene la degeneración neural y la estimación de la presencia de diversos factores neurotrópicos en el hipocampo de modelos animales. Este estudio evalúa el efecto potencial de la AI en la prevención del aprendizaje y la disfunción de la memoria y mejora las alteraciones en el número y el volumen de las neuronas en un modelo de desmielinización, en ratón, inducido con bromuro de etidio (BE). Los ratones se asignaron al azar en el grupo N (dieta normal e inyección salina normal), Grupo A (AI e inyección salina normal), Grupo BEN (dieta normal e inyección BE), Grupo EBF (inyección AI y BE). La prueba de la plataforma oculta se llevó a cabo en función de la longitud del trayecto, la latencia de escape y la velocidad de nado de los ratones. Los estudios estereológicos fueron determinados por la técnica de Cavalieri y la técnica del disector óptico. En el grupo AI disminuyeron significativamente el peso corporal de los ratones, los parámetros bioquímicos, el número total de neuronas y el volumen del hipocampo, y los parámetros de aprendizaje y la memoria de los ratones machos adultos. Sin embargo, el grupo AI en BEF no se mostró tan bien como el grupo A. El grupo EBF mostró una mejora en la memoria espacial significativamente mayor que la del grupo BEN. No hubo diferencias estadísticamente significativas entre los grupos A, BE y BEN en los parámetros estereológicos y de aprendizaje, aunque el grupo EBF mostró latencias de escape más rápidas, y nado en las rutas más rápidas y más cortas que el grupo BEN en estos parámetros. Por lo tanto, como conclusión, el grupo AI mejoró bastante algunos efectos adversos de la BE en los modelos de desmielinización experimental.

Investigation of comet assays under conditions mimicking ocular instillation administration in a three-dimensional reconstructed human corneal epithelial model.

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.

Toxin-Based Models to Investigate Demyelination and Remyelination.

Clinical myelin diseases, and our best experimental approximations, are complex entities in which demyelination and remyelination proceed unpredictably and concurrently. These features can make it difficult to identify mechanistic details. Toxin-based models offer lesions with predictable spatiotemporal patterns and relatively discrete phases of damage and repair: a simpler system to study the relevant biology and how this can be manipulated. Here, we discuss the most widely used toxin-based models, with a focus on lysolecithin, ethidium bromide, and cuprizone. This includes an overview of their respective mechanisms, strengths, and limitations and step-by-step protocols for their use.

LINGO-1 siRNA nanoparticles promote central remyelination in ethidium bromide-induced demyelination in rats.

Multiple sclerosis is among the most common causes of neurological disabilities in young adults. Over the past decade, several therapeutic strategies have emerged as having potential neuroprotective and neuroregenerative properties. We investigated the effect of intranasal administration of LINGO-1-directed siRNA-loaded chitosan nanoparticles on demyelination and remyelination processes in a rat model of demyelination. Adult male Wistar rats were randomly assigned to one of 6 groups (n = 10 each) and subjected to intrapontine stereotaxic injection of ethidium bromide (EB) to induce demyelination. EB-treated rats were either left untreated or received intranasal LINGO-1-directed siRNA-chitosan nanoparticles from day 1 to day 7 (demyelination group) or from day 7 to day 21 (remyelination group) after EB injection. Chitosan nanoparticle (50 µl) was given alone after EB stereotaxic injection for both demyelination and remyelination groups. Two additional groups received 10 µl of saline by stereotaxic injection, followed by intranasal saline as controls for demyelination and remyelination groups (n = 10/group). Behavioural testing was conducted for all rats, as well as terminal biochemical assays and pathological examination of pontine tissues were done. After EB injection, rats had compromised motor performance and coordination. Pathological evidence of demyelination was observed in pontine tissue and higher levels of caspase-3 activity were detected compared to control rats. With LINGO-1-directed siRNA-chitosan nanoparticle treatment, animals performed better than controls. Remyelination-treated group showed better motor performance than demyelination group. LINGO-1 downregulation was associated with signs of repair in histopathological sections, higher expression of pontine myelin basic protein (MBP) mRNA and protein and lower levels of caspase-3 activity indicating neuroprotection and remyelination enhancement.

Remyelination promoting therapies in multiple sclerosis animal models: a systematic review and meta-analysis.

An unmet but urgent medical need is the development of myelin repair promoting therapies for Multiple Sclerosis (MS). Many such therapies have been pre-clinically tested using different models of toxic demyelination such as cuprizone, ethidium bromide, or lysolecithin and some of the therapies already entered clinical trials. However, keeping track on all these possible new therapies and their efficacy has become difficult with the increasing number of studies. In this study, we aimed at summarizing the current evidence on such therapies through a systematic review and at providing an estimate of the effects of tested interventions by a meta-analysis. We show that 88 different therapies have been pre-clinically tested for remyelination. 25 of them (28%) entered clinical trials. Our meta-analysis also identifies 16 promising therapies which did not enter a clinical trial for MS so far, among them Pigment epithelium-derived factor, Plateled derived growth factor, and Tocopherol derivate TFA-12.We also show that failure in bench to bedside translation from certain therapies may in part be attributable to poor study quality. By addressing these problems, clinical translation might be smoother and possibly animal numbers could be reduced.

Mitochondrial genome and functional defects in osteosarcoma are associated with their aggressive phenotype.

Osteosarcoma (OSA) is an aggressive mesenchymal tumor of the bone that affects children and occurs spontaneously in dogs. Human and canine OSA share similar clinical, biological and genetic features, which make dogs an excellent comparative model to investigate the etiology and pathogenesis of OSA. Mitochondrial (mt) defects have been reported in many different cancers including OSA, although it is not known whether these defects contribute to OSA progression and metastasis. Taking a comparative approach using canine OSA cell lines and tumor tissues we investigated the effects of mtDNA content and dysfunction on OSA biology. OSA tumor tissues had low mtDNA contents compared to the matched non-tumor tissues. We observed mitochondrial heterogeneity among the OSA cell lines and the most invasive cells expressing increased levels of OSA metastasis genes contained the highest amount of mitochondrial defects (reduced mtDNA copies, mt respiration, and expression of electron transport chain proteins). While mitochondria maintain a filamentous network in healthy cells, the mitochondrial morphology in OSA cells were mostly "donut shaped", typical of "stressed" mitochondria. Moreover the expression levels of mitochondrial retrograde signaling proteins Akt1, IGF1R, hnRNPA2 and NFkB correlated with the invasiveness of the OSA cells. Furthermore, we demonstrate the causal role of mitochondrial defects in inducing the invasive phenotype by Ethidium Bromide induced-mtDNA depletion in OSA cells. Our data suggest that defects in mitochondrial genome and function are prevalent in OSA and that lower mtDNA content is associated with higher tumor cell invasiveness. We propose that mt defects in OSA might serve as a prognostic biomarker and a target for therapeutic intervention in OSA patients.

Mutagen-induced phytotoxicity in maize seed germination is dependent on ROS scavenging capacity.

Ethidium bromide (EB) and acridine orange (AO) bind to nucleic acids and are thus considered as potential mutagens. In this study, the effects of EB and AO on the germination behaviours of white, yellow, red, and purple maize seeds were investigated. The results indicate that low concentrations of EB (50 µg mL-1) and AO (500 µg mL-1) promote germination, particularly for the white and yellow seeds. However, high concentrations of EB (0.5 mg mL-1) and AO (5 mg mL-1) significantly inhibit germination, with the level of inhibition decreasing in the following order: white > yellow > red > purple. In addition, EB and AO induce H2O2 production in a concentration-dependent manner. The effects of these mutagens on seed germination were partly reversed by dimethyl thiourea, a scavenger of reactive oxygen species (ROS), and diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, while the effects were enhanced by treatment with H2O2 and 3-amino-1,2,4-triazole, a specific inhibitor of catalase. In addition, AO and EB profoundly increased NADPH oxidase activities in germinating seeds. The treatment of seeds with EB and AO did not affect the growth or drought tolerance of the resultant seedlings. The findings suggest that the mechanism of mutagen toxicity is related to the induction of ROS production.

CLUH couples mitochondrial distribution to the energetic and metabolic status.

Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status.

SYBR safeTM efficiently replaces ethidium bromide in Aspergillus fumigatus gene disruption.

Invasive aspergillosis is a disease responsible for high mortality rates, caused mainly by Aspergillus fumigatus. The available drugs are limited and this disease continues to occur at an unacceptable frequency. Gene disruption is essential in the search for new drug targets. An efficient protocol for A. fumigatus gene disruption was described but it requires ethidium bromide, a genotoxic agent, for DNA staining. Therefore, the present study tested SYBR safeTM, a non-genotoxic DNA stain, in A. fumigatus gene disruption protocol. The chosen gene was cipC, which has already been disrupted successfully in our laboratory. A deletion cassette was constructed in Saccharomyces cerevisiae and used in A. fumigatus transformation. There was no statistical difference between the tested DNA stains. The success rate of S. cerevisiae transformation was 63.3% for ethidium bromide and 70% for SYBR safeTM. For A. fumigatus gene disruption, the success rate for ethidium bromide was 100 and 97% for SYBR safeTM. In conclusion, SYBR safeTM efficiently replaced ethidium bromide, making this dye a safe and efficient alternative for DNA staining in A. fumigatus gene disruption.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem.

OBJECTIVE: This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. METHOD: Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. RESULTS AND CONCLUSION: Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

Mitochondrial homeostatic disruptions are sensitive indicators of stress in neurons with defective mitochondrial DNA transactions.

Neurodegeneration and mitochondrial dysfunction are closely linked across many clinical conditions. In genetic diseases that result from defects in mitochondrial DNA (mtDNA) synthesis or maintenance, neurodegeneration is a frequent and major component of the disease pathology. In sporadic neurodegenerative diseases such as Alzheimer's and Parkinson's disease, mtDNA defects have been observed clinically. Mitochondrial stress related to mtDNA dysregulation can produce neuronal dysfunction and death via impaired electron transport chain activity, which results in deficient ATP production and related increases in mitochondrial reactive oxygen species (ROS) production. However, mtDNA dysregulation in post-mitotic neurons may also produce disturbances in mitochondrial homeostasis that are known to impair neuronal function as well. In this study, we used sub-toxic doses of ethidium bromide (EtBr) to induce mtDNA-associated mitochondrial stress in primary cortical neurons and measured several aspects of mitochondrial homeostasis, mitochondrial function and cell death. We found that low-dose EtBr severely depletes mtDNA synthesis and mitochondrial mRNA levels. Furthermore, homeostatic processes are especially disrupted in toxin treated neurons while mitochondrial function is relatively preserved. Mitochondria become fragmented and motility is abolished, while respiration and mitochondrial polarization are partially maintained. Moreover at these doses, cells do not exhibit increased ROS production, clear neurite retraction or loss of viability. These results indicate that mitochondrial homeostasis is a sensitive marker of mtDNA associated stress compared to mitochondria-functional outputs or endpoints related to cellular toxicity. These homeostatic disruptions are expected to contribute to neuronal dysfunction and potentially drive neurodegenerative disease pathology.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Propentofylline treatment on open field behavior in rats with focal ethidium bromide-induced demyelination in the ventral surface of the brainstem.

Propentofylline (PPF) is a xanthine derivative with pharmacological effects that are distinct from those of classic methylxanthines. It depresses the activation of microglial cells and astrocytes, which is associated with neuronal damage during neural inflammation and hypoxia. Our previous studies showed that PPF improved remyelination following gliotoxic lesions that were induced by ethidium bromide (EB). In the present study, the long-term effects of PPF on open field behavior in rats with EB-induced focal demyelination were examined. The effects of PPF were first evaluated in naive rats that were not subjected to EB lesions. Behavior in the beam walking test was also evaluated during chronic PPF treatment because impairments in motor coordination can interfere with behavior in the open field. The results showed that PPF treatment in unlesioned rats decreased general activity and caused motor impairment in the beam walking test. Gliotoxic EB injections increased general activity in rats that were treated with PPF compared with rats that received saline solution. Motor incoordination was also attenuated in PPF-treated rats. These results indicate that PPF reversed the effects of EB lesions on behavior in the open field and beam walking test.

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

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

Toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three popular DNA intercalating fluorescent dyes.

We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells.

Propentofylline reverses delayed remyelination in streptozotocin-induced diabetic rats.

OBJECTIVE: The diabetic state induced by streptozotocin injection is known to impair oligodendroglial remyelination in the rat brainstem following intracisternal injection with the gliotoxic agent ethidium bromide (EB). In such experimental model, propentofylline (PPF) recently showed to improve myelin repair, probably due to its neuroprotective, antiinflammatory and antioxidant effects. The aim of this study was to evaluate the effect of PPF administration in diabetic rats submitted to the EB-demyelinating model. MATERIALS AND METHODS: Adult male rats, diabetic or not, received a single injection of 10 microlitres of 0.1% EB solution into the cisterna pontis. For induction of diabetes mellitus the streptozotocin-diabetogenic model was used (50 mg/kg, intraperitoneal route - IP). Some diabetic rats were treated with PPF (12.5 mg/kg/day, IP route) during the experimental period. The animals were anesthetized and perfused from 7 to 31 days after EB injection and brainstem sections were collected for analysis of the lesions by light and transmission electron microscopy. RESULTS: Diabetic rats injected with EB showed larger amounts of myelin-derived membranes in the central areas of the lesions and considerable delay in the remyelinating process played by surviving oligodendrocytes and invading Schwann cells after the 15th day. On the other hand, diabetic rats that received PPF presented lesions similar to those of non-diabetic animals, with rapid remyelination at the edges of the lesion site and fast clearance of myelin debris from the central area. CONCLUSION: The administration of PPF apparently reversed the impairment in remyelination induced by the diabetic state.