Confirmación de la presencia de células serotoninérgicas en la corteza cerebral fetal en cultivos e in situ / Confirmation of the presence of serotonergic cells in the fetal cerebral cortex in cultures and in situ

Resumen Antecedentes: La aparición temprana de serotonina en el cerebro fetal y sus efectos en la morfogénesis cerebral apoyan su papel neurotrófico. Objetivo: Determinar la presencia de células serotoninérgicas y la expresión de triptófano-5-hidroxilasa (TPH), 5-hidroxitriptamina (5-HT), transportador de serotonina (SERT), receptor 5-HT1A y Pet-1 durante el desarrollo de la corteza cerebral, tanto in situ como en cultivo de tejidos. Material y métodos: Se realizó estudio observacional descriptivo en ratas Wistar preñadas. La presencia del tapón se consideró el inicio de la gestación; en los días 13, 16 y 17 se practicaron cesáreas para obtener los fetos e inmediatamente se disecaron los cerebros para identificar células serotoninérgicas, TPH, 5-HT, SERT, 5-HT1A y Pet-1 en cultivo de tejido e in situ mediante inmunomarcaje detectado en un microscopio confocal. Resultados: Células y terminales serotoninérgicas fueron observadas en el mesencéfalo el día 17 de gestación y en cocultivos de neopalio los días 13 y 16. También se observaron células inmunopositivas a TPH, 5-HT, SERT y Pet-1 en el neopalio en el día 12 del cultivo. Conclusiones: Se confirmó la presencia de células serotoninérgicas y otros elementos del sistema serotoninérgico en la corteza cerebral temprana, la cual puede ser transitoria y participar en los procesos de maduración cortical durante el desarrollo cerebral.

Abstract Background: Early appearance of serotonin in the fetal brain and its effects on brain morphogenesis support its neurotrophic role. Objective: To determine the presence of serotonergic cells and the expression of tryptophan-5-hydroxylase (TPH), 5-hydroxytryptamine (5-HT), serotonin transporter (SERT), 5-HT1A receptor and Pet-1 during the development of the cerebral cortex, both in situ and in tissue cultures. Material and methods: A descriptive, observational study was carried out in pregnant Wistar rats. The presence of the plug was regarded as the beginning of gestation. On days 13, 16 and 17, cesarean sections were performed to obtain the fetuses, and the brains were then immediately dissected to identify the presence of serotonergic cells, TPH, 5-HT, SERT, 5-HT1A and Pet-1 in tissue cultures and in situ by immunostaining detected on a confocal microscope. Results: Serotonergic cells and terminals were observed in the midbrain on day 17 of gestation, and in neopallium cocultures on days 13 and 16. TPH, 5-HT, SERT and Pet-1 immunopositive cells were also observed in the neopallium on day 12 of culture. Conclusions: The presence of serotonergic cells and other elements of the serotonergic system in the early cerebral cortex was confirmed, which may be transient and participate in cortical maturation processes during brain development.

[Anatomopathological findings during development of diabetic cardiomyopathy in rats]. / Hallazgos anatomopatológicos durante el desarrollo de la miocardiopatía diabética en la rata.

BACKGROUND: The diabetic cardiomyopathy occurs in both type 1 and type 2 diabetes mellitus. Hyperglycemia and associated metabolic changes participate in the pathogenesis of this disease. OBJECTIVE: To characterizes various pathological changes occurring during the development of diabetic cardiomyopathy in rats. METHODS: Diabetic rats were used for streptozotocin administration. At 7, 14, 21 and 30 days after toxic administration, the heart was obtained and placed in a Hartman solution and 4% p-formaldehyde. Five-micrometer thick sections were stained with hematoxylin-eosin, Masson trichrome and immunocytochemistry using anti-ß-tubulin antibody. RESULTS: At 14 days after application of streptozotocin, dilated sinusoids with endothelial lining in the myocardium and collagen deposits in the cardiac interstitium and between the Purkinje fibers were observed. At 21 days there was a slight decrease of the arteriolar lumen due to hyperplasia of the medial layer. It is important to note that cardiac sinusoids as well as collagen deposits became more evident at 30 day of the study, as well as a major derangement of the microtubular system of the cardiomyocytes. CONCLUSIONS: Cardiac sinusoids representing fetal vascular pattern and interstitial fibrosis in the myocardium and the microtubular derangement of cardiomyocytes support the fact that the pathophysiological mechanism of diabetic cardiomyopathy begins in the coronary microcirculation due to changes in cardiac metabolism, contributing to the development of myocardial dysfunction in diabetes.

Antecedentes: la miocardiopatía diabética ocurre en ambos tipos de diabetes mellitus y en su patogenia intervienen la hiperglucemia y los cambios metabólicos asociados. Objetivo: caracterizar los diferentes cambios patológicos que aparecen durante la evolución de la miocardiopatía diabética en la rata. Material y métodos: estudio transversal comparativo en dos grupos de ratas diabéticas por la administración de estreptozotocina. A los 14, 21 y 30 días de la administración del tóxico se obtuvieron los corazones, que se colocaron en p-formaldehído al 4%. Se efectuaron cortes de 5 µm y se tiñeron con hematoxilina-eosina, tricrómica de Masson e inmunocitoquímica con anticuerpos anti ß-tubulina. Resultados: a los 14 días de la aplicación de la estreptozotocina se observaron en el miocardio sinusoides dilatadas y depósito de colágena entre las fibras de Purkinje e intersticio cardiaco. A los 21 días disminuyó la luz arteriolar por hiperplasia de la capa media. A los 30 días del estudio se hicieron más evidentes los sinusoides cardiacos y los depósitos de colágena y un importante desarreglo del sistema microtubular de los cardiomiocitos. Conclusiones: los sinusoides cardiacos, que representan un patrón vascular fetal y la fibrosis intersticial en el miocardio y el desarreglo microtubular de los cardiomiocitos, apoyan el hecho de que el mecanismo fisiopatológico de la miocardiopatía diabética se inicia en la microcirculación coronaria debido a cambios en el metabolismo cardiaco que contribuyen a la disfunción miocárdica durante el estado diabético.

Serotonin and dopamine in the hypothalamus of control and malnourished mother rats during pregnancy and lactation and body composition of their offspring.

OBJECTIVE: To determine concentrations of serotonin and dopamine in the hypothalamus of undernourished rats and controls during pregnancy and lactation and body composition of their offspring. METHODS: Malnourished rats along with control rats were used during pregnancy and lactation. At birth of their offspring, control mothers nursed their young and malnourished rats and the undernourished mothers nursed their offspring and control pups. On days 5, 10, 15, and 21 of lactation (at the beginning and end of a feeding), L-tryptophan (L-Trp)-free, bound and total, plasma prolactin (PRL) and milk composition were determined. Serotonin and dopamine were measured in the hypothalamus. Body composition of offspring was determined. RESULTS: Increase of free L-Trp was confirmed in undernourished mothers. Furthermore, hypothalamic serotonin was elevated at the start of suckling and decreased at termination. There was also a decrease in dopamine in the hypothalamus at the beginning and end of suckling followed by an increase of plasma PRL that was greater in control mothers who breastfed malnourished offspring. Interestingly, undernourished offspring consumed more milk and showed a clear recovery of body composition with accumulation of body fat. DISCUSSION: Changes observed in hypothalamic neurotransmitters appear to be closely related to nutritional status and to the response and control of PRL production, possibly to adapt the offspring to the metabolic changes. It was also confirmed that on-demand feeding of undernourished offspring is the main factor involved in nutritional recovery and a predisposition to overweight in the recovered undernourished animals.

Increased expression of tryptophan-5-hydroxylase 1, but not 2, in brainstem as a result of intrauterine malnutrition.

The aim of this study was to determine whether intrauterine malnutrition (IUM) produces a change in the expression of tryptophan-5-hydroxylase (TPH) 1 and/or 2 as the primary mechanism to explain the observed chronic cerebral acceleration of the synthesis of 5-hydroxytryptamine (5-HT). We used an IUM model and controls with ages of 1, 15 and 21 days. The brainstem was obtained to determine L-tryptophan, 5-HT and TPH activity. Expression of TPH1 and TPH2 via specific antibodies for each was also evaluated by immunocytochemistry and Western blot. Malnourished offspring had a significant elevation of L-Trp, TPH activity and 5-HT in the brainstem. Both isoforms (1 and 2) of TPH were expressed from birth in both groups; however, TPH1 expression was significantly higher in offspring with IUM in relation to the controls. Importantly, these malnourished offspring showed reduced expression of TPH2 compared to controls. It was confirmed that IUM produces an increase in 5-HT in the brainstem and also showed increased expression of TPH1 at birth, with decreased expression of TPH2. These findings together allow us to propose that chronic elevation of synthesis of 5-HT observed in the brain of the offspring with IUM is probably due to a change in the expression and activity of TPH1 induced from fetal life.

Association of metabolic syndrome with reduced central serotonergic activity.

The aim of this study was to determine the differences between two groups of adolescents with metabolic syndrome (MetS) and normal controls in relation to brain serotonergic activity through intensity-dependent auditory-evoked potentials (IDAEPs) and plasma free fraction of L-tryptophan. Eighteen adolescents with MetS and thirteen controls were studied. Free fraction, bound and total plasma L-tryptophan, glucose, cholesterol, triglycerides, HDL-cholesterol, albumin and IDAEPs were determined. Glycemia, triglycerides were significantly elevated, and HDL-cholesterol in plasma was significantly reduced. Free fraction and free fraction/total L-tryptophan ratio were decreased. The slope of the amplitude/stimulus intensity function of the N1/P2 component significantly increased in adolescents with MetS. Decrease of free fraction of L-tryptophan in plasma and increase of the slope of the N1/P2 component suggest a low brain serotonin tone. Cortex responses are regulated by serotonergic innervations and may show a different behavior in young patients with MetS. Therefore, the slope of the N1/P2 component along with the free fraction of L-tryptophan in plasma, indicate that in adolescents with MetS the state of serotonergic brain activity is depressed and possibly related to psychiatric disorders.

Dihydropteridine reductase activity in the brainstem of intrauterine growth-restricted rats.

The aim of this study was to determine whether intrauterine growth restriction produces an increase of dihydropteridine reductase activity as a compensatory mechanism that maintains the necessary concentration of cofactor, tetrahydrobiopterin, during accelerated brain serotonin biosynthesis. Intrauterine growth-restricted offspring and controls were used. On days 1, 10, 15 and 21 of life, the brainstem was dissected and l-tryptophan, serotonin, tryptophan-5-hydroxylase and dihydropteridine reductase activities were determined. Intrauterine growth-restricted pups showed a significant increase of l-tryptophan, 5-hydroxytryptamine, tryptophan-5-hydroxylase and also dihydropteridine activity in the brainstem in comparison to normal pups. These results confirm that intrauterine growth restriction produces an increase of serotonin biosynthesis in the brainstem. This is accompanied by an increase in dihydropteridine activity that appears to be a compensatory mechanism to maintain sufficient tetrahydrobiopterin for the donation of electrons during the accelerated synthesis of brain serotonin in intrauterine growth-restricted rats.

Caracterización de células serotoninérgicas en el tejido cardiaco fetal / Characterization of serotonergic cells in fetal heart tissue

Objetivo: Caracterizar morfológica y bioquímicamente células productoras de serotonina durante el desarrollo del tejido cardiaco. Material y métodos: Se utilizaron ratas gestantes de la cepa Wistar. A los días 10, 12, 16 y 20 de gestación se obtuvieron los fetos por cesárea, a los cuales se les disecaron los corazones, que se fijaron para los ensayos de inmunohistoquímica para triptófano- 5-hidroxilasa (Tph), además se efectuó Western Blot para la enzima; se determinó la concentración de serotonina y la actividad de Tph por cromatografía de líquidos de alta resolución. Los resultados fueron analizados mediante U de Mann-Whitney, aceptando un nivel de significación de p < 0.05. Resultados: En los cortes de corazón fetal, desde el día 10 de gestación se observaron células inmunorreactivas para Tph, con metacromasia en su interior. Fibras nerviosas inmunorreactivas para la misma enzima que hacen contacto probablemente con las células serotoninérgicas. La actividad enzimática y la concentración de la serotonina aumentaron con la edad gestacional, además, se encontró la proteína enzimática por Western- Blot en el corazón fetal de 16 días de gestación. Conclusiones: Se demostró la presencia de células productoras de serotonina en el miocardio fetal, cuyo fenotipo corresponde a mastocitos cardiacos, lo cual sugiere que la serotonina puede ser importante en el desarrollo del tejido cardiaco y que también participa en los mecanismos fisiopatológicos de los defectos cardiacos estructurales o en la predisposición a enfermedades cardiovasculares en el adulto.

BACKGROUND: We undertook this study to present biochemical and morphological characterization of serotonergic cells during fetal heart development. METHODS: Wistar rats (10, 12, 16 and 20 days of gestation) were used. After obtaining the fetuses by Cesarean section, the hearts were removed and fixed for immunohistochemical assay of tryptophan-5-hydroxylase (Tph), in addition to Western blot for enzyme. Serotonin concentration and Tph were also evaluated with high-performance liquid chromatography. Results were analyzed using Mann-Whitney U test with a significance level of p <0.05. RESULTS: Metachromatic cells immunoreactive for Tph were observed from day 10 of gestation. Nerve fibers were also labeled, apparently making contact with serotonergic cells. Tph activity was measurable and serotonin levels increased with gestational age. The presence of Tph protein was confirmed by Western blot on day 16. CONCLUSIONS: The present results support the existence of cells located in the fetal myocardium, capable of producing serotonin whose phenotype belongs to cardiac mast cells. Their presence in this tissue strongly suggests that serotonin may play a key role in normal and abnormal development of cardiac tissue.

Characterization of serotonergic cells in fetal heart tissue.

BACKGROUND: We undertook this study to present biochemical and morphological characterization of serotonergic cells during fetal heart development. METHODS: Wistar rats (10, 12, 16 and 20 days of gestation) were used. After obtaining the fetuses by Cesarean section, the hearts were removed and fixed for immunohistochemical assay of tryptophan-5-hydroxylase (Tph), in addition to Western blot for enzyme. Serotonin concentration and Tph were also evaluated with high-performance liquid chromatography. Results were analyzed using Mann-Whitney U test with a significance level of p <0.05. RESULTS: Metachromatic cells immunoreactive for Tph were observed from day 10 of gestation. Nerve fibers were also labeled, apparently making contact with serotonergic cells. Tph activity was measurable and serotonin levels increased with gestational age. The presence of Tph protein was confirmed by Western blot on day 16. CONCLUSIONS: The present results support the existence of cells located in the fetal myocardium, capable of producing serotonin whose phenotype belongs to cardiac mast cells. Their presence in this tissue strongly suggests that serotonin may play a key role in normal and abnormal development of cardiac tissue.