Poster | 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]![]() |
Since the clasical studies of Ramón y Cajal (1913) and Del Rio Hortega (1920) about astrocytes, microglial cells and oligodendrocytes, the interest of glial cells has increased. In this way, in the last 20 years a radical change in the CNS studies has been observed. Actually, the interest is not only on neurons but also in the role of glial cells in the neuronal functions. This fact is been performed by the development of the new technologies as specific stained, cell cultures and autorradiographic techniques which allow a better and bigger knolodge about these cells and the functional role of glia in the noervous system (Raine, 1989; Noremberg, 1994).
In the last years, many atention has been payed to the changes observed in the glial cells during lesions, traumas, degenerative illness and also aging. Damage in the CNSs breaks the physical and functional juntions of the nervous tissues, disrupts in this way, the interactions between neuronal and glial cells (Nieto.Sanpedro, 1998). This fact could be the responsive mechanism of the described reactivity of glial cells, especially for astrocytes and microglial cells in some lesions (Hajos et al., 1990; Landis, 1994; Nieto Sanpedro et al., 1985; Ramírez Expósito y Martínez Martos 1998a, b, 1999).
Astroglial changes are widely recognized to be one of the earliest and most remarkable cellular responses following a wide variety of insults to the CNS. These changes are known as reactive astrogliosis. Reactive astrocytes are characterized by a hypertrophy including pericaryon and processes. In some instances, increased cell size may be related to a requirement for increased astrocytic metabolic activity (Landis, 1994) and the increase in the processes may be related with the formation of the glial scar (Norenber, 1994). However, the most extensively studied aspect of reactive astrocytic is the increase in GFAP inmunoreactivity observed after lesions (Nathanial and Nathanial, 1984; Eng, 1988; Steward et al., 1993; Neary et al., 1994).
By other hand, proliferation of astrocytes after an injury is still uncertain, although it is known that the possibilities of mitosis are limited (Miyake et al., 1988, 1992) evidences suggest that astrocytes may arise in injured brain (Landis, 1994). However, some authors considered that proliferation anly exits in lesions affected BBB (Nieto Sanpedro, 1998; Kimelberg y Noremberg, 1994). All these changes induced by lesions are similar to those observed during aging (Hansen et al., 1987; Bronson et al., 1993; Finch, 1993; Berciano et al., 1995). The role of oligodendrocytes in cerebral lesions is unknown due to the problems of their identification (Giordana et al., 1994; Bartholdi and Schwab, 1998). The quantitative changes in astrocytes and oligodendrocytes population may be related with the neuronal death and axonic and dendritic degeneration (Peters et al., 1991).
Microglial cells are the other glial cell type more affected by lesions. In this situation, microglial cells are able to increase their number by division of their precursor o migration (Kreutzberg, 1996), and resting microglial cells rapidly transform into an activated form with a rounded morphology (Akiyama et al., 1988) and phagocitic activity (Streit, 1988). Reactive microglia also cann regulate the reactive gliosis (Giulian and Baker, 1986; Giulian et al.,1989). Increase in the number of microglial cells could be related with elimination of lipofuscine and death neurons.
The aim of present work is to study the changes of glial cells in different neurodegenerative process, so we analyzed the effects of aging and lesions. The quantitative changes of glial population observed in the different cortical layer of the frontal cortex were analyzed.
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).
Figure 3 shows numerical cell density (mean±SEM) obtained in the different cortical layers of the frontal zone.
The study of the influence of ageing in the glial population of Fr cortex of the rat showed a significant increase (p<0.01) of total glia (TG) in cortical layer V of aged animals. However, the combination of ageing and contralateral lesions induced a significant increase (p<0.01) in all cortical layer except layer VI. In the same way, a significant increase of TG were observed in old and lesioned animals in reration with old animals. This increase was significat in cortical layer I, II-IV (p<0.01) and V (p<0.05).
The study of the different glial cells also showed a significant increase with aging in astrocyte population (A) in cortical layer V. However, aging and lesions together, induce a decrease (p<0.01) in the number of A in cortical layers II-IV and V in relation with old animals. In relation with young controls, old and lesioned animals increased (p<0.01) the A only in cortical layer I.
Ageing produced a significant decrease (p<0.05) in the number of microglia-oligodendrocytes in layer I. Rest of cortical layers presented no changes. The effects of ageing and lesioned induced significant increases (p<0.01) in all the cortical thickness.
In the present work, the quantitative study has been made using micrometer-ocular techniques. These methods have been clasically the most used (O´kusky and Colonnier, 1982; Konigsmark, 1970, Trillo y Gonzalez, 1992) because it sis possible to count directly the number of cells into a grid. However, in the last years, authomatic methods (quicker and less hard) are been developed (Antal et al., 1992; Coggeshall, 1992), but these techniques diferenciate the cell population by their size, so in our study, we can not deifferenciate the glial cells because their size is very similar. Recently, the used of inmunocytochemical techniques has been also questioned because crossed stained may happen.
In this work we have chosen optical microscope techniques based on morphological criteria (O´kusky and colonnier, 1982; Sturrock, 1983; Peter et al., 1991). Frontal cortex have been chose because this area is the target of some projections and its alterations may be responsible of some cognitive disfunction (Kemper,1984; Amenta et al., 1992; 1994).
The reactivity of glial cells in the CNS during aging and different pathologies as ischemic, trauma or degenerative disorders is well known (Hajos et al., 1990; Landis, 1994; Myers et al., 1993; Giulian and Vaca, 1993; Kempsky and Volk, 1994; Ridoux et al., 1994; Leuba and Kraftsik, 1994a, Mieto Sanpedro y Mora, 1994; Streit, 1996). Some authors have suggested that quantitative changes observed in glial cells during these situation may be related with the changes in number of neurons and also with metabolic variations (Sturrock, 1983). The methods used in this study allowed us to differenciated the different glial cell types and so, it si possible to know which glial type participate in gliosis process.
Quantitative studies of glial population during aging are heterogenous and also contradictory. A first group of authors do not find quantitative changes with aging in glial cells (Curcio and Coleman, 1982; Haug et al., 1984). In other studies, the results are different depending to the studied area (Haug et al., 1983), the method used (Cragg, 1975; Sturrock, 1983, Haug et al., 1984; Vincent et al., 1989) and the animal model (Curcio and Coleman, 1982).
Our results showed an increase in the total glial cells during aging in the studied area. The most affected cortical layer is layer V.
Astrocytes are considered as the most affected cells during aging, and significant increases have been described in white matter of mice (Bronson et al., 1993) and in frontal cortex of old rats (Amenta et al., 1994). Simillar variations have been observed in the hippocampus (Landfield et al., 1977; Lolova,1991). Our results are in acccordance with that studies because we found age-related increases in astrocytes.
In present work, the microglial cells and oligodendrocytes were quantified together because these glial cell types are not distinguish by morphological and stained criteria (Vaughan, 1989; Peter et al., 1994). Our results did not show changes with aged, except in cortical layer I. These dates are in accordance with the study of lawson and col (1992). However, althoug no quantitative changes were observed, an increased in the reactivity of microglial cells has been described (ogura et al., 1994; Perry et al., 1993). The presence of reactive microglia during ageing may be related with the neurodegenerative process happened during aging and may be implicated in control of synapsis (Adams and Jones, 1982) and with the remodelation of dendrites (Flood and Coleman, 1981). By contrast other authors have described light increase in microglial cells with aging (Vaughan and Peter, 1974; Peter et al., 1991; 1994).
The effects of induced and cronical lesions in the frontal cortex also have been studied. It is known that cerebral injuries in adult animals produce degeneration (Jones and Cowan, 1983). However, our results are difficult to dicuss because is a specific lesion.
an increase in glial cell number was observed with aging and lesions, because independly, these procceses cause reactive gliosis (Sturrock, 1983; Haug et al., 1984; Landis, 1994; Giordana et al., 1993). The combined effects of aged and lesions showed a high glial reaction. However, the glial cell type implicated in ageing and lesions is different. Aging induced an increase in astrocytes, but lesions, analized in contralateral side, produced an increase in microglia-oligodendrocytes, but astrocytes did not change. It could be possible due to a compensatory effects to the increase produce in ageing ant to the possible decrease during lesions.
Other important results are the different behaviour of cortical layers. Aging only produces changes in cortical layer V, but the combinatory effects of lesions and aging affected to all the thicknes of cortex. Theses results suggest that the effects of lesion are over the aging.
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[Cell Biology & Cytology]![]() |
[Neuroscience]![]() |
[Physiology]![]() |