THE ANATOMICAL RECORD 248:137–141 (1997) Quantitative Morphological Changes in Neurons From the Dorsal Lateral Geniculate Nucleus of Young and Old Rats ALICIA VILLENA,1* FLORENTINA DÍAZ,1 VIRGINIA REQUENA,1 ISABEL CHAVARRÍA,1 FRANCISCA RIUS,2 AND IGNACIO PÉREZ DE VARGAS1 1Department of Normal and Pathological Morphology, Faculty of Medicine, University of Málaga, Málaga, Spain 2Department of Public Health, Faculty of Medicine, University of Málaga, Málaga, Spain ABSTRACT Background: We studied the morphological changes occurring in neurons from the dorsal lateral geniculate nucleus (dLGN) during aging by analysing the size and shape of cell bodies and nuclei. Methods: Male albino Wistar rats, aged 3, 18, 24, and 30 months, were used. After appropriate tissue preparation and following the usual histological procedure, the profiles of 1,920 neuronal bodies and nuclei were drawn using a camera lucida. Data was later recorded and processed with a semiautomatic image analyser. Results and conclusions: We observed that dLGN neurons do not change in size from the age of 3–24 months. Between 24 and 30 months, the soma and nucleus of the cell undergo hypertrophy, 32.8% and 35.6%, respectively, when compared to those from 3-month-old animals (P F 0.01). Furthermore, we found a high correlation between cell body size/nucleus size, which does not disappear with age. The r values (correlation coefficient) were 0.7998, 0.8662, 0.8433 and 0.7304, and R2 (determination coefficient) was equal to 0.6397, 0.7504, 0.7112, and 0.5335. These latter values show that in 63.97%, 75.04%, 71.12%, and 53.35% of cases, respectively, modifications in somata size were accompanied by similar changes in nucleus size, and vice-versa. The study of the shape of the soma and nucleus of the cell revealed that both structures have a rounded-oval configuration that does not change in a significant way from adulthood to old age. Anat. Rec. 248:137–141, 1997. r 1997 Wiley-Liss, Inc. Key words: dLGN; aging; quantitative; size; shape The morphological, neurochemical, and physiological characteristics of the dLGN have been exhaustively studied from postnatal development (Guillery, 1966; Parnavelas et al., 1977; Heumann and Rabinowicz, 1980; Gottlieb et al., 1985; Aggelopoulos et al., 1989; Villena et al., 1989, 1991; Dı́az et al., 1994) to adulthood (Casagrande and Norton, 1991; Grossman et al., 1973; Montero and Zempel, 1986; Pasik et al., 1990). However, data are scarce regarding changes occurring in this and other brain regions during aging. Such data should help to identify which brain areas are more susceptible to aging and also what are the specific changes taking place in each region during senescence. In general, the basic morphological modifications undergone by neurons during aging are known, but the findings are controversial. Thus there is no agreement regarding the different parameters currently under study, e.g., neuronal size, extension of the dendritic tree, or the number of synapses per cell (Curcio and Hinds, 1983; Coleman and Flood, 1986; Markus et al., 1987; Flood and Coleman, 1988; de Lacalle et al., 1991; Crespo et al., 1992; Stroessner-Johnson et al., 1992; r 1997 WILEY-LISS, INC. Flood and Coleman, 1993; Peinado et al., 1993; Rance et al., 1993). Similarly, it is argued whether or not neuronal loss occurs during old age (Diamond et al., 1977; Curcio and Coleman, 1982; Satorre et al., 1985; Sturrock, 1989). All these data are fundamental for understanding the aging process in the brain and needed for interpreting results coming from the application of physiological, biochemical, and pharmacological methods. In this study we have analysed the morphological modifications occurring in the dLGN neurons from adulthood to senescence. We used quantitative methods that enabled us to assess changes in the size and shape of the soma and nucleus of the cell. In this sense, the quantification of somatic and nuclear size provides us with very useful information to evaluate the integrity of the neurons left in the senescent brain. Received 23 July 1996; accepted 23 December 1996. *Correspondence to: Department of Normal and Pathological Morphology, Faculty of Medicine, University of Málaga, 29080-Málaga, Spain. 138 A. VILLENA ET AL. MATERIALS AND METHODS Animals Sixteen male albino rats (Wistar strain) were used in this study. They were subdivided into four groups of 3, 18, 24, and 30 months old (n 5 4 per age group). The mean lifespan of this strain of rats is 27 months (Janicke et al., 1985). Animals were provided with food and water ad libitum and maintained in a temperaturecontrolled room with a 12-hour dark/light cycle. Their weight was between 275 6 21 gm and 410 6 30.8 gm (Table 1). Tissue Preparation Animals were anaesthetised with 8% chloral hydrate (0.1 ml/30 gm weight). Fixation was achieved by intracardiac perfusion with buffered 10% formaldehyde (after a saline wash). The entire brains were then removed and a block containing the dLGN dissected out and placed in the same fixative for 48 hours. Thereafter, they were processed in the standard way for embedding in paraffin. Serial 8 µm coronal sections containing the dLGN were made through the block’s entire length and then stained with gallocyanine (Einarson, 1951). Morphometric Study A morphometric analysis of the size and shape of the neuronal soma and nuclei was carried out using these sections. The outlines of these cells were drawn with a camera lucida attached to a Leitz Orthoplan microscope using a 3100 oil objective. A total of 1,920 neuronal bodies and nuclei profiles were measured (480 per group, i.e., 120 per animal). The data was recorded and processed with a semiautomatic image analyser (Kontron MOP-AM2). We chose only those neuron cell bodies whose nucleoli were clearly visible (Konigsmark, 1970). Neuron cell bodies were distinguished from glia on the TABLE 1. Mean weight (mean 6 SEM) of rats from each age group Age (months) Mean weight (g) 275 6 21 375 6 38.9 410 6 30.8 370 6 10 3 18 24 30 basis of size (neurons are much larger than glia in the dLGN), the presence of a large and relatively pale nucleus, and well-defined Nissl material in the cytoplasm of neurons. The following soma and nuclear parameters were calculated: to evaluate the size (area, perimeter, and maximum diameter) and to evaluate the shape (form factor). Statistical Analysis The data are presented as mean 6 SEM (standard error mean). Statistical analysis was performed first by a computerised Kolmogorov-Smirnov test to evaluate whether or not the data followed a normal distribution. Because this test refuted the hypothesis of a normal distribution in most parameters, the results obtained were compared using the Kruskall-Wallis test (P , 0.05 and P , 0.01). Finally, bivariate linear models were used to describe the statistical relationship between soma area/nuclear area. RESULTS Size Parameters Area, perimeter, and maximum diameter of neuronal cell bodies and nuclei followed similar modifications during aging (Tables 2 and 3). Thus we observed that no significant changes occurred in any parameter from the 3rd to the 24th month except for a slight increase (7.2%, P , 0.05) in the maximum somatic diameter in the 24-month-old rats when compared to the 3-month-olds. However, from the 24th month onward a significant increase (P , 0.01) is observed in each parameter reaching their peak value in the 30th month (Tables 2 and 3). In order to compare the behaviour of the area parameter among the different age groups, we analysed the frequency distribution in the cell body and in the nucleus (Fig. 1). At the age of 3 months, it was observed that in the neuronal soma (Fig. 1A), the main percentage (26.66%) had an average size of between 110–130 µm2; at the age of 18 months, 25% of the soma were between 130–150 µm2; at the 24th month 26.66% were between 150–170 µm2; and finally at the age of 30 months, 27.5% had a size of 170–190 µm2. So, as the animal advanced in age a deviation toward the right occurred in the frequency distribution. In the nuclear area we observed a similar pattern: 35% and 22.5% of the 3- and 18-month- TABLE 2. Size and shape somatic parameters (mean 6 SEM) between 3 and 30 months of age1 Age (months) 3 Area (µm2) Perimeter (µm) Maximum diameter (µm) Form factor 18 24 30 138.06 6 2.45 141.98 6 2.88 150.23 6 2.83* 183.47 6 2.88**,***,**** (100) (102.8) (108.8) (132.8) 44.54 6 0.42 44.90 6 0.45 45.42 6 0.54 50.96 6 0.41**,***,**** (100) (100.8) (101.9) (114.4) 13.73 6 0.19 14.66 6 0.20 14.73 6 0.38 15.73 6 0.21**,***,**** (100) (106.7) (107.2) (114.5) 0.874 6 0.006 0.879 6 0.006 0.892 6 0.006 0.884 6 0.005 (100) (100.5) (102) (101.1) 1Percent of values with respect to 3 months old in parens. *P , 0.05 as compared to 3–24 months old. **P , 0.01 as compared to 3–30 months old. ***P , 0.01 as compared to 18–30 months old. ****P , 0.01 as compared to 24–30 months old. 139 CHANGES IN NEURONS FROM dLGN DURING AGING TABLE 3. Size and shape nuclear parameters (mean 6 SEM) between 3 and 30 months of age1 Age (months) Area (µm2) Perimeter Maximum diameter (µm) Form factor 3 18 24 30 78.61 6 1.79 (100) 33.10 6 0.34 (100) 10.29 6 0.15 (100) 0.922 6 0.005 (100) 80.87 6 2.19 (102.8) 33.58 6 0.42 (101.4) 10.83 6 0.18 (105.2) 0.906 6 0.006 (98.2) 85.03 6 1.73 (108.1) 34.84 6 0.34* (105.2) 10.92 6 0.15 (106.1) 0.900 6 0.006 (97.6) 106.60 6 1.96**,***,**** (135.6) 38.46 6 0.32**,***,**** (116.1) 12.02 6 0.15**,***,**** (116.8) 0.925 6 0.005 (100.3) 1Percent of values with respect to 3 months old in parens. *P , 0.05 as compared to 3–24 months old. **P , 0.01 as compared to 3–30 months old. ***P , 0.01 as compared to 18–30 months old. ****P , 0.01 as compared to 24–30 months old. Fig. 1. Frequency distribution for somatic (A) and nuclear (B) cross-sectional areas. Comparison between the different ages shows a tendency to increasing sizes with advancing age. old rats, respectively, had an average nucleus size of 70–85 µm2; 32.5% of the 24-month-old group had an average between 85–100 µm2; and at the age of 30 months, 36.6% showed a nucleus size of between 100–115 µm2 (Fig. 1B). To determine the degree of dependence between nuclear and somatic size and possible changes as age advances, we calculated the correlation coefficient (r) and the determination coefficient (R2 ) as well as their corresponding regression lines in each age group (Fig. 2A–D). These revealed a high correlation at all ages studied with values of 0.7998, 0.8662, 0.8433, and 0.7304 for r, and 0.6397, 0.7504, 0.7112, and 0.5335 for R2. Therefore, changes in somatic size are accompanied by similar changes in nuclear size, and vice-versa. Shape Parameter We calculated the form factor to detect possible modifications in neuron cell bodies and nuclei shape during the process of aging. We observed that in both cases values were close to 1, but more so in the nucleus (Tables 2 and 3). This shows that both the neuronal cell body and the nucleus have a rounded-oval shape. Aging does not seem to affect this parameter in a significant way (Tables 2 and 3). DISCUSSION The growing interest in the process of aging has encouraged great advances and diversification in neurobiological methodologies, which are applicable not only to humans but to other animal species. Given the difficulty of carrying out this type of research on humans, laboratory animals are used. Among these, rodents present many advantages (Masoro, 1990). Changes found in rodents can lead to an understanding of the processes taking place during human aging (John, 1972). Our investigation was carried out on dLGN neurons from albino rats. The aim was to explore some basic neurobiological questions concerning cell aging and death, in line with earlier studies in which we had already analysed some other aspects of this nucleus during postnatal development and adulthood (Villena et al., 1989, 1991; Dı́az et al., 1994). We performed a neuronal morphological analysis following morphometric methodology. Size parameters such as area, perimeter, and maximum diameter were evaluated. Area was given more importance as it is considered the most useful parameter regarding neuronal size evaluation (Ewart et al., 1989; de Lacalle et al., 140 A. VILLENA ET AL. Fig. 2. Regression lines illustrating the relationship between somatic area/nuclear area at 3 (A), 18 (B), 24 (C), and 30 (D) months old. r 5 correlation coefficient. R2 5 determination coefficient. 1991; Peinado et al., 1993; Rance et al., 1993). We also analysed shape parameters such as form factor with values ranging from 0 to 1, the latter being the definition of a sphere. Our results show that there are no significant changes in somata and nuclear size between the ages of 3 and 24 months. However, between the 24th and the 30th months, an important increase in all these parameters is observed (P , 0.01). Thus the area of the cell body increases from 150.23 6 2.83% µm2 to 183.47 6 2.88 µm2 and that of the nucleus increases from 85.03 6 1.73 µm2 to 106.60 6 1.96 µm2. The total area increase, as compared with the young adult (3 months), is 32.8% and 35.6% for the somata and the nucleus, respectively. This coincides with observations of Ahmad and Spear (1993) in monkey dLGN, who found an increase during aging of 31% and 36% in the neuronal bodies of the magno and parvicellular layers, respectively. The increase observed in the mean somata size could be due to a decrease in the soma of small neurons and/or an increase in larger neurons’ soma as the animal’s age advances (Sturrock, 1989; Stroessner-Johnson et al., 1992). In this regard, by analysing the frequency distribution, we demonstrated that both processes are present. Thus at the 3rd month 14.99% of neurons are small (,110 µm2 ), but at the 30th month this falls to 5%. Furthermore, we observed that at the age of 3 months most of the neuronal population (73.32%) have somata ranging in size from 110–170 µm2, but in the 30th month, 56.66% of them have somata ranging in size from 170–210 µm2. In the 3-month-old animals neuron cell bodies larger than 210 µm2 are not found, but in the 30-month-old rats these neurons represent 19.16%. The analysis of nuclear size revealed the self-same facts. It can be inferred from our study that dLGN neurons undergo a process of hypertrophy during senescence, i.e., from the 24th month onward in the Wistar rat. This phenomenon has been observed in different centres of the central nervous system by other researchers (Flood and Coleman, 1988; de Lacalle et al., 1991; Ahmad and Spear, 1993; Stroessner-Johnson et al., 1992; Rance et al., 1993). Nevertheless, other studies have found just the opposite, i.e., stability and a shrinkage in size (Luine et al., 1986; Peinado et al., 1993; Willot et al., 1994). In some cases, the discrepancies might be due to the varying susceptibility of the different centres of the nerve system to aging, and in others to the species and breeds employed as well as the different methodologies. From previous results (submitted), we also know that during aging there is an increase in volume of dLGN and a decrease in neuronal density and number/mm3. Thus the somatic hypertrophy observed in this study CHANGES IN NEURONS FROM dLGN DURING AGING from the 24th month onward could be explained as a compensatory mechanism to maintain the same total somatic volume in spite of the increase in the total volume in the dLGN. This, according to Hinds and McNelly (1977), Flood et al., (1985), and Coleman and Flood (1986), could be related to an increase in the dendritic tree in order to maintain the existing synaptic connections, despite the increase in the dLGN total volume, or perhaps, to compensate for the decrease in the number of synapses and/or in their efficiency during aging (Ahmad and Spear, 1993). The data concerning the degree of correlation or dependency between the size of the soma and nucleus of the cell and their possible modifications during aging have proved of interest. By analysing the correlation and determination coefficients (r and R2, respectively) we found that there was a high reciprocal dependence between both parameters, which does not disappear in the older animals. In 63.97%, 75.04%, 71.12%, and 53.35% of cases, modifications in somata size were accompanied by a similar change in nuclear size, and vice-versa. These data corroborate that found by de Lacalle et al. (1991) during the same period in the nucleus basalis of Meynert. Finally, when the cell body and the nucleus shape were studied, it was confirmed that dLGN neurons and their nuclei have a rounded-oval shape with a value close to 0.90 for the cell body and slightly higher for the nucleus. It is important to note that this parameter does not change from adulthood to senescence. 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