RESUMEN
AIMS: Perinatal asphyxia is one of the major causes of neonatal death at birth. Survivors can progress but often suffer from long-term sequelae. We aim to determine the effects of perinatal asphyxia on mitochondrial dynamics and whether mesenchymal stem cell secretome (MSC-S) treatment can alleviate the deleterious effects. MATERIALS AND METHODS: Animals were subjected to 21 min of asphyxia at the time of delivery. MSC-S or vehicle was intranasally administered 2 h post-delivery. Mitochondrial mass (D-loop, qPCR), mitochondrial dynamics proteins (Drp1, Fis1 and OPA1, Western blot), mitochondrial dynamics (TOMM20, Immunofluorescence), as well as mitochondrial membrane potential (ΔΨm) (Safranin O) were evaluated at P1 and P7 in the hippocampus. KEY FINDINGS: Perinatal asphyxia increased levels of mitochondrial dynamics proteins Drp1 and S-OPA1 at P1 and Fis1 at P7. Mitochondrial density and mass were decreased at P1. Perinatal asphyxia induced sex-specific differences, with increased L-OPA1 in females at P7 and increased mitochondria circularity. In males, asphyxia-exposed animals exhibited a reduced ΔΨm at P7. MSC-S treatment normalised levels of mitochondrial dynamics proteins involved in fission. SIGNIFICANCE: This study provides novel insights into the effects of perinatal asphyxia on mitochondrial dynamics in the developing brain and on the therapeutic opportunities provided by mesenchymal stem cell secretome treatment. It also highlights on the relevance of considering sex as a biological variable in perinatal brain injury and therapy development. These findings contribute to the development of targeted, personalised therapies for infants affected by perinatal asphyxia.
Asunto(s)
Hipocampo , Células Madre Mesenquimatosas , Mitocondrias , Dinámicas Mitocondriales , Animales , Hipocampo/metabolismo , Hipocampo/patología , Femenino , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratas , Mitocondrias/metabolismo , Asfixia Neonatal/terapia , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patología , Animales Recién Nacidos , Trasplante de Células Madre Mesenquimatosas/métodos , Potencial de la Membrana Mitocondrial , Ratas Sprague-DawleyRESUMEN
We have recently reported that global perinatal asphyxia (PA) induces a regionally sustained increase in oxidized glutathione (GSSG) levels and GSSG/GSH ratio, a decrease in tissue-reducing capacity, a decrease in catalase activity, and an increase in apoptotic caspase-3-dependent cell death in rat neonatal brain up to 14 postnatal days, indicating a long-term impairment in redox homeostasis. In the present study, we evaluated whether the increase in GSSG/GSH ratio observed in hippocampus involves changes in glutathione reductase (GR) and glutathione peroxidase (GPx) activity, the enzymes reducing glutathione disulfide (GSSG) and hydroperoxides, respectively, as well as catalase, the enzyme protecting against peroxidation. The study also evaluated whether there is a shift in the metabolism towards the penthose phosphate pathway (PPP), by measuring TIGAR, the TP53-inducible glycolysis and apoptosis regulator, associated with delayed cell death, further monitoring calpain activity, involved in bax-dependent cell death, and XRCC1, a scaffolding protein interacting with genome sentinel proteins. Global PA was induced by immersing fetus-containing uterine horns removed by a cesarean section from on term rat dams into a water bath at 37 °C for 21 min. Asphyxia-exposed and sibling cesarean-delivered fetuses were manually resuscitated and nurtured by surrogate dams. Animals were euthanized at postnatal (P) days 1 or 14, dissecting samples from hippocampus to be assayed for glutathione, GR, GPx (all by spectrophotometry), catalase (Western blots and ELISA), TIGAR (Western blots), calpain (fluorescence), and XRCC1 (Western blots). One hour after delivery, asphyxia-exposed and control neonates were injected with either 100 µl saline or 0.8 mmol/kg nicotinamide, i.p., shown to protect from the short- and long-term consequences of PA. It was found that global PA produced (i) a sustained increase of GSSG levels and GSSG/GSH ratio at P1 and P14; (ii) a decrease of GR, GPx, and catalase activity at P1 and P14; (iii) a decrease at P1, followed by an increase at P14 of TIGAR levels; (iv) an increase of calpain activity at P14; and (v) an increase of XRCC1 levels, but only at P1. (vi) Nicotinamide prevented the effect of PA on GSSG levels and GSSG/GSH ratio, and on GR, GPx, and catalase activity, also on increased TIGAR levels and calpain activity observed at P14. The present study demonstrates that the long-term impaired redox homeostasis observed in the hippocampus of rats subjected to global PA implies changes in GR, GPx, and catalase, and a shift towards PPP, as indicated by an increase of TIGAR levels at P14.
Asunto(s)
Asfixia Neonatal/complicaciones , Glutatión/metabolismo , Hipocampo/metabolismo , Niacinamida/farmacología , Estrés Oxidativo , Vía de Pentosa Fosfato , Animales , Asfixia Neonatal/metabolismo , Catalasa/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Homeostasis/efectos de los fármacos , Redes y Vías Metabólicas , Estrés Oxidativo/efectos de los fármacos , Vía de Pentosa Fosfato/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Ratas WistarRESUMEN
The hypothesis of enhanced vulnerability following perinatal asphyxia was investigated with a protocol combining in vivo and in vitro experiments. Asphyxia-exposed (AS) (by 21 min water immersion of foetuses containing uterine horns) and caesarean-delivered control (CS) rat neonates were used at P2-3 for preparing triple organotypic cultures (substantia nigra, neostriatum and neocortex). At DIV 18, cultures were exposed to different concentrations of H2O2 (0.25-45 mM), added to the culture medium for 18 h. After a 48-h recovery period, the cultures were either assessed for cell viability or for neurochemical phenotype by confocal microscopy. Energy metabolism (ADP/ATP ratio), oxidative stress (GSH/GSSG) and a modified ferric reducing/antioxidant power assay were applied to homogenates of parallel culture series. In CS cultures, the number of dying cells was similar in substantia nigra, neostriatum and neocortex, but it was several times increased in AS cultures evaluated under the same conditions. A H2O2 challenge led to a concentration-dependent increase in cell death (>fourfold after 0.25 mM of H2O2) in CS cultures. In AS cultures, a significant increase in cell death was only observed after 0.5 mM of H2O2. At higher than 1 mM of H2O2 (up to 45 mM), cell death increased several times in all cultures, but the effect was still more prominent in CS than in AS cultures. The cell phenotype of dying/alive cells was investigated in formalin-fixed cultures exposed to 0 or 1 mM of H2O2, co-labelling for TUNEL (apoptosis), MAP-2 (neuronal phenotype), GFAP (astroglial phenotype) and TH (tyrosine hydroxylase; for dopamine phenotype), counterstaining for DAPI (nuclear staining), also evaluating the effect of a single dose of nicotinamide (0.8 nmol/kg, i.p. injected in 100 µL, 60 min after delivery). Perinatal asphyxia produced a significant increase in the number of DAPI/TUNEL cells/mm3, in substantia nigra and neostriatum. One millimolar of H202 increased the number of DAPI/TUNEL cells/mm3 by ≈twofold in all regions of CS and AS cultures, an effect that was prevented by neonatal nicotinamide treatment. In substantia nigra, the number of MAP-2/TH-positive cells/mm3 was decreased in AS compared to CS cultures, also by 1 mM of H202, both in CS and AS cultures, prevented by nicotinamide. In agreement, the number of MAP-2/TUNEL-positive cells/mm3 was increased by 1 mM H2O2, both in CS (twofold) and AS (threefold) cultures, prevented by nicotinamide. The number of MAP-2/TH/TUNEL-positive cells/mm3 was only increased in CS (>threefold), but not in AS (1.3-fold) cultures. No TH labelling was observed in neostriatum, but 1 mM of H2O2 produced a strong increase in the number of MAP-2/TUNEL-positive cells/mm3, both in CS (>2.9-fold) and AS (>fourfold), decreased by nicotinamide. In neocortex, H2O2 increased the number of MAP-2/TUNEL-positive cells/mm3, both in CS and AS cultures (≈threefold), decreased by nicotinamide. The ADP/ATP ratio was increased in AS culture homogenates (>sixfold), compared to CS homogenates, increased by 1 mM of H202, both in CS and AS homogenates. The GSH/GSSG ratio was significantly decreased in AS, compared to CS cultures. One millimolar of H2O2 decreased that ratio in CS and AS homogenates. The present results demonstrate that perinatal asphyxia induces long-term changes in metabolic pathways related to energy and oxidative stress, priming cell vulnerability with both neuronal and glial phenotype. The observed effects were region dependent, being the substantia nigra particularly prone to cell death. Nicotinamide administration in vivo prevented the deleterious effects observed after perinatal asphyxia in vitro, a suitable pharmacological strategy against the deleterious consequences of perinatal asphyxia.
Asunto(s)
Asfixia Neonatal/tratamiento farmacológico , Neocórtex/efectos de los fármacos , Neostriado/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Niacinamida/farmacología , Sustancia Negra/efectos de los fármacos , Animales , Animales Recién Nacidos , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Femenino , Peróxido de Hidrógeno/metabolismo , Masculino , Neocórtex/metabolismo , Neocórtex/patología , Neostriado/metabolismo , Neostriado/patología , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas Wistar , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Perinatal asphyxia (PA) is associated to delayed cell death, affecting neurocircuitries of basal ganglia and hippocampus, and long-term neuropsychiatric disabilities. Several compensatory mechanisms have been suggested to take place, including cell proliferation and neurogenesis. There is evidence that PA can increase postnatal neurogenesis in hippocampus and subventricular zone (SVZ), modulated by dopamine, by still unclear mechanisms. We have studied here the effect of selective dopamine receptor agonists on cell death, cell proliferation and neurogenesis in organotypic cultures from control and asphyxia-exposed rats. Hippocampus and SVZ sampled at 1-3 postnatal days were cultured for 20-21 days. At day in vitro (DIV) 19, cultures were treated either with SKF38393 (10 and 100 µM, a D1 agonist), quinpirole (10 µM, a D2 agonist) or sulpiride (10 µM, a D2 antagonist) + quinpirole (10 µM) and BrdU (10 µM, a mitosis marker) for 24 h. At DIV 20-21, cultures were processed for immunocytochemistry for microtubule-associated protein-2 (MAP-2, a neuronal marker), and BrdU, evaluated by confocal microscopy. Some cultures were analysed for cell viability at DIV 20-21 (LIVE/DEAD kit). PA increased cell death, cell proliferation and neurogenesis in hippocampus and SVZ cultures. The increase in cell death, but not in cell proliferation, was inhibited by both SKF38393 and quinpirole treatment. Neurogenesis was increased by quinpirole, but only in hippocampus, in cultures from both asphyxia-exposed and control-animals, effect that was antagonised by sulpiride, leading to the conclusion that dopamine modulates neurogenesis in hippocampus, mainly via D2 receptors.
Asunto(s)
Asfixia Neonatal/tratamiento farmacológico , Agonistas de Dopamina/farmacología , Hipocampo/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , 2,3,4,5-Tetrahidro-7,8-dihidroxi-1-fenil-1H-3-benzazepina/farmacología , Animales , Animales Recién Nacidos , Asfixia Neonatal/metabolismo , Asfixia Neonatal/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Antagonistas de Dopamina/farmacología , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Neurogénesis/fisiología , Quinpirol/farmacología , Ratas Wistar , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Nicho de Células Madre/efectos de los fármacos , Nicho de Células Madre/fisiología , Sulpirida/farmacología , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Motivation is an essential aspect in the training process of medical students. The association that motivation can have with learning self-regulation is of utmost importance for the design of curriculum, teaching methods and evaluation. AIM: To describe the motivational aspects of self-directed learning among medical students from a traditional Chilean University. MATERIAL AND METHODS: A qualitative, descriptive study based on grounded theory of Strauss and Corbin. Twenty 4th and 5th year medical students were selected using a maximum variation sampling technique. After obtaining an informed consent, semi-structured interviews and field notes were carried out. Data were analyzed to the level of open coding through Atlas-ti 7.5.2. RESULTS: From the student point of view, personal motivational aspects are linked to the search for information, constant updating, the perception of the physician-patient relationship and interest in subject matters. From the scope of teachers, a main issue is related to their ability to motivate students to develop independent study skills. CONCLUSIONS: Personal motivational aspects facilitate the development of independent study skills, specifically in the search of information. The role of teachers is crucial in promoting these skills and the perception of medical students from their learning process.
Asunto(s)
Educación de Pregrado en Medicina , Aprendizaje , Motivación , Estudiantes de Medicina/psicología , Chile , Humanos , Investigación CualitativaRESUMEN
Background: Motivation is an essential aspect in the training process of medical students. The association that motivation can have with learning self-regulation is of utmost importance for the design of curriculum, teaching methods and evaluation. Aim: To describe the motivational aspects of self-directed learning among medical students from a traditional Chilean University. Material and Methods: A qualitative, descriptive study based on grounded theory of Strauss and Corbin. Twenty 4th and 5th year medical students were selected using a maximum variation sampling technique. After obtaining an informed consent, semi-structured interviews and field notes were carried out. Data were analyzed to the level of open coding through Atlas-ti 7.5.2. Results: From the student point of view, personal motivational aspects are linked to the search for information, constant updating, the perception of the physician-patient relationship and interest in subject matters. From the scope of teachers, a main issue is related to their ability to motivate students to develop independent study skills. Conclusions: Personal motivational aspects facilitate the development of independent study skills, specifically in the search of information. The role of teachers is crucial in promoting these skills and the perception of medical students from their learning process.
Asunto(s)
Humanos , Estudiantes de Medicina/psicología , Educación de Pregrado en Medicina , Aprendizaje , Motivación , Chile , Investigación CualitativaRESUMEN
BACKGROUND: Self-directed learning is a skill that must be taught and evaluated in future physicians. AIM: To analyze the association between self-directed learning, self-esteem, self-efficacy, time management and academic commitment among medical students. MATERIAL AND METHODS: The self-directed learning, Rosemberg self-esteem, general self- efficacy, time management and Utrecht work engagement scales were applied to 297 first year medical students. RESULTS: A multiple regression analysis showed a significant association between self-efficacy, time management and academic commitment with self-directed learning. Self-esteem and satisfaction with studies did not enter in the model. CONCLUSIONS: self-esteem, academic commitment and a good time management were associated with self-directed learning in these students.
Asunto(s)
Evaluación Educacional , Aprendizaje , Autoimagen , Autoeficacia , Estudiantes de Medicina/psicología , Administración del Tiempo/psicología , Adolescente , Adulto , Estudios Transversales , Educación de Pregrado en Medicina/métodos , Femenino , Humanos , Masculino , Satisfacción Personal , Análisis de Regresión , Encuestas y Cuestionarios , Confianza/psicología , Adulto JovenRESUMEN
Background: Self-directed learning is a skill that must be taught and evaluated in future physicians. Aim: To analyze the association between self-directed learning, self-esteem, self-efficacy, time management and academic commitment among medical students. Material and methods: The self-directed learning, Rosemberg self-esteem, general self- efficacy, time management and Utrecht work engagement scales were applied to 297 first year medical students. Results: A multiple regression analysis showed a significant association between self-efficacy, time management and academic commitment with self-directed learning. Self-esteem and satisfaction with studies did not enter in the model. Conclusions: self-esteem, academic commitment and a good time management were associated with self-directed learning in these students.
Asunto(s)
Adolescente , Adulto , Femenino , Humanos , Masculino , Adulto Joven , Evaluación Educacional , Aprendizaje , Autoimagen , Autoeficacia , Estudiantes de Medicina/psicología , Administración del Tiempo/psicología , Estudios Transversales , Educación de Pregrado en Medicina/métodos , Satisfacción Personal , Análisis de Regresión , Encuestas y Cuestionarios , Confianza/psicologíaRESUMEN
BACKGROUND: Understanding how autonomous students are capable of regulating their own learning process is essential to develop self-directed teaching methods. AIM: To understand how self-directed medical students approach learning in medical schools at University of Concepción, Chile. MATERIAL AND METHODS: A qualitative and descriptive study, performed according to Grounded Theory guidelines, following Strauss & Corbin was performed. Twenty medical students were selected by the maximum variation sampling method. The data collection technique was carried out by a semi-structured thematic interview. Students were interviewed by researchers after an informed consent procedure. Data were analyzed by the open coding method using Atlas-ti 7.5.2 software. RESULTS: Self-directed learners were characterized by being good planners and managing their time correctly. Students performed a diligent selection of contents to study based on reliable literature sources, theoretical relevance and type of evaluation. They also emphasized the discussion of clinical cases, where theoretical contents can be applied. This modality allows them to gain a global view of theoretical contents, to verbalize knowledge and to obtain a learning feedback. CONCLUSIONS: The learning process of autonomous students is intentional and planned.
Asunto(s)
Educación de Pregrado en Medicina/estadística & datos numéricos , Aprendizaje , Estudiantes de Medicina/estadística & datos numéricos , Adulto , Chile , Femenino , Humanos , Masculino , Investigación Cualitativa , Facultades de MedicinaRESUMEN
Oxygen interruption leads to death when re-oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of biochemical events for restoring function at the cost of improper homeostasis. The effects observed long after perinatal asphyxia (PA) have been explained by over-expression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD(+) during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a therapeutic strategy. We studied the effects of nicotinamide and theophylline on PARP-1 activity assayed in brain and peripheral (heart) rat tissue 1-24 h after birth, as well as on changes in behaviour and monoamine neurotransmission in adult rats. PA was induced by immersing rat foetuses into a water bath for 0 or 21 min. After resuscitation, the pups were treated with nicotinamide (0.8 mmol/kg, i.p.), theophylline (0.14 mmol/kg, i.p.) or saline (0.9% NaCl) and nurtured by surrogate dams, pending behavioural and microdialysis experiments, or euthanised after birth for assaying PARP-1 activity. To estimate the in vivo distribution of a single dose of nicotinamide or theophylline into brain and peripheral compartment, a series of animals were implanted with microdialysis probes, one into the brain and other subcutaneously, 1 h after birth, assaying the drugs with a HPLC-UV system. Nicotinamide, but not theophylline prevented the long-term effects induced by PA. Only nicotinamide produced a consistent decrease in PARP-1 activity in brain and heart, whether assayed in control or asphyxia-exposed pups. The present results support the idea that the long-term effects induced by PA imply PARP-1 over-activation.
Asunto(s)
Asfixia/metabolismo , Conducta Animal/efectos de los fármacos , Niacinamida/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Teofilina/farmacología , Complejo Vitamínico B/farmacología , Animales , Animales Recién Nacidos/metabolismo , Asfixia Neonatal/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Corazón/efectos de los fármacos , Humanos , Recién Nacido , Microdiálisis , Modelos Animales , Miocardio/metabolismo , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Asphyxia during delivery produces long-term deficits in brain development, including hippocampus. We investigated hippocampal plasticity after perinatal asphyxia, measuring postnatal apoptosis and neurogenesis. Asphyxia was performed by immersing rat fetuses with uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Caesarean-delivered pups were used as controls. The animals were euthanized 1 week or 1 month after birth. Apoptotic nuclear morphology and DNA breaks were assessed by Hoechst and TUNEL assays. Neurogenesis was estimated by bromodeoxyuridine/MAP-2 immunocytochemistry, and the levels and expression of proteins related to apoptosis and cell proliferation were measured by Western blots and in situ hybridization, respectively. There was an increase of apoptosis in CA1, CA3, and dentate gyrus (DG) and cell proliferation and neurogenesis in CA1, DG, and hilus regions of hippocampus 1 week after asphyxia. The increase of apoptosis in CA3 and cell proliferation in the suprapyramidal band of DG was still observed 1 month following asphyxia. There was an increase of BAD, BCL-2, ERK2, and bFGF levels in whole hippocampus and bFGF expression in CA1 and CA2 and hilus at P7 and P30. There was a concomitant decrease of phosphorylated-BAD (Ser112) levels. The increase of BAD levels supports the idea of delayed cell death after perinatal asphyxia, whereas the increases of BCL-2, ERK2, and bFGF levels suggest the activation of neuroprotective and repair pathways. In conclusion, perinatal asphyxia induces short- and long-term regionally specific plastic changes, including delayed cell death and neurogenesis, involving pro- and antiapoptotic as well as mitogenic proteins, favoring hippocampal functional recovery.
Asunto(s)
Animales Recién Nacidos/fisiología , Apoptosis/fisiología , Asfixia/patología , Diferenciación Celular/fisiología , Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Animales , Animales Recién Nacidos/anatomía & histología , Asfixia/genética , Asfixia/metabolismo , Proliferación Celular , Femenino , Hipocampo/citología , Neuronas/citología , Embarazo , Ratas , Ratas WistarRESUMEN
There is clinical and experimental evidence indicating that neurocircuitries of the hippocampus are vulnerable to hypoxia/ischemia occurring at birth, inducing, upon re-oxygenation/re-circulation, delayed neuronal death, but also compensatory mechanisms, including neurogenesis. In the present report, perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath at 37 degrees C for 20 min. Some pups were delivered immediately after the hysterectomy to be used as non-asphyxiated caesarean-delivered controls. The pups were sacrificed after seven days for preparing organotypic hippocampal cultures. The cultures were grown on a coverslip in a medium-containing culture tube inserted in a hole of a roller device standing on the internal area of a cell incubator at 35 degrees C, 10% CO2. At days in vitro (DIV) 25-27, cultures were fixed for assaying cell proliferation and neuronal phenotype with antibodies against 5-bromo-2'deoxyuridine (BrdU) and microtubule associated protein-2 (MAP-2), respectively. Confocal microscopy revealed that there was a 2-fold increase of BrdU-positive, but a 40% decrease of MAP-2-positive cells/mm3 in cultures from asphyxia-exposed, compared to that from control animals. Approximately 30% of BrdU-positive cells were also positive for MAP-2 (approximately 4800 cells), mainly seen in the dentate gyrus of the hippocampus, demonstrating a 3-fold increase of postnatal neurogenesis, when the total amount of double-labelled cells seen in cultures from asphyxia-exposed animals is compared to that from control animals.
Asunto(s)
Asfixia/fisiopatología , Hipocampo/fisiopatología , Neuronas/fisiología , Animales , Animales Recién Nacidos , Neuronas/citología , Técnicas de Cultivo de Órganos , Fosfoproteínas Fosfatasas/efectos de los fármacos , Fosfoproteínas Fosfatasas/metabolismo , RatasRESUMEN
We have investigated the idea that nicotinamide, a non-selective inhibitor of the sentinel enzyme Poly(ADP-ribose) polymerase-I (PARP-1), provides neuroprotection against the long-term neurological changes induced by perinatal asphyxia. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. The effect of perinatal asphyxia on neurocircuitry development was studied in vitro with organotypic cultures from substantia nigra, neostriatum and neocortex, platted on a coverslip 3 days after birth. After approximately one month in vitro (DIV 25), the cultures were treated for immunocytochemistry to characterise neuronal phenotype with markers against the N-methyl-D-aspartate receptor subunit 1 (NR1), the dopamine pacemaker enzyme tyrosine hydroxylase (TH), and nitric oxide synthase (NOS), the enzyme regulating the bioavailability of NO. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyctic and caesarean-delivered pups 24, 48 and 72 h after birth. It was found that nicotinamide treatment prevented the effect of perinatal asphyxia on several neuronal parameters, including TH- and NOS-positive neurite atrophy and NOS-positive neuronal loss; supporting the idea that nicotinamide constitutes a therapeutic alternative for the effects produced by sustained energy-failure conditions, as occurring during perinatal asphyxia.
Asunto(s)
Asfixia Neonatal/metabolismo , Asfixia Neonatal/patología , Ganglios Basales/metabolismo , Ganglios Basales/patología , Fármacos Neuroprotectores/farmacología , Niacinamida/farmacología , Animales , Asfixia Neonatal/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Humanos , Recién Nacido , Neocórtex/metabolismo , Neocórtex/patología , Neostriado/metabolismo , Neostriado/patología , Neuritas/patología , Fármacos Neuroprotectores/administración & dosificación , Niacinamida/administración & dosificación , Óxido Nítrico Sintasa/metabolismo , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The present report summarizes studies combining an in vivo and in vitro approach, where asphyxia is induced in vivo at delivery time of Wistar rats, and the long term effects on hippocampus neurocircuitry are investigated in vitro with organotypic cultures plated at postnatal day seven. The cultures preserved hippocampus layering and regional subdivisions shown in vivo, and only few dying cells were observed when assayed with a viability test at day in vitro 27. When properly fixed, cultures from asphyxia-exposed animals showed a decreased amount of microtubule-associated protein-2 immunocytochemically positive cells (approximately 30%), as compared with that from controls. The decrease in microtubule-associated protein-2 immunocytochemistry was particularly prominent in Ammon's horn 1 and dentate gyrus regions (approximately 40%). 5-Bromo-2'deoxyuridine labeling revealed a two-fold increase in cellular proliferation in cultures from asphyxia-exposed, compared with that from control animals. Furthermore, confocal microscopy and quantification using the optical disector technique demonstrated that in cultures from asphyxia-exposed animals approximately 30% of 5-bromo-2'deoxyuridine-positive cells were also positive to microtubule-associated protein-2, a marker for neuronal phenotype. That proportion was approximately 20% in cultures from control animals. Glial fibrillary acidic protein-immunocytochemistry and Fast Red nuclear staining revealed that the core of the hippocampus culture was surrounded by a well-developed network of glial fibrillary acidic protein-positive cells and glial fibrillary acidic protein-processes providing an apparent protective shield around the hippocampus. That shield was less developed in cultures from asphyxia-exposed animals. The increased mitotic activity observed in this study suggests a compensatory mechanism for the long-term impairment induced by perinatal asphyxia, although it is not clear yet if that mechanism leads to neurogenesis, astrogliogenesis, or to further apoptosis.
Asunto(s)
Asfixia/fisiopatología , Proliferación Celular , Hipocampo/citología , Neuronas/citología , Fenotipo , Animales , Animales Recién Nacidos , Compuestos Azo/metabolismo , Bromodesoxiuridina/metabolismo , Recuento de Células/métodos , Supervivencia Celular , Embrión de Mamíferos , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Microscopía Confocal/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas WistarRESUMEN
The intraspecific variability of Triatoma dimidiata Latreille, a major vector of Chagas disease, was studied in four departments of Guatemala. Insects were collected from either domestic and sylvatic habitats, and their cuticular hydrocarbon pattern and head morphology were analyzed using ordination and classification techniques. A significant discrimination was obtained both with morphometric and hydrocarbon analyses. Insects from northern departments were easily differentiated from southern conspecifics. Distinctive hydrocarbon pattern and head shape were detected for insects collected from caves in the north central region of the country, posing concern about their taxonomic status.
Asunto(s)
Reduviidae/anatomía & histología , Reduviidae/química , Animales , Enfermedad de Chagas/transmisión , Cromatografía de Gases , Femenino , Guatemala , Hidrocarburos/análisis , Insectos Vectores , Masculino , Especificidad de la EspecieRESUMEN
The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after three days for preparing organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features and cell viability were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for immunocytochemistry using antibodies against the N-methyl-D-aspartate receptor subunit 1 (NR1) and tyrosine hydroxylase (TH). While in vitro survival was similar in cultures from both asphyxiated and control animals, differences were observed when the neuronal phenotype was assessed. Compared to controls, the total number of NR1-positive neurons in substantia nigra, as well as the number of secondary to higher level branching of TH-positive neurites from asphyxiated pups were decreased, illustrating the vulnerability of the dopaminergic systems to perinatal asphyxia.
Asunto(s)
Asfixia/patología , Encéfalo/metabolismo , Feto/patología , Neuritas/patología , Animales , Asfixia/metabolismo , Encéfalo/patología , Supervivencia Celular , Femenino , Feto/metabolismo , Inmunohistoquímica/métodos , Masculino , Neuritas/metabolismo , Técnicas de Cultivo de Órganos/métodos , Embarazo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after 3 days to prepare organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for histochemistry using fast red for cell nucleus labelling and antibodies against tyrosine hydroxylase for dopaminergic neurons. Compared to controls, cultures from asphyxiated pups revealed a diminished integration quantified during 21 DIV. After immunocytochemistry and camera lucida reconstruction, tyrosine hydroxylase-positive neurons showed a decreased number of neurites from secondary and higher level branching, demonstrating a vulnerability of the dopaminergic systems after perinatal asphyxia.
Asunto(s)
Asfixia/fisiopatología , Neocórtex/crecimiento & desarrollo , Neostriado/crecimiento & desarrollo , Neuritas/fisiología , Sustancia Negra/crecimiento & desarrollo , Animales , Animales Recién Nacidos , Dopamina/fisiología , Femenino , Vías Nerviosas/crecimiento & desarrollo , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas WistarRESUMEN
Asphyxia during birth can cause gross brain damage, but also subtle perturbations expressed as biochemical or motor deficits with late onset in life. Thus, it has been shown that brain dopamine levels can be increased or decreased depending upon the severity of the insult, and the region where the levels are determined. In this study, perinatal asphyxia was evoked by immersing pup-containing uterus horns removed by hysterectomy in a water bath at 37 degrees C for various periods of time from 0 to 20 min. After the insult, the pups were delivered, given to surrogate mothers, treated with nicotinamide, further observed and finally, 4 weeks later, killed for monoamine biochemistry of tissue samples taken from substantia nigra, neostriatum and nucleus accumbens. The main effect of perinatal asphyxia was a decrease in dopamine and metabolite levels in nucleus accumbens, and a paradoxical increase in the substantia nigra. Nicotinamide (100 mg/kg i.p., once a day for 3 days, beginning 24 h after the perinatal asphyctic insult) prevented the effect of asphyxia in nucleus accumbens. Furthermore, striatal dopamine levels were increased by nicotinamide in asphyctic animals. No apparent changes were observed in substantia nigra. A prominent unexpected effect of perinatal asphyxia alone was on the levels of the metabolite of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid (5-HIAA), which were increased in substantia nigra and decreased in both neostriatum and accumbens. However, nicotinamide increased 5-HIAA levels in all regions, which appeared to be related to the extent of the asphyctic insult. These results suggest that nicotinamide is a useful treatment against the long-term consequences produced by perinatal asphyxia on brain monoamine systems, and that there is a therapeutic window following the insult, providing a therapeutic opportunity to protect the brain.
Asunto(s)
Asfixia/tratamiento farmacológico , Asfixia/metabolismo , Ganglios Basales/metabolismo , Monoaminas Biogénicas/metabolismo , Niacinamida/farmacología , Animales , Animales Recién Nacidos , Ganglios Basales/efectos de los fármacos , Cesárea/métodos , Femenino , Niacinamida/uso terapéutico , Embarazo , RatasRESUMEN
The frequency of oestrus cycles in female mice was significantly reduced by the implantation of a pellet of subcutaneous ketoconazole (50 mg every 6 days). The effect was more pronounced after 22 days than after 13 days and it was probably related with the progressive reduction in steroid synthesis induced by the inhibition of key steroidogenic P450 cytochromes by the drug. In addition, the influence of ketoconazole on the incidence of seizures after the administration of intraperitoneal pentylenetetrazol was evaluated in female mice. Pentylenetetrazol produced a higher percentage of seizures during dioestrus than during oestrus. Pretreatment with ketoconazole significantly increased the percentage of animals with induced seizures in oestrus but not in dioestrus as compared to controls, probably through reduced progesterone levels. The reduced seizure threshold confirm the modulatory role exerted by progesterone on central nervous system excitability, and may be relevant in epileptic patients undergoing antifungal therapy.
Asunto(s)
Antifúngicos/farmacología , Ciclo Estral/efectos de los fármacos , Cetoconazol/farmacología , Convulsiones/fisiopatología , Animales , Convulsivantes/farmacología , Femenino , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos , Pentilenotetrazol/farmacología , Convulsiones/inducido químicamente , Factores de TiempoRESUMEN
1. The analgesic effect of clonixinate of L-lysine (Clx) in the nociceptive C-fiber reflex in rat and in the writhing test in mice is reported. 2. Clx was administered by three routes, i.v., i.t. and i.c.v., inducing a dose-dependent antinociception. 3. The antinociceptive effect of Clx was 40-45% with respect to the control integration values in the nociceptive C-fiber reflex method. 4. The writhing test yielded ED50 values (mg/kg) of 12.0 +/- 1.3 (i.p.), 1.8 +/- 0.2 (i.t.) and 0.9 +/- 0.1 (i.c.v.) for Clx administration. 5. Ondansetron was not able to antagonize the antinociception response of Clx in the algesiometric tests used. 6. Chlorophenilbiguanide did not produce any significative change in the analgesic effect of Clx in the nociceptive C-fiber reflex method. 7. It is suggested that the mechanism of action of the central analgesia of Clx is not mediated by 5-HT3 subtype receptors.