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.

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 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. Arch Endocrinol Metab. 2015;59(1):47-53 .

Decreased astrocytic GFAP expression in streptozotocin-induced diabetes after gliotoxic lesion in the rat brainstem / Expressão astrocitária diminuída de GFAP no diabetes induzido por estreptozotocina após lesão gliotóxica no tronco encefálico de ratos

OBJECTIVE: The aim of this study was to evaluate the effect of diabetic hyperglycemia on astrocyte function, estimated by means of glial fibrillary acidic protein - GFAP - immunohistochemical expression. MATERIALS AND METHODS: Adult male rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 microlitres 0.1% EB solution or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB or 0.9% saline solution were also injected in non-diabetic rats. Animals were anesthetized and perfused through the heart 15 and 31 days after EB or saline injection, and brainstem sections were collected for ultrastructural analysis and GFAP immunohistochemical staining. RESULTS: The GFAP brown-stained areas were evaluated by colorimetry using a computerized image analysis system and the results have shown that diabetes hindered the increase of GFAP astrocyte expression in the EB-injected group compared to non-diabetic animals. However, diabetes did not affect GFAP response in the saline-injected group or in control animals. CONCLUSION: Streptozotocin-induced diabetic condition reduced astrocytic GFAP expression following gliotoxic injury.

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da hiperglicemia na função astrocitária, estimada pela expressão imuno-histoquímica da proteína glial fibrilar ácida - GFAP. MATERIAIS E MÉTODOS: Ratos machos adultos receberam uma injeção intravenosa única de estreptozotocina (50 mg/kg) e foram submetidos, 10 dias após, à injeção de 10 microlitros de solução de BE 0,1% ou de salina 0,9% na cisterna pontina. Dez microlitros de BE 0,1% ou salina 0,9% foram também injetados em ratos não diabéticos. Os animais foram anestesiados e perfundidos por via intracardíaca aos 15 e 31 dias pós-injeção de BE ou salina, e amostras de tronco encefálico foram coletadas para estudo ultraestrutural e análise imuno-histoquímica para a GFAP. RESULTADOS: Utilizando um sistema computadorizado de análise de imagens, os resultados das áreas coradas em marrom pela GFAP, medidas por colorimetria, mostram que o diabetes reduziu o aumento de expressão dessa proteína no grupo injetado com BE em comparação aos animais não diabéticos, mas não alterou a resposta no grupo injetado com salina ou nos controles diabéticos. CONCLUSÃO: O estado diabético induzido pela estreptozotocina reduziu a expressão astrocitária de GFAP após dano gliotóxico.

Ethidium bromide-induced demyelination in the sciatic nerve of diabetic rats / Desmielinização induzida pelo brometo de etídio no nervo ciático de ratos diabéticos

This study aims to observe the process of myelin loss and repair following the injection of the gliotoxic agent ethidium bromide (EB) in the sciatic nerve of rats previously induced to diabetes mellitus by streptozotocin. Injection of EB was also done in non-diabetic rats. The animals were euthanatized from 3 to 31 days after intraneural injection and nerve sections were collected for ultrastructural study. In non-diabetic rats, Schwann cells (CS) showed signs of intoxication 3 days after, with cytoplasmic vacuolization and rejection of their myelin sheaths. Myelin debris were removed by macrophages in the endoneurium and mast cells were abundant in the lesions. From 14 days following EB injection, supernumerary CS were seen in the expanded endoneurium as well as thin myelin sheaths indicating remyelination. Diabetic rats presented a more extensive myelin vesiculation and segmentar demyelination, with delayed activities from both macrophages and remyelinating SC. No mast cells were noted.

O estudo visa à observação do processo de perda e reparo mielínico pós-injeção do gliotóxico brometo de etídio (BE) no nervo ciático de ratos previamente induzidos a diabetes mellitus pela estreptozotocina. Injeção de BE foi igualmente realizada em ratos não-diabéticos. Os animais foram eutanasiados dos 3 aos 31 dias pós-injeção intraneural, com colheita de amostras neurais para estudo ultra-estrutural. Nos animais não-diabéticos, as células de Schwann (CS) mostraram sinais de intoxicação a partir dos 3 dias pós-gliotóxico, com vacuolização citoplasmática e rejeição de suas bainhas de mielina. Restos mielínicos eram removidos por macrófagos no interior do endoneuro e mastócitos eram abundantes nas lesões. A partir dos 14 dias, CS supranumerárias foram encontradas no endoneuro expandido, além de finas bainhas de mielina indicativas de remielinização. Os ratos diabéticos apresentaram vesiculação mielínica e desmielinização segmentar mais extensas, bem como ausência de mastócitos e atraso na atividade macrofágica e na função remielinizante das CS.

Marcação imuno-histoquímica da resposta macrofágica e astrocitária no tronco encefálico de ratos Wistar submetidos ao modelo gliotóxico do brometo de etídio e tratados com ciclofosfamida / Immunohistochemical staining of the macrophagic and astrocytic response in the brainstem of Wistar rats submitted to the ethidium bromide gliotoxic model and treated with cyclophosphamide

O gliotóxico brometo de etídio (BE) foi utilizado para o estudo da resposta macrofágica e astrocitária sob imunossupressão com ciclofosfamida (CY). Investigou-se a imunorreatividade astrocitária à proteína glial fibrilar ácida (GFAP) e à vimentina (VIM), e a imunorreatividade macrofágica ao ED1 após injeção do BE. Foram utilizados ratos Wistar adultos injetados na cisterna basal com salina a 0,9 por cento (grupo I), BE a 0,1 por cento (grupo II) e BE a 0,1 por cento, imunossuprimidos com CY (grupo III). Fragmentos do tronco encefálico foram colhidos do 1° ao 21° dia pós-injeção para estudo imuno-histoquímico da GFAP, VIM e ED1. Nos grupos II e III, observou-se imunorreatividade aumentada para GFAP e re-expressão de VIM. No grupo II, células ED1-positivas foram observadas a partir do 2° dia e no grupo III, a partir do 3° dia. Aos 14 dias pós-injeção, havia mais células ED1-positivas no grupo III. A CY aparentemente não alterou a resposta astrocitária.

The gliotoxic ethidium bromide (EB) was used to study morphologically the macrophagic and astrocytic response under immunosuppression by cyclophosphamide (CY). Astrocyte immunoreactivity to glial fibrillary acidic protein (GFAP) and vimentin (VIM) and macrophagic immunoreactivity to ED1 were investigated after EB injection. Male Wistar rats were injected with 0.9 percent saline solution (group I), 0.1 percent BE (group II) and 0.1 percent EB associated with CY treatment (group III). Brainstem samples were collected from the 1st to the 21st day post-injection for GFAP, VIM and ED1 immunostaining. In groups II and III, it was observed increased immunoreactivity to GFAP and reexpression of VIM. In group II, ED1-positive cells were noted after the 2nd day and in group III, after the 3rd day. On the 14th day post-injection, it was observed a greater quantity of ED1- positive cells in group III than in group II. Apparently, CY did not change the astrocytic response pattern.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 æL 0.1 percent (w/v) EB or 0.9 percent saline solution into the cisterna pontis. Ten microliters of 0.1 percent EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Reversion mutation in dark variants of luminous bacteria and its application in gene toxicant monitoring / 华中科技大学学报（医学）（英德文版）

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.

Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system

Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB) is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 mul of either 0.1 percent ethidium bromide in normal saline (19 rats) or 0.9 percent saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5 percent glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuos and were found in only 34 percent of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.