Poster
# 24
Poster |
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6th Internet World Congress for Biomedical Sciences |
María Jesús Ramírez-Expósito(1), José Manuel Martínez-Martos(2)
(1)Unit of Physiology. University of Jaen - Jaén. Spain
(2)Unit of Physiology. University of Jaén - Jaén. Spain
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[Cell Biology & Cytology] |
[Neuroscience] |
[Physiology] |
Twenty male Wistar rats (269±27 g body weight and 3 months old) were used in this study. The animals were randomly divided into two groups of ten rats each. All the animals had free access to fed and water and were housed at a constant temperature of 25º C with lights on from 7:00 am to 7:00 pm. One group was stereotaxically lesioned in the left frontal cortex with a cronically implanted plastic needle (external diameter of 0.20 mm). The rats were anaesthetized by an intraperitoneal injection of Equithesin (2 ml/Kg body weight). Stereotaxic coordinates for the implant from bregma were: anterior, 2.7 mm; lateral, 0.8 mm; and dorsal -5.0 mm from the dura (Paxinos and Watson, 1985). Seven days after, all the animals were sacrificed. The rats were anaesthetized by an intraperitoneal injection of equithesin and perfused through the ascending aorta with PBS buffer (pH=7.4), followed by a fixative containing 4% p-formaldehyde and 0.5 % glutaraldehyde in PBS buffer (pH=7.4). The brains were removed and immersed in 4% paraformaldehyde in the same buffer for 4 hours at room temperature. After that, the brains were transversally sectioned and the parts containing the frontal lobe were used for quantitative analysis.
1 mm thickness coronal sections (Bregma 2.7; Interaural 1.7) containing the Fr area (Zilles and Wree, 1985) of the right hemisphere were obtained with a vibratome (Agar Scientific). Right Fr1 area (contralateral to the lesion) was carefully sectioned from these slides, osmificated, dehydrated in an ascending ethanol series, immersed in propylene oxide and embedded in Epon. Following, the tissue blocks were sectioned with an ultramicrotome (Reichert-Jung) to obtain 1 µm thickness sections oriented in a perpendicular plane to the pial surface. In this way, sections passed through the entire thickness of the cerebral cortex. To ensure this topic, the section plane was parallel to the lengths of the apical dendrites of the pyramidal neurons. Over these semithin sections previously stained with toluidine blue, the neuronal population were quantified. Rest of the 1 mm thickness sections were embedded in paraffin and coronal sections of 15 µm thickness were obtained and stained with cresyl violet to verify anatomically the Fr zone.
Counts to determine the numerical density of neurons per unit of volume of tissue were made using micrometer ocular techniques (Konismark, 1970). To determine the neuronal density, the semithin sections were visualized with a 40X objetive and a 10X ocular fitted with a micrometer grid of 200 x 200 µm2. The cells profiles were drawn with a camera lucida (O´Kusky and Colonnier, 1982). The cells intercepted by the right vertical and top grid bars were included in the counts; those intercepted by the left vertical and bottom bars were not. The grid was lowered successively through all cortical laminae and this procedure was repeated extending from pial surface to white matter in six semithin sections, separated between them 50 µm, of each animals. The cortical layers were grouped as I, II-IV V and VI. Under our conditions it was difficult to distinguish between layers II, III and IV. The cell nucleolus was chosen as test object (Trillo and Gonzalez, 1992; Amenta et al.,1994; Ramos et al., 1995).
Attending to the levels of hyperchromasia, three neuronal types of neurons were differentiated (Figure 1): 1) normal neurons (NN), without hyperchromasia with toluidine blue staining. The nucleus and the nucleolus were perfectly observed (Ong and Garey, 1991); 2) light dark neurons (LDN); with light hyperchromasia but with optical microscopy no degeneration marks were observed, and 3) strong dark neurons (SDN); strongly stained with deformed or contracted aspect. The limit between soma and nucleus is difficult to observe (Ong and Garey, 1991; Gallyas et al., 1992b). Total neurons (TN) were considered as the addition of NN, LDN and SDN.
The number of neurons were expressed as number of neurons/106 µm3 (mean±SEM).
An image analysis equipment (Videoplan, Kontron) was used for the cytomorphometric study. This study included the areas of soma, nucleus and nucleolus and the form index of soma and nucleus. The profiles of these structures were drawn with a digital pencil in the same semithin section used for the counts. The three types of neurons were measured in a proportional number to the ratio of each neuronal types in the total neurons. A systematic study across the cortical thickness of the frontal cortex was made using the immersion objetive. The area measurements were expressed in µm2 by calibration of the Videoplan system to obtain real measurements. The form indexes were obtained by the ratio between maximum and minimum diameters, so they are an adimensional parameters. Values near one showed round cells or nucleus, but values higher than one show elliptic cells or nucleus. One-way analysis of variance (ANOVA) with the Newman-Keuls post hoc test and umpaired Student t test was used to compare different groups. All experiments were done in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
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[Cell Biology & Cytology] |
[Neuroscience] |
[Physiology] |
