Protective Effect of Tilia americana var. mexicana Against Kainic Acid-induced Damage in Brain, Liver, and Kidney: Behavioral and Biochemical Changes.

Tiliaamericana var. mexicana (Tilia) possesses anticonvulsant, antioxidant, neuroprotective, and hepatoprotective activities. The spectrum of anticonvulsant activity in status epilepticus models has not been sufficiently explored. We evaluated the effects of ethyl acetate (EAc), and methanol (ME) extracts on kainic acid (KA)-induced seizures by measuring rats'behavior (severity and latency) and lipoperoxidation in different brain areas (cerebellum, brain hemispheres, cortex, and medulla), kidneys, and liver. Male Wistar rats were administered KA (10 mg/kg, i.p.) after three days of pretreatment with Tilia extract (100 mg/kg). The EAc and ME Tilia extracts significantly decreased the severity of phase 1 and phase 2 seizures, respectively. The ME Tilia extract increased the latency to seizure (27 ± 2 min) compared to the control (13 ± 2 min). The ME and EAc Tilia extracts significantly prevented the increased lipid peroxidation caused by KA-induced seizures in the cerebellum, brain hemispheres, cortex, medulla, liver, and kidneys. The vehicle olive oil (OO) also showed anticonvulsant effects, decreasing the severity of seizures to phase 3 and lipoperoxidation levels in the cerebellum, brain hemispheres, cortex, medulla, liver, and kidneys. The anticonvulsant activity of Tilia is mediated by antioxidant effects in central and systemic areas that involve synergistic interactions among the chemical constituents of these extracts (glucosides of quercetin and kaempferol), while vehicle OO showed the same effects, probably due to its constituent oleuropein.

Exposure to Sub-Lethal Doses of Permethrin Is Associated with Neurotoxicity: Changes in Bioenergetics, Redox Markers, Neuroinflammation and Morphology.

Permethrin (PERM) is a member of the class I family of synthetic pyrethroids. Human use has shown that it affects different systems, with wide health dysfunctions. Our aim was to determine bioenergetics, neuroinflammation and morphology changes, as redox markers after subacute exposure to PERM in rats. We used MDA determination, protein carbonyl assay, mitochondrial O2 consumption, expression of pro-inflammatory cytokines and a deep histopathological analysis of the hippocampus. PERM (150 mg/kg and 300 mg/kg body weight/day, o.v.) increased lipoperoxidation and carbonylated proteins in a dose-dependent manner in the brain regions. The activities of antioxidant enzymes glutathione peroxidase, reductase, S-transferase, catalase, and superoxide dismutase showed an increase in all the different brain areas, with dose-dependent effects in the cerebellum. Cytokine profiles (IL-1ß, IL-6 and TNF-α) increased in a dose-dependent manner in different brain tissues. Exposure to 150 mg/kg of permethrin induced degenerated and/or dead neurons in the rat hippocampus and induced mitochondrial uncoupling and reduction of oxidative phosphorylation and significantly decreased the respiratory parameters state 3-associated respiration in complex I and II. PERM exposure at low doses induces reactive oxygen species production and imbalance in the enzymatic antioxidant system, increases gene expression of pro-inflammatory interleukins, and could lead to cell damage mediated by mitochondrial functional impairment.

Ketogenic Diet Provided During Three Months Increases KCC2 Expression but Not NKCC1 in the Rat Dentate Gyrus.

Ketogenic diet, a high fat and low carbohydrate diet, has been used as a non-pharmacological treatment in refractory epilepsy since 1920. In recent years, it has demonstrated to be effective in the treatment of numerous neurological and non-neurological diseases. Some neurological and neuropsychiatric disorders are known to be caused by gamma-aminobutyric acid (GABA)-mediated neurotransmission dysfunction. The strength and polarity of GABA-mediated neurotransmission are determined by the intracellular chloride concentration, which in turn is regulated by cation-chloride cotransporters NKCC1 and KCC2. Currently, it is unknown if the effect of ketogenic diet is due to the modulation of these cotransporters. Thus, we analyzed the effect of a ketogenic diet on the cation-chloride cotransporters expression in the dentate gyrus. We estimated the total number of NKCC1 immunoreactive (NKCC1-IR) neuronal and glial cells by stereology and determined KCC2 labeling intensity by densitometry in the molecular and granule layers as well as in the hilus of dentate gyrus of rats fed with normal or ketogenic diet for 3 months. The results indicated that ketogenic diet provided during 3 months increased KCC2 expression, but not NKCC1 in the dentate gyrus of the rat. The significant increase of KCC2 expression could explain, at least in part, the beneficial effect of ketogenic diet in the diseases where the GABAergic system is altered by increasing its inhibitory efficiency.

Dose-Dependent Behavioral and Antioxidant Effects of Quercetin and Methanolic and Acetonic Extracts from Heterotheca inuloides on Several Rat Tissues following Kainic Acid-Induced Status Epilepticus.

Kainic acid (KA) has been used to study the neurotoxicity induced after status epilepticus (SE) due to activation of excitatory amino acids with neuronal damage. Medicinal plants can protect against damage caused by KA-induced SE; in particular, organic extracts of Heterotheca inuloides and its metabolite quercetin display antioxidant activity and act as hepatoprotective agents. However, it is unknown whether these properties can protect against the hyperexcitability underlying the damage caused by KA-induced SE. Our aim was to study the protective effects (with regard to behavior and antioxidant activity) of administration of natural products methanolic (ME) and acetonic (AE) extracts and quercetin (Q) from H. inuloides at doses of 30 mg/kg (ME30, AE30, and Q30 groups), 100 mg/kg (ME100, AE100, and Q100 groups), and 300 mg/kg (ME300, AE300, and Q300 groups) against damage in brain regions of male Wistar rats treated with KA. We found dose-dependent effects on behavioral and biochemical studies in the all-natural product groups vs. the control group, with decreases in seizure severity (Racine's scale) and increases in seizure latency (p < 0.05 in the ME100, AE100, Q100, and Q300 groups and p < 0.01 in the AE300 and ME300 groups); on lipid peroxidation and carbonylated proteins in all brain tissues (p < 0.0001); and on GPx, GR, CAT, and SOD activities with all the treatments vs. KA (p ≤ 0.001). In addition, there were strong negative correlations between carbonyl levels and latency in the group treated with KA and in the group treated with methanolic extract in the presence of KA (r = -0.9919, p = 0.0084). This evidence suggests that organic extracts and quercetin from H. inuloides exert anticonvulsant effects via direct scavenging of reactive oxygen species (ROS) and modulation of antioxidant enzyme activity.

Docosahexaenoic acid protection in a rotenone induced Parkinson's model: Prevention of tubulin and synaptophysin loss, but no association with mitochondrial function.

Rotenone, a classic mitochondrial complex I inhibitor, leads to dopaminergic neuronal death resulting in a Parkinson's-like-disease. Docosahexaenoic acid (DHA) has shown neuroprotective effects in other experimental models of Parkinson's disease, but its effect on the rotenone-induced parkinsonism is still unknown. We tested whether DHA in vivo exerts a neuroprotective effect on rotenone-induced parkinsonism and explored the mechanisms involved, including mitochondrial function and ultrastructure as well as the expression of tubulin and synaptophysin. We pretreated eighty male Wistar rats with DHA (35 mg/kg/day) for seven days and then administered rotenone for eight days. We then measured rearing behavior, number of dopaminergic neurons, tyrosine hydroxylase content, tubulin and synaptophysin expression, mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential, ATP production activity and mitochondrial ultrastructure. We found that in vivo DHA supply exerted a neuroprotective effect, evidenced by decreased dopaminergic neuron cell death. Although we detected rotenone induced mitochondrial ultrastructure alterations, these were not associated with mitochondrial dysfunction. Rotenone had no effect on mitochondrial complex I, respiratory control ratio, mitochondrial transmembrane potential or ATP production activity. DHA also prevented a rotenone-induced decrease in tubulin and synaptophysin expression. Our results support the neuroprotective effect of DHA on rotenone-induced parkinsonism, and a possible effect on early stage Parkinson's disease. This protective effect is not associated with mitochondrial function improvement, but rather with preventing loss of tubulin and synaptophysin, proteins relevant to synaptic transmission.

Status epilepticus: Using antioxidant agents as alternative therapies.

The epileptic state, or status epilepticus (SE), is the most serious situation manifested by individuals with epilepsy, and SE events can lead to neuronal damage. An understanding of the molecular, biochemical and physiopathological mechanisms involved in this type of neurological disease will enable the identification of specific central targets, through which novel agents may act and be useful as SE therapies. Currently, studies have focused on the association between oxidative stress and SE, the most severe epileptic condition. A number of these studies have suggested the use of antioxidant compounds as alternative therapies or adjuvant treatments for the epileptic state.

Overview of Nrf2 as Therapeutic Target in Epilepsy.

Oxidative stress is a biochemical state of imbalance in the production of reactive oxygen and nitrogen species and antioxidant defenses. It is involved in the physiopathology of degenerative and chronic neuronal disorders, such as epilepsy. Experimental evidence in humans and animals support the involvement of oxidative stress before and after seizures. In the past few years, research has increasingly focused on the molecular pathways of this process, such as that involving transcription factor nuclear factor E2-related factor 2 (Nrf2), which plays a central role in the regulation of antioxidant response elements (ARE) and modulates cellular redox status. The aim of this review is to present experimental evidence on the role of Nrf2 in this neurological disorder and to further determine the therapeutic impact of Nrf2 in epilepsy.

El pirofosfato de tiamina reduce el daño celular inducido por hipoxia en el cerebro de ratas neonatas / Thiamine pyrophosphate reduces hypoxia-induced cellular damage in neonatal rat brain

La encefalopatía por hipoxia es causa de discapacidad y requiere de nuevas estrategias terapéuticas. El pirofosfato de tiamina (PPT) es un cofactor esencial de enzimas fundamentales en el metabolismo de la glucosa, cuya disminución puede conducir a la falla en la síntesis de ATP y a la muerte celular. El objetivo de este estudio fue determinar si la administración de PPT, puede reducir el daño celular en un modelo de hipoxia neonatal en ratas. Animales de 11 días de edad fueron tratados con PPT (130 mg/kg) en dosis única o solución salina, una hora antes del protocolo de hipoxia o al término de ésta. Los cerebros fueron colectados para la evaluación del daño celular. Además, se tomaron muestras sanguíneas para evaluar los indicadores gasométricos de presión de dióxido de carbono (PaCO2) y de oxígeno (PaO2) en sangre arterial y pH. Los resultados muestran que la administración de PPT previa a la inducción de hipoxia, reduce el daño celular y restablece los indicadores gasométricos. Estos datos indican que el uso de PPT reduce el daño inducido por la hipoxia en animales neonatos.

Hypoxic encephalopathy is a leading cause of disability and requires new therapeutic strategies. Thiamine pyrophosphate (TPP) is an essential cofactor of fundamental enzymes involved in glucose metabolism. TPP reduction may lead to ATP synthesis failure and cell death. The objective of this study was to determine if TPP administration can reduce cellular damage in a model of neonatal hypoxia in rats. Eleven day old animals were treated with TPP (130 mg/kg) as a single dose or with saline solution one hour before the hypoxia protocol or after ending the protocol. The brains were collected to evaluate cellular damage. Blood samples were also collected to evaluate arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2) and acidity (pH). The results showed that TPP administration previous to hypoxia induction reduces cellular damage and reestablishes arterial blood gases. These data indicate that TPP use reduces the damage induced by hypoxia in neonatal animals.

The age-dependent change in olfactory periglomerular neuronal populations is not affected by interrupting subventricular neuroblast migration in adult rats.

The olfactory bulb (OB) is rich in the number and variety of neurotransmitter and neuropeptide containing cells, in particular in the glomerular layer. Several reports suggest that numbers of some periglomerular phenotypes could change depending on age. However, it is unclear whether the different classes of periglomerular interneurons are modified or are maintained stable throughout life. Thus, our first objective was to obtain the absolute number of cells belonging to the different periglomerular phenotypes at adulthood. On the other hand, the olfactory bulb is continously supplied with newly generated periglomerular neurons produced by stem cells located in the subventricular zone (SVZ) and rostral migratory stream. Previously, we demonstrated that the implantation of a physical barrier completely prevents SVZ neuroblast migration towards the OB. Then, another objective of this study was to evaluate whether stopping the continuous supply of SVZ neuroblasts modified the different periglomerular populations throughout time. In summary, we estimated the total number of TH-IR, CalB-IR, CalR-IR and GAD-IR cells in the OB glomerular layer at several time points in control and barrier implanted adult rats. In addition, we estimated the volume of glomerular, granular and complete OB. Our main finding was that the number of the four main periglomerular populations is age-dependent, even after impairment of subventricular neuroblast migration. Furthermore, we established that these changes do not correlate with changes in the volume of glomerular layer.

Time-place learning is altered by perinatal low-protein malnutrition in the adult rat.

Malnutrition produces changes in the central nervous system (CNS) of mammals during development, related to the intensity and timing of the malnutrition insult during the pre- or postnatal period. Protein malnutrition produces irreversible changes in hippocampal formation and some brain stem nuclei. The suprachiasmatic nucleus (SCN) is dramatically altered by low-protein diets during the gestational and perinatal periods. Also, it is known that circadian oscillators regulate physiological, behavioral, and cognitive processes and there is evidence that the time-place learning process exhibits a daily temporal distribution. The aim of this study was to determine the effects of chronic, prenatal, or postnatal malnutrition on daily patterns of the time-place learning process in the adult rat. Forty Sprague-Dawley male 90-day-old rats, were divided into four groups: 10 well nourished controls (Co), 10 chronically (CM), 10 prenatally malnourished (PrM), and 10 postnatally malnourished (PtM) rats. Efficiency in time-place learning was tested by using a behavioral T-maze. Each rat was assayed for 10 trials before considering the final probe of efficiency. Each trial was 60 seconds long, final efficiency was measured by the amount of time the rat took to reach the end of an arm containing a water pot. Each rat was tested in 2-hour spans until completion of a full 24-hour cycle. A Cosinor analysis was used to evaluate acrophase and percentage of rhythmicity. The obtained results suggest that time-place learning process is influenced by the circadian clock. The severity and timing of prenatal or chronic protein malnutrition modifies the acrophase and rhythmicity of the learning circadian pattern, which can impact important cognitive functions.

Ketogenic diet does not change NKCC1 and KCC2 expression in rat hippocampus.

In control rats, we examined the effects of ketogenic diet on NKCC1 and KCC2 expression levels in hippocampus. Neither the number of NKCC1 immunoreactive cells nor the intensity of labeling of KCC2 was found to modify in hippocampus of the rats after ketogenic diet treatment. These results indicate that ketogenic diet by itself does not modify the expression of these cation chloride cotransporters.

Brain plasticity, signal transduction and epigenesis: a missing link revealed

It has been long thought that the brain reorganizes itself in response to environmental needs. Sensory experiences coded in action potentials are the mean by which information on the surroundings is introduced into neuronal networks. The information approaching the brain in the form of electrochemical codes must then be translated in biochemical, epigenetic and genetic ones. Only until recently we have begun understanding the underpinning of such informational transformations and how this process is expressed as neuronal plastic responses. Central for our comprehension of this matter is the finding that signals transduction cascades can modify gene expression by remodeling the chromatin through epigenetic mechanisms. Hence, chromatin remodeling seems to be the process by which experiences are “imprinted”.

The mossy fiber system of the hippocampal formation is decreased by chronic and postnatal but not by prenatal protein malnutrition in rats.

We tested in 70-day-old Sprague-Dawley rats, whether malnutrition imposed during different periods of hippocampal development produced deleterious effects on the total reference volume of the mossy fiber system. Animals were treated under four nutritional conditions: (a) well nourished; (b) prenatal protein malnourished; (c) chronic protein malnourished and (d) postnatal protein malnourished. Timm's stained material was used in coronal hippocampal sections (40 microm) to estimate--using the Principle of Cavalieri--the total reference volume of the mossy fiber system in each experimental group. Our results show that chronic and postnatal protein malnourished, but not prenatal malnourished rats, decrease the mossy fiber system and the total reference volume of the mossy fiber system are selectively vulnerable to the type of dietary restriction. Thus, chronic and posnatal protein malnutrition produce deleterious effects, but only rats under prenatal protein malnutrition were able to reorganize synapses in this plexus. These findings raise the possibility that chronic malnutrition, as a long-term stressful factor, might be an important paradigm to test structural hippocampal changes that produce physiological and pathophysiological effects, or the possibility to recover its function for nutritional rehabilitation.

Prenatal protein malnutrition decreases mossy fibers-CA3 thorny excrescences asymmetrical synapses in adult rats.

Prenatal protein malnutrition has deleterious effects on hippocampal structure and function that likely result from decreased synapse number. We thus evaluated long-term effects of prenatal protein malnutrition on the mossy fibers-CA3 thorny excrescences asymmetrical synapses in 220-day-old rats. Protein malnourished rats born from pregnant dams fed with 6% casein diet were cross-fostered to lactating control rats at birth. Control animals were fed with a 25% casein diet. Timm's stained material was used to estimate the total reference volume of the mossy fiber system suprapyramidal bundle by means of stereology. The mossy fiber-CA3 asymmetrical synapse numerical density was obtained by electron microscopy, using the physical disector method. The total number of mossy fiber-CA3 asymmetrical synapses was determined on the basis of the total reference volume of the mossy fiber system suprapyramidal bundle and the mossy fiber-CA3 asymmetrical synapse numerical density. Prenatal protein malnutrition produced long-lasting, significant decreases in the volume of the mossy fiber system suprapyramidal bundle and in the numerical density of mossy fiber-CA3 asymmetrical synapse, suggesting a reduction in the total number of this synapse type. Hence, prenatal protein malnutrition induces long lasting deleterious effects on the progression of developmental programs controlling synaptogenesis and/or synaptic consolidation, likely by affecting a myriad of cellular processes.

Disminución del número de espinas dendríticas en las neuronas piramidales de la corteza motora de ratas bajo estrés prenatal / Diminution of dedritic spines in the piramidal neurons of the motor curtex in rats with prenatal stress

En el presente estudio se analizó el efecto a ratas gestantes sometidos a estrés por inmovilización. Sobre las neuronas piramidales de la capa V motora certica de la progenie, se usaron ratas hembras de la cepa Wistar que se distribuyeron al azar en dos grupos: control y experimental (n=5). Las ratas del grupo experimental fueron sometidos a estrés por inmovilización durante 2 a 6 horas diarias a lo largo de toda la gestación; las ratas grupo control se mantuvieron en condiciones normales de bioterio. Las crías de cada grupo fueron sacrificadas a los 14 y 21 días de edad. Para extraer su cerebro y obtener bloques de corteza motora que se procesaron con la técnica de Golgi Rápido. Se cuantifico el número de espinas dendríticas en segmentos de 50 micras, de una sección de 250 m de la dendrita axonal de neuronas piramidales. En el grupo experimental de 14 días, hubo reducción significativa en el número de espinas dendríticas en los segmentos de 50 a 100 y de 100 a 150 micras respecto al grupo control. Estos hallazgos sugieren que las deficiencias en la capacidad de aprendizaje, comportamiento adaptativo y de alteraciones de la actividad locomotora, reportadas en animales descendientes de madres sometidas a estrés durante la gestación pueden ser resultado de la reducción en la complejidad neuronal.

Efectos del estrés prenatal sobre la ramificación neuronal en la corteza visual de ratas neonatas / Prenatal stress of effects on neuronal branching in the visual cortex of the pup rats

Se evaluó el efecto del estrés por inmovilización aplicado a ratas gestantes, sobre la morfología de las neuronas piramidales en la corteza visual de machos descendientes de dichas hembras, a los 14 y 21 días posnatales. Ratas hembra de la cepa Wistar fueron sometidas a estrés por inmovilización forzada durante toda la gestación, en periodos que variaron entre 2 y 6 horas por día. En neuronas del área visual, impregnadas con el método de Golgi se cuatificó el grado de ramificación dendrítica. Los resultados muestran disminución de ramificaciones dendríticas sobre todo en ratas de 21 días. Las deficiencias en la capacidad de aprendizaje y comportamiento adaptativo observadas en animales descendientes de madres sometidas a estrés durante la gestación podrían explicarse con base a estos resultados

Apertura palpebral prematura experimental y desarrollo neuronal en la rata recién nacida / Experimental premature eyelid openingand neuron development in new-born rats

Los extractos de glándula submaxilar (EGS) de ratón induce apertura prematura de los párpados en ratas recién nacidas. En animales sometidos a ciclos alternos de luz-obscuridad y con EGS, las neuronas de la corteza visual exhiben, en la dendrita principal, más espinas y un mayor grado de ramificación que los controles, en tanto que en animales mantenidos en obscuridad total y con EGS, únicamente disminuyen las espinas dendríticas, mientras que el grado de ramificación es mayor comparado con los controles.

Efectos del estrés por inmovilización sobre la corteza motora en la rata / The effects of immobilization-Stress on rat motor cortex

El presente estudio analiza el efecto del estrés por inmovilización aplicado a ratas gestantes sobre las neuronas piramidales de la V capa de la corteza motora de sus descendientes. Se usaron ratas hembra de la cepa Wistar que se distribuyeron al azar en dos grupos: control y experimental (con cinco ratas cada uno). El grupo experimental fue sometido a estrés por inmovilización en períodos que variaron de 2 a 6 horas diarias durante la gestación, el grupo control se mantuvo en condiciones normales de bioterio. Las crías de cada grupo fueron sacrificadas a los 14 y 21 días de edad. Se extrajo el cerebro y se colectaron bloques de la corteza motora que se procesaron con la técnica de Golgi Rápido. Se analizaron las neuronas piramidales de esta área cuantificando el número de intersecciones de las ramificaciones dendríticas con ocho círculos concéntricos. Los resultados mostraron en ambas edades reducción significativa del número de ramificaciones dendríticas en todos los círculos concéntricos en el grupo experimental en comparación con el grupo control. Estos hallazgos indican reducción de la complejidad neuronal que puede ser responsable de deficiencias en la capacidad de aprendizaje, comportamiento adaptativo y de alteraciones de la actividad locomotora reportadas en animales descendientes de madres sometidas a estrés durante la gestación.

Efecto del tolueno en espinas dendríticas descendientes de ratas tratadas en la adolescencia o la gestación / Effects in dendritic spines in rats with a chronic exposure to tolueno

La inhalación crónica de tolueno causa lesiones microscópicas en el sistema nervioso central, tales como acanalamieto del tallo de las neuronas piramidales, hipertrofia neurofibrilar. Tres ratas Wistar jóvenes fueron expuestas a tolueno durante 21 días antes del apareamiento. Otras tres ratas hembras inhalaron vapores de tolueno desde el día 5 hasta el día 21 de la gestación. Después del tratamiento durante 15 minutos/día doce crías de cada grupo se sacrificaron a los 14 y 21 días de edad. Se determinó el número de espinas de la dendrita principal en segmento de 50 micras, de neuronas piramidales de la corteza visual en preparaciones de Golgi. Se encontró reducción significativa del número de espinas dendríticas en los grupos expuestos a tolueno en comparación con animales mantenidos bajo condiciones normales de laboratorio o del grupo sometido a las mismas condiciones pero sin tolueno.