Automated quantification of dopaminergic immunostained neurons in substantia nigra using freely available software.

Computerized techniques for image analysis are critical for progress in cell biology. The complexity of the data in current methods eliminates the need for manual image analysis and usually requires the application of multiple algorithms sequentially to the images. Our aim was to develop a software for immunohistochemical analysis of brain dopaminergic neurons combining several computational approaches to automatically analyze and quantify their number in the substantia nigra after a neurotoxic injury. For this purpose, we used a Parkinson's disease animal model to test our application. The dopaminergic neurotoxin, 6-hydroxydopamine, was administered in adult male rats to damage dopaminergic neurons in substantia nigra and to induce hemiparkinsonism. The lesion was corroborated by behavioral evaluation in response to apomorphine and amphetamine. The animals were euthanized and their brains processed for tyrosine hydroxylase immunohistochemistry for dopamine neuron identification. Neurons positive for tyrosine hydroxylase were evaluated in substantia nigra by light microscopy. The images were used to show quantification applicability. To test our software counting accuracy and validity, automatic dopamine neuron number was correlated with the data obtained by three independent observers. Several parameters were used to depict neuronal function in dataset images from control and lesioned brains. In conclusion, we could perform an automated quantification of dopaminergic neurons and corroborate the validity and accuracy of a freely available software.

"Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation".

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.

Alpha and beta noradrenergic mediation of NMDA glutamatergic effects on lordosis behaviour and plasmatic LH concentrations in the primed female rat.

In previous studies we have found that blockade of NMDA (N-Methyl-D-Aspartic-Acid)-type glutamatergic receptor with intracerebroventricular (ICV) selective drugs induces an inhibition of lordosis in ovariectomized (OVX) estrogen primed rats receiving progesterone or luteinizing hormone releasing hormone (LHRH). By the opposite way, stimulation with NMDA in OVX estrogen primed rats induced a significant increase of lordosis. In the present study the action of an alpha1-noradrenergic antagonist, HEAT (BE 2254/2-beta-4-Hydroxyphenyl-Ethyl-Aminomethyl-1-Tetralone), and Metoprolol, a beta-noradrenergic antagonist, were studied injecting them ICV previously to NMDA administration in treated OVX estrogen primed rats. In experiment 1, the enhancing effect on lordosis induced by NMDA at high dose (1 microg) was abolished by HEAT administration (P < 0.001 for 3 and 6 microg), and the LH plasma levels were decreased only with the higher dose (P < 0.05), suggesting that behavioral effects are quite more sensitive to the alpha-blockade than hormonal effects. In experiment 2, enhancing effects on lordosis behavior were not observed with neither the NMDA at low dose (0.5 microg) nor the metoprolol alone (5.71 microg), but a synergism was observed when both were simultaneously administered (P < 0.001). The LH plasma levels were increased by Metoprolol alone (P < 0.05), and powered by the combination with NMDA at low dose (P < 0.01 vs. SAL and NMDA alone); no differences were observed with Metoprolol. LH increase was observed with Metoprolol even without behavioural modifications. These findings strongly suggest that facilitatory and inhibitory effects of NMDA in this model are mediated by alpha- and beta-adrenergic transmission in both, behavioral and hormonal effects.

Differential responses in central dopaminergic activity induced by apomorphine in IPL nude rat.

The IPL nude rat, derived by spontaneous mutation from the Sprague-Dawley strain, presents alterations in the prolactin synthesis and secretion due to an increased dopaminergic inhibition. However, there are no reports concerned to central dopamine activity. The corpus striatum is a brain area involved in the development of stereotyped behavior after the activation of mesolimbic and/or nigro-striatal dopamine pathways. In order to identify possible mesolimbic and/or nigro-striatal dysfunctions in the IPL nude rat, we study the spontaneous oral behaviors and the effects of apomorphine-induced dopaminergic activation on stereotyped behavior and neurochemical changes. Males from both strains were injected with saline or apomorphine (2 and 5 mg/kgs.c.) and evaluated during 30 min in a stereotypes oral tests. The corpus striatum and nucleus accumbens were used to measure dopamine (DA), 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) by HPLC. The concentrations were expressed as synthesis rate (DA/DOPA) and turnover rate (DOPAC/DA). We observed that the spontaneous gnaw movements were significantly different between the untreated IPL nude and Sprague-Dawley (SD) rats. Apomophine injection decreased the amount of stereotyped gnawing in IPL nude rats at the two doses used, but it induced an increase in SD rats. Apomorphine also caused an enhancement in the number of biting and sniffing without modifying the licking behavior. In addition, modifications of the dopaminergic activity were also observed. Synthesis rate in the striatum of IPL nude rats was higher than in SD rats after the injection of saline. Apomorphine caused a reduction of the synthesis rate in both strains. Turnover rate was significantly lower in the striatum of IPL nude rats than in the SD rats injected with saline. Apomorphine caused an increase in the turnover rate in both strains. Contrary to observed in the striatum, the 2 mg/kg dose of apomorphine caused a significant increase in the DA synthesis rate in nucleus accumbens, while 5 mg/kg decrease it in both strains. The DA turnover rate in the same area was lower in IPL nude than in SD rats after saline injection. Apomorphine enhances the DA turnover rate in both strains. We conclude that the modifications of the oral spontaneous and induced stereotypical patterns observed in the IPL nude rats could be related to the differential responses in dopaminergic activity in the two brain areas examined.

Propuesta de caracterización y tipificación de proteínas monoclonales aplicable al laboratorio clínico / Proposal of characterization and typification of monoclonal proteins applicable to clinical laboratory

En el presente trabajo se incorporan y ordenan procedimientos para obtener la mayor información posible a nivel laboratorio clínico en la indagación de proteínas "M" y la definición de su clase (cadenas pesadas) y tipo (cadenas livianas). Se propone una secuencia de las siguientes técnicas: electroforesis, inmunoelectroforesis, inmunofijación, inmunodifusión radial cuantitativa, tratamientos despolimerizantes e inmunosustracción. La inmunosustracción, descripta por W. A. White y col., consiste en la remoción de la inmunoglobulina en estudio mediante un antisuero específico en presencia de polietilenglicol, separación del inmunocomplejo precipitado y posterior evaluación del sobrenadante mediante inmunofijación; se identifica la cadena liviana que pertenece a la inmunoblobulina sustraída por su ausencia al fijar con el antisuero correspondiente. Es particularmente útil cuando se desea establecer la correspondencia liviana/pesada en presencia de bandas homogéneas múltiples, identificar bandas menores enmascaradas en un fondo policlonal y excluir la presencia coincidente de cadenas livianas libres. Se aconseja incorporar el tratamiento despolimerizante con 2-mercaptoetanol previamente a la cuantificación de IgM por inmunodifusión radial ya que la comparación previa entre controles tratados y no tratados reveló una diferencia muy significativa cuando se analizó mediante contraste de diferencias entre pares homólogos (n = 12, test t = 37,2 p <0,01)

Actividad amilásica y relación lecitina-esfingomielina en líquido amniótico como parámetros de madurez pulmonar fetal

La medida de la actividad amilásica en líquidos amnióticos (L.A.) ha sido propuesta como prueba "screening" para determinar madurez fetal, Nosotros hemos reunido y examinado 272 muestras de L.A. para dosar amilasa y evaluar la relación lecitina-esfingomielina. La amilasa del L.A. mostró una correlación pobre con respecto a la relación lecitina-esfingomielina (r: 0.240). El análise de la amilasa como prueba "screening" para determinar la necesidad de evaluar la relación lecitina-esfingomielina mostró una sensibilidad del 54% y una especificidad del 88%. Se estudiaron tres grupos de gestantes: Un grupo con amilasa inferior a 200 U/L, un grupo intermedio con valores entre 200 y 300 U/L y un grupo de pacientes con amilasa superior a 300 U/L. Solamente el 46% del grupo con baja amilasa, tenía relación lecitina-esfingomielina inmadura. El grupo con cifras de amilasas altas, mostró la mejor correlación entre amilasa y relación lecitina-esfingomielina, pero un 12% de estas muestras dieron lugar a una relación lecitina-esfingomielina inmadura o "borderline". Se concluye que la amilasa del L.A. no debe ser usada como prueba "screening" para determinar la necesidad de recurrir a la evaluación de la relación lecitina-esfingomielina

Estudio crítico de la actividad tromboplástica del líquido amniótico en diversas semanas gestacionales / Critical study of the tromboplastic activity of the amniotic fluid in different gestational weeks

Se determinó la actividad tromboplástica del LA en setenta y ocho embarazos normales y patológicos por el método de Quick, en una etapa, modificado por Yaffe, para la medida del tiempo de protombina. Se comprobó el aumento de la actividad tromboplástica con el progreso de la gestación, siendo el coeficiente de correlación r = -0,76. No se observaron diferencias entre los valores de actividad tromboplástica entre embarazos normales y patológicos. En tres de cuatro casos de embarazos postérmino, la actividad tromboplástica del LA fue inferior a 45 s. Nuestros resultados corroboraron los enunciados por Yaffe y difieren de los de Phillips y Davidson