Poster
# 25
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] |
Three groups of male Wistar rats have been used in this study. The first goup was formed by 10 young animals (269±27 g body weight and 3 months old). Second and third groups were formed each one by 10 old animals (534±23 g body weight and 22-24 months old). All the animals had free access to fed and water and were housed at a constant temperature of 25 ºC with lights on from 7:00 am to 7:00 pm. One of the old groups was stereotaxically lesioned in the left frontal cortex with a cronically implanted pastic neddle 8 external diameter of 0.20 mm). 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 sacrified. The rats were anaesthetized by an intraperitoneal injection of equithesin and perfused through the ascending aorta with PBS buffer (pH=7.4). The brains were removed and immersed in 4% p-formaldehyde and 0.5% glutaraldehyde in PBS buffer (pH=7.4). The brains were removed and immersed in 4% p-formaldehide 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 y Wree, 1985) of the right hemisphere were obtained with a vibratome (Agar Scientific). Right Fr area (contralateral to the lesion) was carefully sectioned from these slides, osmificated, dehydrated in an ascending ethanol series, osmificated, dehydrated in an ascending ethanol series, immersed in propylene oxide and embedded in Epon. Followingg, 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, sectioned passed throught the entire thickness of the cerebral cortex (Figure 1). 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 glial 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 cresysl violet to verify anatomically the Fr1 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 100X objetive and a 10X ocular fitted with a micrometer grid of 160x80 µm2. The cells profiles were drawn with a camera lucida (O´Kusky and Colonnier, 1982). The cells intercepted by the left vertical and bottom bars were not considered. The grid was lowered successively throug 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 layer were grouped as I, II-IV, V and VI. Under our conditions it was difficult to distinguish between layer II, III and IV. The cell nucleus was chosen as test object (Trillo and González, 1992; Amenta et al., 1994; Ramos et al., 1995).
To performed the counts, microglia and oligodendrocytes were considered together, because in semithin sections stained with toluidine blue is difficult to deferenciate those glial cells.
The criteria to diferenciate astrocytes and microglia-oligodendrocytes were described by Ling and col (1973); Sturrock (1983). Briefly, astrocytes are cells with big nucleus big and slowly stained nucleus. A dark and continous nuclear ring is observed under the nuclear membrane. Microglia-oligodendrocytes were characterized as cells with a small and dark nuceus (Figure 2). Total glia was considered ad the addition of astrocyte and microglia-oligodendrocyte population.
The number of glial cells were expresed as number of cells/106 µm3 (mean±SEM).
One way analysis of variance (ANOVA) with the Newman-Keuls post hoc test and umpaired Sudent 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] |
