Graciela Garcia

Central nervous system myelin deficit in rats exposed to 2,4-dichlorophenoxyacetic acid throughout lactation

Our results show that 2,4-dichlorophenoxyacetic acid (2,4-D) exposure through mothers milk during the period of rapid myelination (from the 15th to the 25th postnatal days) results in a myelin deficit in the pups brain and demonstrates the vulnerability of the developing central nervous system (CNS) to 2,4-D. After 100 mg/kg 2,4-D administration to dams, brains of male and female rats show a significant diminution of myelin markers such as monohexosylceramide as well as phospholipids and free fatty acids (FFA) and an increase of cholesteryl esters. Histological studies revealed myelin deficit in some brain regions after 2,4-D treatment. These data indicate that 2,4-D, through the mothers milk, alters the myelination process during a specific postnatal period.

2,4-dichlorophenoxyacetic acid through lactation induces astrogliosis in rat brain

Comparison of astroglial immunoreactivity in mesencephalon, cerebellum, and hippocampus of 25-d-old rat pups exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through the mothers milk was made using a quantitative immunohistochemical analysis. A glial reaction was detected at the level of serotonergic nuclei and extreme astrogliosis in the hippocampus and cerebellum. A quantitative analysis of reactive astrocytes was performed by using GFAP and S-100 protein as specific markers. The study showed a significant increase in their number, size, number of processes, and density of immunostaining in 2,4-D-exposed animals. Exposure to 2,4-dichlorophenoxyacetic acid on the first days of life modifies the astroglial cytoarchitecture in parallel to previously described neuronal changes.

Vanadium (V)-Induced Neurotoxicity in the Rat Central Nervous System: A Histo-Immunohistochemical Study

As vanadium was found to induce oxidative stress in the central nervous system, the morphological alterations of neurons and astroglial cells in adult rat central nervous system after vanadium exposure was studied, using histological markers of cellular injury. Animals were intraperitoneally injected with 3 mg/kg body weight of sodium metavanadate for 5 consecutive days. NADPH diaphorase histochemistry and heat shock protein (hsp) 70, glial fibrillary acidic protein (GFAP), and S-100 immunohistochemistry were performed in floating sections of several brain areas. NADPHd staining was higher in the molecular and granular layers of the cerebellar cortex, and small NADPHd-stained interneurons were observed in hippocampal sections in V(+5)-exposed animals. hsp 70 immunostaining showed the presence of reactive neurons in cerebellum of treated animals. GFAP and S-100 immunohistochemistry showed enlarged astrocytes in cerebellum and hippocampus in the V(+5)-exposed animals. The histological markers used showed that the main areas affected by vanadium-mediated free-radical generation were the hippocampus and the cerebellum.

Morphological Alterations of Central Nervous System (CNS) Myelin in Vanadium (V)‐Exposed Adult Rats

In the present work we show morphological data of the in vivo susceptibility of CNS myelin to sodium metavanadate [V(+5)] in adult rats. The possible role of vanadium in behavioral alterations and in brain lipid peroxidation was also investigated. Animals were injected intraperitoneally (i.p.) with 3 mg/kg body weight (bw) of sodium metavanadate [1.25 V/kg bw/day] for 5 consecutive days. Open field and rotarod tests were performed the day after the last dose had been administered and then animals were sacrificed by different methods for histological and lipid peroxidation studies. The present results show that intraperitoneal administration of V(+5) to adult rats resulted in changes in locomotor activity, specific myelin stainings and lipid peroxidation in some brain areas. They support the notion that CNS myelin could be a preferential target of V(+5)‐mediated lipid peroxidation in adult rats. The mechanisms underlying this action could affect the myelin sheath leading to behavioral perturbations.

CHANGES IN SEROTONIN-IMMUNOREACTIVITY IN THE DORSAL AND MEDIAN RAPHE NUCLEI OF RATS EXPOSED TO 2,4-DICHLOROPHENOXYACETIC ACID THROUGH LACTATION

Comparison of serotonin-immunoreactive (SER-IR) neurons in nucleus raphe dorsalis (NRD) and median raphe nucleus (MRN) of 25-d-old rat pups exposed to 70 mg/kg/d 2,4-dichloro-phenoxyacetic acid through mothers milk and control pups was made using an immunohistochemical analysis. Significant 2,4-D-treatment-related increase in size and density of SER-IR neuronal somata as well as in fiber length were observed. We postulate that exposure to 2,4-dichlorophenoxyacetic acid on the first day of life would modify the synthesis of 5-HT or the maturation of the brain serotonergic system.

ROS formation and antioxidant status in brain areas of rats exposed to sodium metavanadate

In the present work, in vivo ROS formation and the activity of antioxidant enzymes in the hippocampus and the cerebellum of sodium metavanadate (NaVO₃) treated rats were studied. Rats were i.p. injected with 3 mg/kg bw/day (V₁ group) or with 7.2 mg/kg bw/day of NaVO₃ (V₂ group) for 5 consecutive days. Results show that after only 5 days of NaVO₃ exposure, reactive oxygen species formation and alteration of the oxidative defence system were observed. Vanadium-induced OH production was detected in cerebellum at the high dose. This result was confirmed by in situ ROS histochemical staining. Neither Cat nor Cu-Zn SOD activities showed changes while GSH/GSSG ratio, in both brain areas, was significantly decreased in NaVO₃-treated groups. The present work indicates that the NaVO₃ dose and the particular brain area constitution would be critical in the cellular and molecular oxidative mechanism of this element.

Morphological study of 5-HT neurons and astroglial cells on brain of adult rats perinatal or chronically exposed to 2,4-dichlorophenoxyacetic acid.

2,4-D is a chlorophenoxyherbicide used worldwide. We have studied the morphological alterations of 5-HT neurons and glial cells in the mesencephalic nuclei of adult rats exposed to 2,4-D both perinatally (during pregnancy and lactation) and chronically (during pregnancy, lactation and after weaning) with quantitative methods. Pregnant rats were daily exposed to 70 mg/kg of 2,4-D from gestation day (GD) 16 to post-natal day (PND) 23 through diet. After weaning, pups were assigned to one of two sub-groups: T1 (fed with untreated diet until PND 90) and T2 (maintained with 2,4-D diet until PND 90). Brain sections were immunocytochemically stained using polyclonal anti-5-HT, anti-GFAP and anti-S-100 protein antibodies as cells markers. 2,4-D exposure during pregnancy and lactancy (T1 group) produced an increase in 5-HT neuronal area and immunoreactivity (IR) in the mesencephalic nuclei studied. However, with the chronical 2,4-D exposure (T2 group) only the 5-HT neuronal area from the dorsal raphe nucleus (DRN) was increased, suggesting an adaptable response of 5-HT neurons in median raphe nucleus (MRN). The presence of reactive astrocytes in mesencephalic nuclei and in hippocampus were also different for the two 2,4-D exposure designs, showing the existence of a correspondence between neuronal changes and astrogliosis. Results support evidences that 2,4-D alters the serotoninergic system and that 5-HT neurons of each mesencephalic nuclei show different responses to the 2,4-D exposure designs which are parallel to astrogliosis.

Neonatal Hypomyelination by the Herbicide 2,4-Dichlorophenoxyacetic Acid. Chemical and Ultrastructural Studies in Rats

The purpose of this study was to determine whether 2,4-dichlorophenoxyacetic acid (2,4-D), which is an herbicide used to control the growth of broadleaf weeds, had a direct or an indirect (mediated by undernutrition) hypomyelinating effect. We also proposed to analyze the effect of 2,4-D on undernourished (UN) pups. Four experimental rat groups were used: well-nourished (WN) pups, litters with eight offsprings; UN pups, litters with fourteen offsprings; WN pups whose mother received 70 mg/kg/day of 2,4-D from postnatal day (PND) 9 to 21 (WN70 pups); and UN pups whose mother received 70 mg/kg/day of 2,4-D from PND 9 to 21 (UN70 pups). In this work, we demonstrated that (1) myelin proteins (analyzed by Western blot and/or immunohistochemical study) showed a significant decrease in WN70, UN, and UN70 with respect to control group; (2) there is a good correlation between these myelin-specific protein expression with the degree of myelin compaction detected by electron microscopy in groups exposed to 2,4-D; (3) a decreased and normal number of myelin sheets were detected in UN and 2,4-D exposed pups, respectively; and (4) undernourishment sensitized pups to the hypomyelinating effect of 2,4-D. According to this and besides the fact that WN70 group have no body weight changes, these results are indicating that 2,4-D and undernourishment are two independent hypomyelinating factors.

Dopamine-β-Hydroxylase Immunohistochemical Study in the Locus Coeruleus of Neonate Rats Exposed to 2,4-Dichlorophenoxyacetic Acid Through Mother's Milk

Dopamine-β-hydroxylase (DβH), the enzyme that synthesizes noradrenaline from dopamine, was studied in the locus coeruleus (LC) of neonate rats exposed to 2,4-dichlorophenoxyacetic acid (2,4-D) through lactation for 14 days (from PND 9 to 22). Pups (22 days old) were anesthetized and fixed by transcardiac perfusion. Control and treated serial sections from brain stem—which correspond with the LC according to the Paxinos and Watson atlas—were simultaneously processed by an immunohistochemistry method for the DβH detection. Using an image analysis system, the immunostaining optical density (OD) was measured as an estimation of the enzyme content, and an OD significant decrease in the LC of 2,4-D–exposed animals was observed. As tyrosine hydroxylase levels in the LC are regulated by serotonin and in a previous study we demonstrated that this neurotransmitter was increased in 2,4-D–exposed pups, an indirect effect through serotonergic inhibition could be involved in the decreased DβH synthesis in the LC of these pups.

Medición Comparada de Procesos Odontoblásticos y Canalículos Dentinarios

El complejo dentinopulpar comparte el odontoblasto cuyo cuerpo esta ubicado en la parte externa de la pulpa dentaria. De su polo apical se desprende el proceso odontoblastico que se introduce en el canaliculo dentinal atravesando la dentina originando en su recorrido multiples colaterales hasta su finalizacion. Describir el proceso fue ardua tarea de investigadores que combinaron tecnicas histologicas para preservarlo en el interior de los canaliculos utilizando microscopio electronico de transmision o scanning para visualizarlo. En un trabajo previo observamos ambos tejidos unidos por los odontoblastos y los procesos coloreados. El objetivo actual fue medir micrometricamente la longitud de procesos y canaliculos para verificar si ambas son similares tanto en corona como en raiz. Se utilizaron treinta dientes sanos, extraidos por razones ortodoncicas de ambos sexos, cuyas edades oscilaron entre 6 y 18 anos. Se descubrio la pulpa dental, se fijo y se dividieron en partes iguales. Las mitades sin pulpa se prepararon con tecnica por desgaste. Las mitades que conservaron la pulpa se desmineralizaron aplicandoseles Colagenasa tipo II y coloreandolos con la tecnica de Schmorl. Con microscopio optico y una cuadricula calibrada con micrometro objetivo se midio primero el ancho de los campos histologicos y luego la longitud de procesos y canaliculos dentinales. Los resultados fueron analizados con pruebas de chi2, t de Student y test exacto de Fischer con un nivel de significacion del 5%. Observamos que el ancho de los campos histologicos coloreados tuvo una retraccion del 92% respecto al mismo campo en el desgaste y que la longitud de canaliculos siempre fue mayor que la de procesos, si bien se hallaron cuatro coincidencias de longitudes en corona y una en raiz. Estos resultados sugieren que la longitud del proceso aun sigue siendo tema controversial. La precausion profesional en los tratamientos odontologicos es el unico medio para evitar danarlo.