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] |
In the last years, studies performed in all the neurobiological fields had produced important knowledge about the neurodegenerative processes that are responsible for cell damaging and neuronal death. These processes seem to be originated by similar mechanisms, including deficiencies in energy levels (Lees,93), release of excitatory amino acids, particularly glutamic acid (Choi and Harley, 1993) and hyperproduction of free radicals such as nitric oxide (Lees, 1993; Moreno and Prior, 1992). All these disorders may be responsible of several pathologies such as those described in induced lesions, cerebrovascular accidents and neurodegenerative illness (Reinikainen et al., 1990; Lees, 1993). However, all these mechanism may occurs simultaneously, producing finally an increase in the intracellular free Ca2+ concentration (Choi, 1992; Khachaturian, 1994; Sun et al., 1997) that probably is the major responsible for cell damage and death.
It is known that neurodegeneration could produce quantitative and morphological changes in neurons and glial cells. Although traditionally, several types of degenerative disorders have been associated with neuronal loss, quantitative studies performed after induced lesions are very scarce; practically, all these studies are focused in the quantitative analysis of neuronal population after ischemic-reperfusion processes (Kudo et al.,1993; Hanyu et al., 1993). Neuronal loss in the cortex and in the CA1 hippocampal area have been described, but this neuronal death is selective and seems to be dependent of the duration of the lesion (Hanyu et al.,1993). Other induced lesions performed are aspiration of cortical areas, finding in this report selective neuronal loss too (Loopuijt et al.,1995).
In addition to the quantitative changes, the neurodegenerative processes also can induce structural changes in neurons; in fact, the presence of pyknotic neurons in the histological studies of the CNS is very well known. These neurons are characterized by their high affinity for several histological stains, like toluidine blue, cresil violet and acid fuschine. This affinity is the responsible of the dark appearance, so in the last years, these neurons were called dark neurons (Cammermeyer,1961; Cohen and Pappas,1969; Mugnaini,1965; Garey and Powell,1971; Cragg, 1975; Ong and Garey, 1991; Gallyas et al, 1992a; 93) hyperchromatic, cromophilic, contracted (Cammermeyer,1961) and more recently argyrophilic (Gallyas et al.,1992a) or collapsed neurons (Gallyas et al.,1992a; Czurko and Nishino, 1993). In semithin sections stained with toluidine blue, these neurons exhibit a retried soma and nucleus and strong staining. Occasionally, the nucleus is stained stronger than the nucleolus (Gallyas et al.,1990a). Although the structural characteristics of these neurons are well known, the quantitative studies performed are also scarce.
The aim of the present work is to study the quantitative and cytomorphometric changes in neurons in the Fr cortex of the rat during a neurodegenerative process induced by a stereotaxic lesion in the contralateral frontal cortex.
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).
shows numerical cell density (mean±SEM) obtained in the different cortical layers of the Fr zone. No significant differences were observed in the different cortical layers in the number of TN. The study of the different neuronal types considered showed a significant (p<0.001) decrease in the number of NN in layer II-IV, V and VI of lesioned animals although no differences were observed for layer I. By contrast, in all layers, lesioned animals showed a significant increase (p<0.001) of SDN. No changes were observed in the number of LDN with the lesion.
The study of the influence of contralateral lesions in the neuronal soma, nucleus and nucleolus area are showed in figure 3. Neuronal soma area showed a significant (p<0.001) increase in the TN of lesioned animals. Considering the different neuronal types, the soma area of NN were higher (p<0.001) in lesioned animals than in controls. No changes were observed in the soma area of LDN and SDN with the lesion. In all the studied animals, the soma area of LDN and SDN is significantly lower (p<0.001) than the soma area of NN. No differences were observed in the soma form index of TN, NN, LDN and SDN between lesioned and control animals (figure 4). Taken together, SDN presented a higher soma form index (p<0.001) than LDN, and these higher than NN (p<0.001).
The study of the nucleus area (figure 3) showed a lesion-dependent increase (p<0.001) in NN, whereas no significant differences were observed in nucleus area of LDN and SDN between lesioned and control rats. However in all the studied animals, the nucleus area of LDN and SDN were significantly (p<0.001) lower than nucleus area of NN. The nucleus area of TN did not change with the contralateral lesions. No lesion-dependent differences were observed in the nucleus form index in NN, LDN, SDN and NT. Taken together, SDN presented a higher (p<0.001) nucleus form index than LDN and these, higher (p<0.001) than NN.
The study of the nucleolus area showed a significant increase (p<0.001) in NN of lesioned animals. No changes were detected in LDN and SDN with the lesion. The nucleolus area of LDN and SDN was significantly lower than nucleolus area of NN.
It is known that injures produced in the adult mammalian brain are able to induce degeneration in the nervous tissue, even in remote places from the lesions (Jones and Cowan, 1983). However, the discussion of our results about induced lesions is difficult because the literature about this type of lesions is scarce.
Our results showed no loss of neurons in the contralateral frontal cortex of the lesioned rats. In contrast, most of the quantitative studies of neuronal population have been performed with lesions due to ischemic damage, which produces important neuronal loss (Hanyu et al., 1993). The most affected zones are CA1 hippocampal areas, cortical layers II, V and VI, the Purkinje cells in the cerebellum and striatal neurons (Pulsinelli et al., 1982; Gallyas et al.,1992c). In this situation, the neuronal loss is dependent on the duration of the ischemic period and the time between repeated ischemics (Tomida et al.,87; Nakano et al.,89; kato y Kogure,90; Hanyu et al.,93). However, this neuronal death is progressive, as has been described in gerbils (Kirino, 1982; Lin et al., 1990; Ito et al., 1987) and in rats (Nakano et al., 1990). In the same way, different neuronal loss areas have been described in cortex and hippocampus after chronical cerebral hypoperfusions (Kudo et al., 1993). All these studies were performed in ipsilateral levels. Waite and col. (1992) described a decrease in neuronal population and in the ventrobasal volume after contralateral lesions in the infraorbital nerve. Also, lesions by aspiration of the frontal or parietal cortex made in cat fetuses, produced loss of about 25% of neurons in the ipsilateral levels but the changes in contralateral side were insignificant (Loopuijt et al.,1995).
Although our results showed no changes in contralateral neuronal populations, some degenerative marks like presence of dark neurons, have been observed. Traditionally, these neurons have been related with poor fixative processes (Cammermeyer, 1961; Mugnaini, 1965; Jones y Powell, 1970), with postmortem manipulation of the tissue and with perfusion with hypertonic fluids (Cammermeyer, 1979). More recent studies revealed that these neurons are generated in vivo as an acute or delayed consequence of several pathological situations and lesions (Kleiner et al., 1986; Gallyas et al., 1992a; 1993; Ong and Garey, 1993; Czurko and Nishino, 1993). However, although the morphological characteristics are perfectly known, only one hypothesis explains their appearance (Gallyas et al., 1992a): an initial and localized damage is extending to all neuronal structure, producing a global deorganization. This theory could explain the presence of dark neurons with similar morphological characteristics in processes with different etiology.
In our study, the increase in SDN is parallel to a reduction in NN, but no neuronal loss have been detected, so it could be possible that the NN are replaced by SDN. Furthermore, we distinguished a third neuronal population (LDN). No changes were observed in the number of these neurons with a light hyperchromasia and without estructural changes. This stability could indicate that these neurons are not an intermediate between NN and SDN. We unknown exactly their significance although it may be possible that they appear spontaneously in all the animals, and it could be possible too, that these neurons could be consequence of manipulation of the tissue, although the animals used in this study were perfused and fixed.
The distribution of the different neuronal types considered in our study, across the cortical thickness showed a decrease in the number of NN and an increase in SDN in practically all the layers. It could be consequence of the inespecifity of the lesion. Clarke and Nussbaumer (1987) observed an increase in the SDN population in some cerebral areas after global ischemic processes. These increase was especially significant in cortical layers II, III and V in ipsi and contralateral hemispheres.
By other hand, it is known that neuronal degeneration is accompanied with an atrophy proccess characterized by a decrease in soma, nucleus and nucleolus areas (Finch, 93). In accordance with our results, the SDN could be considered atrophy neurons. In fact, these neurons presented slower values of soma, nucleus and nucleolus areas. Finally, the NN of lesioned animals showed increases in these parameters and it could be related with some reaction capacity. Other significative results are the values obtained for form index. SDN presented values higher than one; it could be indicative to the structural deorganization and atrophy.
In summary, the quantitative and morphological changes reported here may be responsible for some of the neurophysiological impairments associated with cortical lesions.
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[Cell Biology & Cytology] |
[Neuroscience] |
[Physiology] |
