First study on response of astrocytes in alevines of red-bellied pacu (Piaractus brachypomus) to subchronic exposure to chlorpyrifos and trichlorfon.

Background and Aim: Organophosphate pesticides (OPs) used in agricultural production pose environmental and public health risks whenever non-target organisms are exposed to them. Oxon-type OPs, such as trichlorfon (TCF) and chlorpyrifos (CPF), are frequently used in Colombia and have been detected in water bodies in the vicinity of croplands; however, their effect on aquatic organisms, especially fish, is largely unknown. The neurotoxicity of OPs includes inhibition of esterase enzymes, neuronal damage, and increased glial reactivity. This study aimed to assess the astrocytic response in the brain tissue of juvenile red-bellied pacu (Piaractus brachypomus) exposed to TCF and CPF. Materials and Methods: A 25-day subchronic assay was conducted in which juvenile red-bellied pacu were exposed to CPF and TCF. After 25 days of exposure, the fish were killed and brain samples were collected and processed for immunohistochemistry to assess the morphology and reactivity of astrocytes; glial acidic fibrillary protein was used as a biomarker. Results: The brain samples from animals under subchronic exposure to OPs for 25 days showed higher cellular density as well as changes in astrocyte phenotype characterized by shortening of cytoplasmic projections, hypertrophy, and ameboid morphology compared to those from nonexposed animals. Similarly, astrocyte hyperreactivity was detected in the optic tectum and medial longitudinal fasciculus of the exposed group. Conclusion: Immunoreactivity of brain glial cells under subchronic exposure to OPs measured through immunohistochemical tests as well as OPs-induced neuropathology may be useful as a biomarker for monitoring environmental pollution. The results also indicate that P. brachypomus is a suitable biomonitoring model for studying neurotoxicological and neurodegenerative diseases.

Molecular characterization of HEPCIDIN-1 (HAMP1) gene in red-bellied pacu (Piaractus brachypomus).

Hepcidins are cysteine-rich peptides, which participate in iron metabolism regulation, the inflammatory and antimicrobial response. This study characterizes the hepcidin-1 (HAMP1) gene, its transcript expression in different tissues, as well as its regulation in a model of brain injury in Piaractus brachypomus. Bioinformatic analysis was carried out to determine conserved domains, glycosylation sites and protein structure of HAMP1, and probability that HAMP1 corresponds to an antimicrobial peptide (AMP). Relative gene expression of the P. brachypomus HAMP1 gene was determined by qPCR from cDNA of several tissues, a brain injury model, an organophosphate sublethal toxicity model and anesthetic experiment using the 2-ΔΔCt method. HAMP1 ORF encodes for a 91 aa pre-prohepcidin conformed for a prodomain with 42 aa and mature peptide of 25 aa. Mature domain was determined as an AMP. HAMP1 transcript is expressed in all the tissues, being higher in the spleen and liver. HAMP1 mRNA level was upregulated in the brain injury group, as well as in the olfactory bulb, optic chiasm and telencephalon of red-bellied pacu brain exposed to an organophosphate. In anesthetic experiment, HAMP1 mRNA level was upregulated in the liver and gills. HAMP1 gene of P. brachypomus may be involved in the inflammatory, antimicrobial, hypoxia and stress oxidative response.

Molecular characterization of myelin basic protein a (mbpa) gene from red-bellied pacu (Piaractus brachypomus).

BACKGROUND: Myelin basic protein (MBP) is one of the most important structural components of the myelin sheaths in both central and peripheral nervous systems. MBP has several functions including organization of the myelin membranes, reorganization of the cytoskeleton during the myelination process, and interaction with the SH3 domain in signaling pathways. Likewise, MBP has been proposed as a marker of demyelination in traumatic brain injury and chemical exposure. METHODS: The aim of this study was to molecularly characterize the myelin basic protein a (mbpa) gene from the Colombian native fish, red-bellied pacu, Piaractus brachypomus. Bioinformatic tools were used to identify the phylogenetic relationships, physicochemical characteristics, exons, intrinsically disordered regions, and conserved domains of the protein. Gene expression was assessed by qPCR in three models corresponding to sublethal chlorpyrifos exposure, acute brain injury, and anesthesia experiments. RESULTS: mbpa complete open reading frame was identified with 414 nucleotides distributed in 7 exons that encode 137 amino acids. MBPa was recognized as belonging to the myelin basic protein family, closely related with orthologous proteins, and two intrinsically disordered regions were established within the sequence. Gene expression of mbpa was upregulated in the optic chiasm of the chlorpyrifos exposed fish in contrast to the control group. CONCLUSIONS: The physicochemical computed features agree with the biological functions of MBP, and basal gene expression was according to the anatomical distribution in the tissues analyzed. This study is the first molecular characterization of mbpa from the native species Piaractus brachypomus.

Biochemical events related to glial response in spinal cord injury / Eventos bioquímicos de respuesta glial en la fisiopatología de la lesión de médula espinal

Abstract Introduction: Spinal cord injury (SCI) is a devastating event with physical, psychological and socioeconomic implications. Morphophysiological changes are observed in the tissue close to the injury, which allow determining the functional recovery of the medullary segment and the effector organs that depend on the injured axonal tracts. Objective: To describe the most relevant sequential biochemical events of glial cells response after SCI. Materials and methods: A search of scientific publications released in the past 18 years was carried out in PubMed and Science Direct databases, with the terms spinal cord injury (SCI), SCI pathophysiology, SCI inflammation, microglia in SCI, glial scar and chondroitin sulfate proteoglycans (CSPG). Results: The pathophysiological processes resulting from SCI are determinant for the neurological recovery of patients. Activation of glial cells plays an important role in promoting bioactive molecules and the formation of physical barriers that inhibit neural regeneration. Conclusion: Knowledge of neurobiological changes after SCI allows a greater understanding of the pathophysiology and favors the search for new therapeutic alternatives that limit the progression of the primary injury and minimize secondary damage, responsible for neurological dysfunction.

Resumen Introducción. La lesión de la médula espinal (LME) es un evento devastador con implicaciones físicas, psicológicas y socioeconómicas. En el tejido cercano a la lesión se instauran cambios morfofisiológicos que determinan la recuperación funcional del segmento medular y de los órganos efectores dependientes de los tractos axonales lesionados. Objetivo. Describir los eventos bioquímicos secuenciales más relevantes de la respuesta de las células gliales posterior a la LME. Materiales y métodos. Se realizó una búsqueda de publicaciones científicas de los últimos 18 años en las bases de datos PubMed y ScienceDirect, bajo los términos en inglés spinal cord injury (SCI), SCI pathophysiology, SCI inflammation, microglia in SCI, glial scar y chondroitin sulfate proteoglycans (CSPG). Resultados. Los procesos fisiopatológicos que se producen después de la LME determinan la recuperación neurológica de los pacientes. La activación de las células gliales juega un papel importante, ya que promueve la producción de moléculas bioactivas y la formación de barreras físicas que inhiben la regeneración neural. Conclusión. El conocimiento de los cambios neurobiológicos ocurridos tras la LME permite una mayor comprensión de la fisiopatología y favorece la búsqueda de nuevas alternativas terapéuticas que limiten la progresión de la lesión primaria y que minimicen el daño secundario responsable de la disfunción neurológica.

Sex steroid hormones as neuroprotective elements in ischemia models.

Among sex steroid hormones, progesterone and estradiol have a wide diversity of physiological activities that target the nervous system. Not only are they carried by the blood stream, but also they are locally synthesized in the brain and for this reason, estradiol and progesterone are considered 'neurosteroids'. The physiological actions of both hormones range from brain development and neurotransmission to aging, illustrating the importance of a deep understanding of their mechanisms of action. In this review, we summarize key roles that estradiol and progesterone play in the brain. As numerous reports have confirmed a substantial neuroprotective role for estradiol in models of neurodegenerative disease, we focus this review on traumatic brain injury and stroke models. We describe updated data from receptor and signaling events triggered by both hormones, with an emphasis on the mechanisms that have been reported as 'rapid' or 'cytoplasmic actions'. Data showing the therapeutic effects of the hormones, used alone or in combination, are also summarized, with a focus on rodent models of middle cerebral artery occlusion (MCAO). Finally, we draw attention to evidence that neuroprotection by both hormones might be due to a combination of 'cytoplasmic' and 'nuclear' signaling.

Perspective of synaptic protection after post-infarction treatment with statins.

Stroke is the second most common cause of death in people over 45 years of age in Colombia and is the leading cause of permanent disability worldwide. Cerebral ischemia is a stroke characterized by decreased blood flow due to the occlusion of one or more cerebral arteries, which can cause memory problems and hemiplegia or paralysis, among other impairments. The literature contains hundreds of therapies (invasive and noninvasive) that exhibit a neuroprotective effect when evaluated in animal models. However, in clinical trials, most of these drugs do not reproduce the previously demonstrated neuroprotective property, and some even have adverse effects that had not previously been detected in animal experimentation.Statins are drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis. Several studies have shown that statin therapy in an animal model of focal cerebral ischemia reduces infarct volume, as well as markers of neurodegeneration, activates neuronal survival pathways, and improves performance on learning and memory tests. Given the implied therapeutic benefit and the limited understanding of the mechanism of action of statins in brain repair, it is necessary to address the biochemical and tissue effects of these drugs on synaptic proteins, such as NMDA receptors, synaptic adhesion proteins, and cytoskeletal proteins; these proteins are highly relevant therapeutic targets, which, in addition to giving a structural account of synaptic connectivity and function, are also indicators of cellular communication and the integrity of the blood-brain barrier, which are widely affected in the long term post-cerebral infarct but, interestingly, are protected by statins when administered during the acute phase.

Toxicity of trichlorfon exposure using an experimentally induced model with Silver Pacu (Piaractus brachypomus) / Efectos toxicológicos generados por la exposición a triclorfón en un modelo inducido experimentalmente en cachama blanca (Piaractus brachypomus) / Efeitos toxicológicos no peixe Pirapitinga (Piaractus brachypomus) gerado pela exposição a triclorfon em um modelo induzido experimentalmente

The trichlorfon (TCF) is an organophosphate insecticide widely used in agricultural systems and livestock production. However, its indiscriminate use has generated serious problems affecting organisms in aquatic ecosystems that are not direct targets of the chemical. Therefore, the aim of this study was to assess acute toxicity (LC50) and chronic subletal of TCF in cachama blanca fingerlings (Piaractus brachypomus). Acute toxicity was evaluated at 96 hours, using four concentrations of TCF (0.01, 0.1, 0.25, 0.5 mg/L) and a control group, each group consisted of 8 animals (2.5 ± 0.5 g). The LC50 value calculated using the Trimmed Spearman-Karber software was 0.18 mg/L (p<0,05). The evaluation of the effect of chronic exposure to sublethal TCF on the immune response of P. brachypomus was performed by testing respiratory burst, showing significant increase in animals exposed to 0.029 mg / L during the first 10 days of exposure compared to control. Likewise, the plasma bactericidal capacity was evaluated in TCF exposed fish using Aeromonas hydrophila, finding greater of colony forming units on day 20 and 30 of exposure to the concentration 0.018 mg/L as opposed to the control. In addition, a histopathological study was performed, finding gill lesions compatible with epithelial cell hypertrophy and lamellar detachment in fish exposed to 0,05 mg/L on day 30 of exposure. This work showed the cachama blanca as a biomarker in contaminations associated with these xenobiotics.

El Triclorfón (TCF) es un insecticida organofosforado ampliamente utilizado en sistemas de producción agrícola y pecuario; sin embargo, su uso indiscriminado ha generado serios problemas en ecosistemas acuáticos afectando organismos que no son blanco directo del compuesto químico. Por este motivo, el presente estudio tuvo como objetivos evaluar la toxicidad aguda (CL50) y crónica subletal del TCF en alevinos de cachama blanca (Piaractus brachypomus). La toxicidad aguda se evaluó a 96 horas, empleando cuatro concentraciones de TCF (0,01, 0,1, 0,25, 0,5 mg/L) y un grupo control, cada grupo consto de 8 animales (2,5 ± 0,5 g). El valor de CL50 calculado mediante el software Trimmed Spearman-Karber fue 0,18 mg/L (p<0,05). La evaluación del efecto de la exposición crónica subletal a TCF sobre la respuesta inmune de P. brachypomus se realizó mediante el ensayo de explosión respiratoria, evidenciándose aumento significativo en animales sometidos a 0,029 mg/L durante los primeros 10 días de exposición en comparación con el control. Asimismo, se evalúo la capacidad bactericida del plasma de los peces expuestos a TCF frente Aeromonas hydrophila, presentándose mayor conteo de unidades formadoras de colonia para el día 20 y 30 de exposición a la concentración de 0,018 mg/L a diferencia del control. Aunado a esto, se realizó estudio histopatológico, hallándose lesiones branquiales compatibles con hipertrofia de células epiteliales y desprendimiento lamelar en los peces expuestos a 0,05 mg/L para el día 30 de exposición. Este trabajo demuestra que la cachama blanca se constituye en un bioindicador de contaminación asociado a estos xenobióticos.

O triclorfom (TCF) é um inseticida organofosforado amplamente utilizado em sistemas de produção agrícola e pecuária; no entanto, seu uso indiscriminado tem levado a sérios problemas que afetam os organismos nos ecossistemas aquáticos que não são alvos diretos da substância química. Portanto, o presente estudo teve como objetivo avaliar a toxicidade aguda (CL50) e crônica subletal de TCF em alevinos de cachama blanca (Piaractus brachypomus). A toxicidade aguda foi avaliada em 96 horas, usando quatro concentrações de TCF, com um grupo control, cada grupo com 8 animais (2,5 ± 0,5 g). O valor de CL50 calculado com o software de Spearman-Karber foi de 0,18 mg/L (p<0,05). A avaliação do efeito da exposição crônica a concentrações subletais de TCF na resposta imune de P. brachypomus foi realizada pelo teste de explosão respiratória, mostrando um aumento significativo nesta, em animais expostos a 0,029 mg/L, durante os primeiros 10 dias de exposição. Também, a capacidade bactericida do plasma dos peixes expostos a TCF foi avaliada frente a Aeromonas hydrophila, os quais apresentaraõ maior unidades formadoras de colônias no dia 20 e 30 de exposição a concentração de 0,018 mg/L, ao contrario do controle. Alem disso, um estudo histopatológico foi realizado, emcontrando lesões de brânquia compatíveis com hipertrofia e desprendimento de células epiteliais nas lamelas de peixes expostos a 0,05 mg/L no dia 30 de exposição. este trabalho mostra ao cachama blanca como um biomarcador de contaminação asociado com estes xenobióticos.

Neurotoxic potettial of trichlorfon to multiple sublethal doses in wistar rats / Potencial neurotóxico del Triclorfón a dosis múltiples subletales en ratas wistar

The organophosphates used for pest control induce sensory, motor and psychiatric disturbances after chronic exposure. The ester type is the cause of the intermediate syndrome and delayed neuropathy, in which the white and gray matter in the brain are severely affected.The aim of this study was to evaluate the effect of multiple sublethal doses of Trichlorfon on neurons, astrocytes and myelinated tissue in a rat model of brain neurotoxicity. Trichlorfon (metrifonate) was administered to adult Wistar rats at doses of 11 or 22 λg/kg by oral gavage every seven days for four or eight weeks (four experimental groups) and a control group (placebo). One week after the last dose, animals were euthanized and the brains perfused, removed and cut into coronal segments of 50 λm of thickness by using a vibratome. The sections were analyzed by immunohistochemistry, using markers of neuronal survival, astrocytic reactivity and the myelin basic protein. Neuronal and astrocytic reactivity were significantly reduced in Trichlorfon-treated animals relative to controls, whereas myelin reactivity was significantly increased, with abnormal distribution of myelin in white matter. The results suggest a neurotoxic damage of Trichlorfon on neuronal and astrocyte functional balance and abnormal myelin formation consequent to the cell damage.

Los organofosforados usados para control de plagas inducen trastornos sensoriales, motores y psiquiátricos por exposición crónica, siendo los de tipo éster, causa del síndrome intermedio y de la neuropatía retardada, que afectan severamente la sustancia blanca y gris del cerebro. El objetivo del presente trabajo fue evaluar el efecto del organofosforado Triclorfón sobre neuronas, astrocitos y tejido mielinizado en un modelo murino de neurotoxicidad encefálica a dosis múltiples subletales. Se suministró a ratas Wistar, triclorfón (metrifonato) a dosis de 11 y 22 λg/kg mediante sondaje esofagogástrico, cada siete días durante cuatro y ocho semanas en cuatro grupos experimentales y un grupo control (placebo). Una semana después de la última dosis, los animales fueron sacrificados y los cerebros perfundidos, extraídos y cortados en segmentos coronales de 50 λm de grosor mediante vibrátomo. Los cortes fueron analizados por inmunohistoquímica , usando marcadores de supervivencia neuronal, de reactividad astrocitaria y de la proteína base mielina. La reactividad neuronal y astrocitaria se redujo significativamente en los animales tratados con triclorfón en relación a los controles, mientras la reactividad de la mielina se incrementó significativamente, con distribución anormal en la sustancia blanca. Los resultados sugieren un daño neurotóxico del Triclorfón sobre el equilibrio funcional neuronal y astrocitario, con formaciones anómalas de mielina consecuente al daño celular.

Regulatory effect of Dimethyl Sulfoxide (DMSO) on astrocytic reactivity in a murine model of cerebral infarction by arterial embolization.

INTRODUCTION: The pathophysiology of cerebral ischemia is essential for early diagnosis, neurologic recovery, the early onset of drug treatment and the prognosis of ischemic events. Experimental models of cerebral ischemia can be used to evaluate the cellular response phenomena and possible neurological protection by drugs. OBJECTIVE: To characterize the cellular changes in the neuronal population and astrocytic response by the effect of Dimethyl Sulfoxide (DMSO) on a model of ischemia caused by cerebral embolism. METHODS: Twenty Wistar rats were divided into four groups (n= 5). The infarct was induced with α-bovine thrombin (40 NIH/Unit.). The treated group received 90 mg (100 µL) of DMSO in saline (1:1 v/v) intraperitoneally for 5 days; ischemic controls received only NaCl (placebo) and two non-ischemic groups (simulated) received NaCl and DMSO respectively. We evaluated the neuronal (anti-NeuN) and astrocytic immune-reactivity (anti-GFAP). The results were analyzed by densitometry (NIH Image J-Fiji 1.45 software) and analysis of variance (ANOVA) with the Graph pad software (Prism 5). RESULTS: Cerebral embolism induced reproducible and reliable lesions in the cortex and hippocampus (CA1)., similar to those of focal models. DMSO did not reverse the loss of post-ischemia neuronal immune-reactivity, but prevented the morphological damage of neurons, and significantly reduced astrocytic hyperactivity in the somato-sensory cortex and CA1 (p <0.001). CONCLUSIONS: The regulatory effect of DMSO on astrocyte hyperreactivity and neuronal-astroglial cytoarchitecture , gives it potential neuroprotective properties for the treatment of thromboembolic cerebral ischemia in the acute phase.

INTRODUCCIÓN: La fisiopatolog ía de la isquemia cerebral es fundamental para el diagnóstico oportuno, la recuperación neurológica, la instauración temprana del tratamiento y el pronóstico del evento isquémico. Los modelos experimentales de isquemia cerebral, permiten evaluar los fenómenos de respuesta celular y la posible neuroprotección por fármacos. OBJETIVO: caracterizar los cambios celulares en la población neuronal y la respuesta astrocitaria por efecto del dimetilsulfóxido (DMSO) en un modelo de isquemia por embolia cerebral. Métodos: Se utilizaron 20 ratas Wistar distribuidas en cuatro grupos (n =5). Se indujo embolia cerebral con α- trombina bovina (40 NIH/Unit.) y el grupo tratado recibió 90 mg (100 µL) de DMSO en NaCl (1:1 v/v) intraperitoneal por 5 días; el control isquémico, recibió solamente NaCl (placebo) y los dos grupos no isqu émicos (simulados) recibieron NaCl y DMSO respectivamente. Se evaluó la inmunoreactividad neuronal (anti-NeuN) y astrocitaria (anti-GFAP). Los resultados fueron analizados por densitometría (Fiji-Image J NIH 1.45 software) y análisis de varianza (ANOVA) con el programa Graph pad Prism 5. RESULTADOS: La embolia cerebral produjo lesiones reproducibles y confiables en corteza e hipocampo (CA1), similares a las de los modelos focales. El DMSO no revirtió la pérdida de la inmunoreactividad neuronal postisquemia, pero previno el daño morfológico neuronal y redujo significativamente la hiperreactividad astrocitaria en CA1 y corteza somatosensoria (p <0.001). CONCLUSIONES: El efecto regulatorio del DMSO sobre la hiperreactividad astrocitaria y la citoarquitectura neuronal - astroglial, le confiere características potencialmente neuroprotectivas para el tratamiento de la isquemia cerebral tromboembólica en fase aguda.

p120 catenin/αN-catenin are molecular targets in the neuroprotection and neuronal plasticity mediated by atorvastatin after focal cerebral ischemia.

Atorvastatin (ATV), a 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, exerts beneficial effects on stroke through several pleiotropic mechanisms. However, its role following cerebral ischemia is not completely understood yet. We evaluated the effect of ATV treatment on the synaptic adhesion proteins after a transient middle cerebral artery occlusion (t-MCAO) model in rats. Ischemic male Wistar rats were treated with 10 mg/kg ATV. The first dose was 6 hr after reperfusion, then every 24 hr for 3days. Our findings showed that ATV treatment produced an increase in pAkt ser473 and a decrease in pMAPK 44/42 protein levels 12 and 24 hr postischemia in the cerebral cortex and the hippocampus. However, p120 catenin and αN-catenin became drastically increased throughout the temporal course of postischemia treatment (12-72 hr), mainly in the hippocampus. Neurological recovery was observed at 48 and 72 hr, supported by a significant reduction of infarct volume, neuronal loss, and glial hyperreactivity after 72 hr of postischemia treatment with ATV. ATV treatment also up-regulated the association of p120(ctn) , αN-catenin to PSD-95, accompanied by a reduction of RhoA activation and the recovery of MAP2 immunoreactivity, these being significantly affected by the focal cerebral ischemia. Our findings suggested that p120(ctn) and αN-catenin synaptic adhesion proteins are crucial molecular targets in ATV-mediated neuroprotection and neuronal plasticity after focal cerebral ischemia.