- Email: [email protected]
Age-related changes in basic fibroblast growth factor-immunoreactive cells of rat substantia nigra
Mechanisms of Ageing and Development 82 (1995) 73-89
ELSEVIER
andllBwlqnmnt
A.ge-related changes in basic fibroblast growth factor-immunoreactive cells of rat substantia nigra Ivanka S. Lolova*a, Stephan R. Lolovb aInstitute of Physiology, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., bl. 23, 11 I3 Sofia, Bulgaria bInstitute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., bl. 23, 1113 Sofia, Bulgaria
Received 3 March 1995; accepted 9 May 1995
Abstract
Immunohistochemistry and computer-assisted image analysis were used to examine the age-related changes in bFGF-immunoreactivity in rat substantia nigra (SN). Distribution pattern, number, size and staining intensity of bFGF-immunoreactive (bFGF-ir) cells in pars compacta and pars reticulata of 3-, 12- and 26-month-old rats were compared. The overall distribution of bFGF-immunoreactivity was similar in the three age groups, but changes in the morphological appearance of bFGF-ir somata and processes occurred in aging. The results demonstrated a significantly reduced number of bFGF-ir cells in pars compacta (by 56.87%) and pars reticulata (by 30.4%) in 26-month-old rats compared to 3-month-old rats. The reduction of the cell number did not occur smoothly and equally in the two parts of SN. The quantitative analysis clearly indicated a significant decrease in the size of bFGF-ir neurons in pars compacta (by 18.1%) and pars reticulata (by 14.15%) of 26-month-old rats compared to 3-month-old rats. Compared to 3-month-old rats, a 19.77% and 17.83% increase in the staining intensity was observed in the remaining bFGF-ir neurons of pars compacta and pars reticulata, respectively in 26-month-old rats. Since there was no correlation between the decreased size and increased staining, it is most probable that the intensification of the staining intensity of bFGF-ir neurons was a compensatory response to the cell death. Keywords: Aging; bFGF-immunoreactivity; ysis
Substantia nigra; Computer-assisted
* Corresponding Author, e-mail: [email protected]. 0047-6374/95/$09.50
Q 1995
SSDlOO47-6374(95)01599-U
Elsevier Science Ireland Ltd. All rights reserved
image anal-
14
IS.
Lolova, S.R. Lolov 1 Mech. Ageing Dev. 82 (1995) 73-89
1. Introdbction Acidic and basic fibroblast growth-factors (aFGF, bFGF) are the most thoroughly studied members of the fibroblast growth factor family. bFGF and FGF receptors are widely distributed in various brain regions, including substantia nigra (SN) [l-6]. bFGF exerts neutrophic effects on the neurons of SN. It stimulates the histogenesis, survival and differentiation of the mesencephalic dopaminergic neurons in vitro and in vivo [7-91. bFGF protects and increases the repair of MPTP- and 6-hydroxydopamine-lesioned nigrostriatal neurons [lo- 121. Neuronal atrophy and loss occur in many brain regions during normal aging. The extent of neuronal loss in SN is unclear [13]. Some authors have reported an age-related loss of neurons in SN of rodents and humans [14-171. According to other authors, there is no reduction in the number and size of dopaminergic neurons in SN of aged animals [l&19]. There is a hypothesis that an inadequate trophic support could be a contributing factor for the neuronal atrophy and loss in normal and pathological aging [20]. Changes in bFGF immunoreactivity have been observed in the brains of patients with Alzheimer’s, Parkinson’s and Huntington’s diseases [21-241. However, little is known of bFGF-immunoreactive (bFGF-ir) neurons during normal aging [17,25,26]. We failed to find any quantitative analysis of bFGF-immunostaining of rat SN cells in advanced age. In the present study, we compared the distribution pattern of bFGF-immunoreactivity and the number, size and staining intensity of bFGF-ir neurons in SN of 3-, 12- and 26-month-old rats using a computerized image analysis system. The results showed a reduction in the number and size, but an increase in the staining intensity of bFGF-ir neurons of SN in aging. 2. Materials and methods 2.1. Immunohistochemistry Thirty-three male Wistar rats aged 3 months (n = 1l), 12 months (n = 11) and 26 months (n = 11) were used. The mean life span of the rats was 22-24 months. Under deep ether narcosis, the rats were perfused transcardially with phosphate buffered saline (PBS) followed by a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 0.15% picric acid in 0.1 M phosphate buffer (PB), pH 7.4. Midbrains were postfixed in the same fixative without glutaraldehyde for one night at 4°C and then soaked in 0.1 M PB containing 20% sucrose for 2 days at 4°C. Coronal sections, 30 pm thick, through the rostrocaudal extent of SN were cut on a freezing microtome (Reichert-Jung, Austria) and washed in 0.1 M PBS with 0.3% Triton X-100 (PBST) overnight at 4°C. Free-floating sections were incubated with rabbit-bFGF-antiserum (Sigma, St. Louis, MO) diluted 1:400 in PBST containing 1% bovine serum albumin (PBST-BSA) for 48 h at 4°C. Then the sections were incubated in swine anti-rabbit IgG (DAKO) diluted 1:20 and in rabbit peroxidase-
I.S. Lolova, S.R. Lolov) Mech. Ageing Dev. 82 (1995) 73-89
15
antiperoxidase complex (PAP, DAKO) diluted 1:200 according to the double amplification procedure of the PAP technique [27]. Prior to each incubation the sections were washed in PBST. The peroxidase activity was revealed with 0.02% 3-3’, 5-S’-diaminobenzidine and 0.05% hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.6. Control sections were incubated with rabbit IgG (0.1 pg/ml) or normal rabbit serum (1:lOOO) instead of the primary antibody. The sections were mounted on chrome-gelatin-coated slides and air-dried for 24 h. The immunostaining was intensified in 0.005% osmium tetroxide for 30 s. The sections were dehydrated and embedded in Entellan. In leach experiment, sections from one animal of each age group were processed simultaneously. 2.2. Quantitative analysis To assess the effects of aging on bFGF-immunoreactivity in SN, we compared the following parameters in 3-, 12- and 26-month-old rats: the number of bFGFir and Nissl-stained cells and their relationship; the rostrocaudal distribution of bFGF-ir and Nissl-stained cells; and the size and staining intensity (optical density, OD) of bFGF-ir cells. The parameters were examined in pars compacta and pars reticulata of SN. All quantitative analyses were performed on an image analysis system consisting of an Jenaval photomicroscope, a Hitachi videocamera, an IBM computer and morphometry software developed in the Biophysical Institute, Bulgarian Academy of Sciences (Dr B. Balev). 2.2. I. Determination of the volume of pars compacta and pars reticulata, the density and the true number of the N&l-stained and bFGF-ir cells For estimation of the true number of Nissl-stained and bFGF-ir cells, it was necessary to measure the volume of pars compacta and pars reticulata of SN as well as the cell density. Volume. Substantia nigra of nine rats (three in each age group) was cut serially from the most rostra1 to the most caudal levels. All sections were collected in subsets with five sections. Every first section of the subsets was stained by Nissl and every second section was immunostained for bFGF. The cross-sectional areas of both pars compacta and pars reticulata were traced bilaterally by a pointing device on all Nissl-stained sections sequentially starting with the most rostra1 and ending with the most caudal section with a x 3.2 objective lens. These areas and the section thickness (30 pm) were used for volume estimation by a formula based on the Simpson rule for integration [281. Cell density. To count the cell density, an image analyzer was used with a x 25 objective lens. With a threshold value determined empirically, the image was converted from tonal to binary form. Structures with areas less than 50 pm2 were automatically erased to avoid counting positive glial cells. Two sections at rostral, middle and caudal levels stained by Nissl and two adjacent bFGF-immunostained
76
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
sections at the same levels of pars compacta and pars reticulata of each rat were measured. The levels along the rostrocaudal axis of SN were determined according to the atlas of Pellegrino et al. [29]. The averaged values from the measurements of the two sections at each level were used. The number of cells per unit volume was counted according to the equations for a polydispersed system described by Royet LW. True cell number. Estimates of the true number of Nissl-stained and bFGF-ir cells in each pars compacta and pars reticulata were calculated by multiplying the number of cells per unit volume by the volume of the individual pars. On the basis of the cell sectional areas, the numbers of small (50- 150 pm’), medium (150-300 pm2) and large (300-600 pm2) size groups of Nissl-stained and bFGF-ir cells were automatically determined. 2.2.2. Rostrocaudal distribution of N&l-stained pacta and pars reticulata
and bFGF-ir cells in pars com-
To assess whether the distribution of Nissl-stained and bFGF-ir cells along the rostrocaudal axis of SN was changed in aging, we compared the cell density at rostral, middle and caudal levels in the three age groups. 2.2.3. Determination of the size and optical density of bFGF-ir neurons The size and OD of bFGF-ir neurons were measured at the middle level of SN with a x 40 objective. Pilot measurements revealed that there were no significant differences in size and OD of bFGF-ir neurons at the rostral, middle and caudal levels of pars compacta and pars reticulata in 3-month-old rats. Therefore, the measurements at the middle level, where the oculomotor nerve emerges, reflected accurately the size and OD of bFGF-ir neurons in the overall SN. Twenty randomly selected neurons of pars compacta and 20 neurons in the lateral subdivision of pars reticulata in every rat (n = 33) were measured. Neurons cut through the level of the nucleus or the stem of the processes were traced by a pointing device. Every cell was measured three times and the averaged values of the size and OD were used. The OD of bFGF-ir cells was expressed on an arbitrary gray scale ranging from 0 to 1 (0, OD of the glass slide; 1, maximal staining intensity). Recordings were also made of the adjacent background in the cerebral peduncle and the values were subtracted from the cellular values. To establish if there was a correlation between the size and optical density of bFGF-ir neurons, we determined the correlation coefficients. The accuracy of the estimates measured as mean coefficient of variation was 6.1% and 10.96%, respectively for neuronal size and OD determination. 2.2.4. Statistical analysis
All calculations were made for each animal before the means and standard errors of the means were determined for an age group. The statistical differences were analyzed by ANOVA test followed by Newman-Keuls test.
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
71
3. Results 3.1. Distribution of bFGF-immunoreactivity
in substantia nigra
The overall distribution of bFGF-immunoreactivity in SN was similar in the three age groups. bFGF reaction material was observed as a diffuse brown staining within the somata and processes of pars compacta and pars reticulata (Fig. 1). No reaction product was found in the neuronal nuclei. The SN cell somata in 3- and 12-monthold rats were relatively uniformly immunostained. In contrast, some somata in 26-month-old rats displayed an intensification of the immunostaining in comparison with the adjacent somata and the somata of SN in the younger rats. While an even distribution of the reaction product throughout the cell somata in pars compacta and pars reticulata was found in 3- and lZmonth-old rats, a perinuclear or spot-like accumulation of this product was seen in 26-month-old rats. The cell processes became short, thin and sparse in 26-month-old rats. The immunostaining of the neuropil of 3-month-old rats was punctiform and at higher magnification varicose fibers were seen around the cell somata and blood vessels. With advancing age, varicose fibers became more and more dark, swollen and wrinkled. 3.2. Volume of substantia nigra No significant differences in the volume of the left and right pars compacta and pars reticulata in 3-month-old rats were found. Thus, the results included the measurements of the volume of both sides. ANOVA demonstrated a significant influence of age on the volume of SN. The volume of whole SN in 26-month-old rats was significantly decreased [F(2,15) = 5.46, P < 0.051 compared to 3-month-old (by 26.7%) and 12-month-old (by 22.05%) rats. Table 1 shows that this decrease was mainly due to a significant reduction [F(2,15) = 12.21, P < O.OOl] of the volume of pars compacta in 26month-old rats compared to 3-month-old (by 51.49%) and 12-month-old (by 38.75%) rats. A 17.13% reduction in the volume of pars reticulata in 26-month-old rats as compared to 3-month-old rats was not significant. 3.3. Number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata Pars compacta. A significant effect of age on the number of Nissl-stained [F(2,6) = 7.80, P < 0.051 and bFGF-ir [F(2,6) = 8.58, P c 0.051 cells was found by ANOVA. The total number of Nissl-stained cells in 26-month-old rats was significantly decreased compared to 3-month-old (by 51%) and 12-month-old (by 41%) rats (Table 2). An even more pronounced reduction in the total number of bFGF-ir cells was observed in 26-month-old rats compared to 3-month-old (by 56.87%) and lZmonth-old (by 48.49%) rats. The decrease in the total number of Nissl-stained (by 16.7%) and bFGF-ir (by 15.78%) cells in lZmonth-old rats as compared to 3-month-old rats was not significant. As shown in Table 2, the number
78
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
of NM-stained cells in small, medium and large size groups was significantly decreased (by 45.31%, 61% and 67.14%, respectively) in 26-month-old rats compared to 3-month-old rats. Also, a significant reduction in the number of bFGF-ir cells in small, medium and large size groups (by 55.36%, 56.42% and 71.08%,
Fig. 1. bFGF-immunoreactivity in pars compacta (A$) and pars reticulata (3,D) of SN in 3-month-old (A,B) and 26-month-old (C,D) rats. The arrows show few short, thin and poorly visible processes. The arrowheads point to dark swollen fibers and varicosities in C and D in contrast to the fine fibers in B. Magnification: x 400.
IS.
Ldova,
S.R. Ldov 1 Mech. Ageing Dev. 82 (1995) 73-89
Table 1 Volume (mm3) of the whole SN, pars compacta and pars reticulata of the three age groups Age (months)
Side”
Whole SN
Pars compacta
Pars reticulata
3
L R L R L R
3.119 4.089 2.160 3.009 4.064 3.395
1.147 1.104 0.834 0.677 1.334 0.944
2.632 2.985 1.926 2.332 2.730 2.451
Mean S.E.M.
3.516 0.227
1.007 0.096
2.509 0.149
L R L R L R
3.919 4.027 2.705 3.062 2.906 3.171
1.037 1.005 0.720 0.675 0.667 0.716
2.942 3.022 1.985 2.381 2.239 2.455
Mean S.E.M.
3.308 0.229
0.803 0.069
2.505 0.165
L R L R L R
2.801 2.171 2.109 1.908 3.007 2.864
0.530 0.545 0.306 0.414 0.647 0.519
2.211 2.232 1.803 1.494 2.360 2.345
Mean S.E.M.
2.578 0.185
0.494 0.048
2.084 0.145
12
26
“L, left side; R, right side.
respect:ively) was established in 26-month-old rats compared to 3-month-old rats. In 3- and lZmonth-old rats, bFGF-ir cells accounted for 91% of the Nissl-stained cells. In 26-month-old rats, this percentage was slightly (by 9%) reduced. Pars reticufata. A decrease in the total number of Nissl-stained (by 21.3%) and bFGF-ir (by 30.4%) cells of 26-month-old rats compared to 3-month-old rats was observed. The number of Nissl-stained cells in small, medium and large size groups was also reduced (by 12.2%, 40.35% and 49.3%, respectively) compared to 3-monthold rats, but the difference was significant only in the medium size group [F(2,6) = 5.82, P < 0.051. The number of bFGF-ir cells in small, medium and large size groups of 26-month-old rats was decreased by 31.56%, 23.38% and 43%, respectively compared to 3-month-old rats. In 3-month-old rats, the vast majority of Nissl-stained cells exhibited bFGF-immunoreactivity. In 12- and 26-month-old rats, bFGF-ir cells accounted for 89% of the Nissl-stained cells. It should be noted that some cells in the medium and large size groups were not bFGF-ir. However, their amount was relatively small and obviously did not influence the percentage of bFGF-ir of the Nissl-stained cells.
I.S. Lo/ova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
80
Table 2 shows that the decrease in the number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata in aging was more pronounced in medium and large than in small size groups. To estimate whether the decrease in the number of medium and large sized cells was accompanied by a relative increase in the number of small sized cells, the ratios of small to medium and large sized cells in each population were calculated. In fact, during aging this ratio of Nissl-stained cells
Table 2 Number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata of rat SN of three age groups
Age
Nissl-stained cells (size group)
bFGF-ir cells (size group)
(months) Total
Small
Medium
Large
Total
Small
Medium
Large
34 105 23 182 33 988
22 857 16449 23 084
8312 5458 8861
2111 1045 1492
29 457 21 280 32 083
24 970 14 667 26 475
3953 5431 4819
484 975 740
Mean S.E.M.
30 425 3622
20 797 2175
7544 1055
1549 309
27607 3253
22038 3711
4734 429
733 142
12
30 665 22731 22 632
19 901 19 568 15 184
8244 2842 6401
1755 322 953
29 168 19 705 20 881
24 618 15 987 16 324
4175 3284 3980
325 383 380
Mean S.E.M.
25343 2661
18218 1520
5829 1585
1010 415
23 251 2978
18 976 2823
3813 270
362 19
26
17 333 11 125 16 388
12 632 8945 12 532
3693 1905 3232
729 224 575
11 577 8835 15 305
10 025 7057 12 430
2194 1497 2499
113 201 323
Mean S.E.M.
14 948 1931
11 370 1213
2943 536
509 150
11 906 1875
9838 1554
2063 296
212 61
3
78 427 56 355 70 135
56 650 39 825 47 227
16 930 13 598 19 514
4477 2981 2912
79615 56712 69 858
61467 40747 55 548
14 660 14 094 12 585
2876 1480 1668
Mean S.E.M.
68 306 6437
47 901 4869
16 681 1712
3456 511
68 729 6635
52 587 6162
13 780 619
2008 437
12
78 468 62649 63 120
55 242 45215 44 443
17 015 14 174 16 239
5436 2859 2014
69 728 51907 63 012
55 620 41 567 46 670
12 563 9428 13 298
1476 772 2307
Mean S.E.M.
68079 5196
48 300 3478
15 809 848
3436 1029
61 549 5196
47 952 4107
11 763 1187
1518 444
26
65 113 46 029 50 136
48349 38 809 38 033
12 557 6484 10 810
3742 435 1082
48 686 41930 52938
39 334 31 775 36 864
8594 9220 13 861
608 781 2049
Mean S.E.M.
53 760 5799
41 730 3317
9950 1805
1753 1012
47 851 3205
35 991 2225
10 558 1661
1146 454
3
Pars compacta
Pars reticulata
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
81
Table 3 Percentage of the mean values of small, medium and large size cells of the total number in the three age groups and their ratios
Age
bFGF-ir cells (size group)
Nissl-stained cells (size group)
(months) Small
Medium
Large
Ratio
Small
Medium
Large
Ratio
Pars compacta
3 12 26
68.4 71.9 76.1
24.8 23.0 19.7
5.1 4.0 3.4
2.32 3.41 3.45
79.8 81.6 82.6
17.1 16.4 17.3
2.7 1.6 1.8
4.23 4.53 4.30
Pars reticulata
3 12 26
70.1 70.9 77.6
24.4 23.2 18.5
5.1 5.0 3.3
2.38 2.52 3.92
76.5 77.9 75.2
20.0 19.1 22.1
2.9 2.5 2.4
3.34 3.68 3.26
These ‘data were derived from the data presented in Table 2.
increased in the two parts of SN, being significant in pars reticulata [F(2,15) = 6.01, P < 0.011 (Table 3). However, the ratio of small to medium and large sized bFGF-ir cells remained unchanged with aging. 3.4. Rostrocaudal distribution of NM-stained and par’s reticulata
and bFGF-ir cells in pars compacta
The quantitative analysis showed that in 3-month-old rats, Nissl-stained and bFGF-ir cells were distributed evenly in the rostrocaudal extent of pars compacta and pars reticulata (data not shown). In 26-month-old rats, the density of Nisslstained cells was decreased only at the caudal level of pars compacta and pars reticulata compared to the same level in 3- and lZmonth-old rats. Compared to 3and 12,-month-old rats, a reduction of the density of bFGF-ir cells in pars compacta and pars reticulata in 26-month-old rats was observed at the three levels, being more pronounced at the middle level than at the rostra1 and caudal levels. 3.5. Size and staining intensity of bFGF-ir neurons in substantia nigra In contrast to automatic binary processing of the cell number, the discrimination between bFGF-ir neurons and bFGF-ir glial cells by manual tracing in the determination of the cell size and OD was much more precise. Age had a significant influence on the size and OD of bFGF-ir neurons in SN (Table 4). The size of bFGF-ir neurons was significantly decreased in both pars compacta [F(2,30) = 9.56, P < 0.001, ANOVA] and in pars reticulata [F(2,30) = 8.43, 1’ < 0.01, ANOVA] of 26-month-old rats compared to 3-month-old rats (by 18.1% and 14.15%, respectively). No differences in the size of bFGF-ir neurons between 3- and lZmonth-old rats were found. A significant increase in the OD of bFGF-ir neurons was observed in pars compacta [F(2,30) = 4.18, P c 0.05, ANOVA] and pars reticulata [F(2,30) = 4.64, P c 0.05, ANOVA] of 26-monthold rats (by 19.77% and 17.83%, respectively) compared to 3-month-old rats. The OD of bFGF-ir neurons in 3- and lZmonth-old rats did not differ significantly.
82
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
Table 4 Area (pm2) and optical density (arbitrary units) of bFGF-ir reticulata in the three age groups
neurons in pars compacta and pars
Age (months)
Pars reticulata
Pars compacta
3
Mean S.E.M. 12
Mean S.E.M. 24
Mean S.E.M.
Area
OD
Area
OD
158.3 162.7 174.9 186.1 154.3 184.4 162.6 160.3 205.5 187.2 193.0
0.23 0.40 0.26 0.26 0.36 0.39 0.31 0.36 0.24 0.33 0.27
201.6 162.4 179.2 213.1 187.0 205.7 182.4 178.9 201.6 227.0 221.1
0.24 0.38 0.21 0.26 0.34 0.39 0.33 0.28 0.25 0.33 0.34
175.4 5.1
0.31 0.02
196.4 6.0
0.30 0.02
180.4 186.1 141.5 184.1 154.6 189.2 169.1 144.9 154.3 213.2 202.5
0.29 0.31 0.31 0.33 0.29 0.38 0.37 0.34 0.34 0.36 0.21
176.2 199.7 169.4 215.0 193.1 223.1 207.0 194.6 184.9 225.8 228.1
0.29 0.31 0.27 0.33 0.23 0.35 0.31 0.30 0.32 0.33 0.24
174.5 7.1
0.32 0.01
201.5 6.1
0.30 0.01
146.8 132.1 137.0 121.0 137.9 171.7 139.3 121.0 168.7 147.1 157.6
0.30 0.40 0.41 0.37 0.34 0.40 0.35 0.34 0.43 0.45 0.31
164.4 176.6 157.5 189.2 151.8 173.1 175.5 132.8 187.7 144.8 201.0
0.29 0.36 0.42 0.29 0.32 0.38 0.40 0.33 0.46 0.37 0.33
143.7 5.1
0.37 0.01
168.6 6.2
0.36 0.02
Each figure indicates mean value per animal from the measurement of 20 cells (n = 660 in pars compacta and n = 660 in pars reticulata for the three age groups).
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
pars
compacta 40
03
months
al2
months
a26
months
30 g ._ z ‘; g20 % sp 10
ZOO-250
250-300
300-350
350-400 sq.pm
pars
reticulata 40
30 0” ._ 2 ‘; g20 B S?
10
0
50-100
100-150
150-200
200-250250-300
300-350350-400 sq.pm
area
of
IleUrOns
Fig. 2. A histogram of the size distribution of bFGF-ir neurons in pars compacta and pars reticulata of SN in three age groups.
The histogram of bFGF-ir neurons showed a shift towards smaller surfaces in pars compacta and pars reticulata of 26-month-old rats compared to 3- and 12-month-old rats (Fig. 2).
84
IS. L&ova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
There was no correlation between the size and OD of bFGF-ir neurons in pars compacta (r = - 0.16, r = - 0.16, r = - 0.08, respectively) and pars reticulata (r = - 0.13, r = - 0.0004, r = - 0.025, respectively) in 3-, 12- and 26-month-old rats. Therefore, the increase in the OD was not due to the decrease in the size of bFGF-ir neurons. 4. Discussion The present results demonstrate an extensive distribution of bFGF-immunoreactivity in the cell bodies and neuropil of rat SN. It is well known that pars compacta consists mainly of dopaminergic cells [30] while pars reticulata contains predominantly GABA-ergic neurons [31]. Although a double immunostaining was not performed in this study, the large percentage of bFGF-ir of the Nissl-stained cells in the two parts of SN indicates that bFGF is localized in the dopaminergic and GABA-ergic cells. Our results are consistent with the data that the majority of dopaminergic neurons of the ventral midbrain of rats and humans are bFGF-positive [1,2,4,32]. It is suggested that scattered bFGF-ir cells in pars reticulata are GABA-ergic ones [2,5]. The percentage, herein described, of bFGF-ir of the Nissl-stained cells shows that bFGF-immunoreactivity is localized in small, medium and some large sized GABA-ergic cells of pars reticulata. This finding is in agreement with studies reporting a trophic action of bFGF not only on dopaminergic, but also on GABA-ergic neurons of rat SN [7,11]. Neuronal atrophy and loss are considered to be the main characteristics of the aging brain, but opinions still differ concerning their extent in SN of rodents [13]. One of the main findings of our quantitative analysis is the significant reduction in the number of Nissl-stained and bFGF-ir cells in rat SN during normal aging. This age-related decrease in the number of cells per nucleus is due not only to the volume reduction of SN. We estimate also a significant reduction in the density of bFGF-ir cells at the rostral, middle and caudal levels, and of Nissl-stained cells at the caudal level of SN. The quantitative analysis pointed out that the number of Nissl-stained and bFGF-ir cells in SN did not decrease smoothly with aging. The reduction was pronounced and significant between 12 and 26 months of age, but slight and insignificant between 3 and 12 months. Also, the reduction in the number of Nissl-stained and bFGF-ir cells was not the same in the two parts of SN. In pars compacta, the decrease in the number of both Nissl-stained and bFGF-ir cells began earlier and it was more pronounced than that in pars reticulata. These results are in accordance with data about a slow and gradual age-related loss of neurons in SN of rats, mice and humans demonstrated by different methods of staining and counting [14-17,33-351. Some authors, however, have not found any significant loss of dopaminergic neurons in SN of aged rats and mice [l&19,36]. When considering these controversies, it should be born in mind that species- and genotype-specific factors are also responsible for age-related neuronal atrophy and loss [13]. The reduction of the number of bFGF-ir cells could be explained by a true loss of these cells, by a decrease in bFGF content or by both. Some of the results
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
85
support the first explanation. The reduction of bFGF-ir cells in pars compacta is parallel in time and extent to the reduction of the number of Nissl-stained cells. Furthermore, the morphological appearance and quantitative analysis demonstrated a significant increase in the staining intensity of bFGF-ir neurons in pars compacta and pars reticulata in 26-month-old rats. These findings are in keeping with olbservations about age-related changes in the number of bFGF-ir and tyrosine hydroxylase-ir neurons of pars compacta of humans and mice SN [17,33]. The possib:ility that other nigral cells lose bFGF before their death should also be considered. Thus, we could explain why the decrease in the number of bFGF-ir cells in pars reticulata precedes the reduction of Nissl-stained cells. Moreover, the percentage of bFGF-ir of the Nissl-stained cells should not alter in aging if a part of the cells dies without loss of bFGF content. The quantitative analysis showed that this percentage decreased, though slightly, in pars compacta and pars reticulata. Therefore, our studies provided evidence for the presence of a true cell loss and aldditional loss of bFGF content in some cells of pars compacta and pars reticulata of rat SN with advancing age. To Idetermine whether the estimated cell loss was accompanied by an actual cell atrophy, we had to analyze size groups in the total cell population and their ratios. In the: current literature there are much discrepancies concerning the size of the neurons in rat SN (see [37] for review). Also, the discrimination of the small neurons and large glial cells on the basis of the cytological characteristics is impossible by computer-assisted image analysis. The cell sizes have been used as criteria, but the threshold for discrimination is different in the published reports. In human and mouse SN, the cells with an area smaller than 134 pm* and 100 pm*, respectively have been determined as glial cells [23,33]. However, in rat SN, Chadi et al. [32] have identified the neuronal profiles with an area larger and the glial profiles with an area smaller than 30 pm* and this threshold has been validated by computerized automatic and manual counts of both cell types. We divided the Nissl-stained and bFGF-ir cells into three size groups based on the studies of Nissl and immunohistochemical preparations from rat SN [32,38]. Similar to the criteria used lbyabove-cited authors, we discarded cells with an area smaller than 50 pm* as glial cells. Yet, it was possible that the small size group included both small neurons and large glial cells. We found that the Nissl-stained and bFGF-ir cells in the small size group represented the largest percentage in the total population, followed by the cells in the medium size group, and the cells in the large one. If there is a neuronal atrophy in aging, the ratio of the cell number in small to the cell number in the medium and large size groups should be increased. We observed that such an increase in Nissl-stained cells occurred in pars compacta of lZmonth-old rats a.nd in pars reticulata of 26-month-old rats. In contrast, this ratio of bFGF-ir cell population remained unchanged in the two parts of SN with aging. It is difficult to explain this discrepancy in the observations of Nissl-stained and bFGF-ir cells, but participation of hyperplastic glial cells exhibiting bFGF immunoreactivity cannot be excluded. Further studies by a double immunostaining and computer-assisted[ image analysis will contribute to the elucidation of this issue. Using a more precise manual discrimination between neurons and glial cells, we established a shift
86
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
of the size histogram of bFGF-ir neurons to smaller surfaces indicating cell atrophy. Finally, some morphological features of bFGF-ir cells in 26-month-old rats such as shrinkage of the somata and processes, and uneven distribution of the reaction product provide further evidence for a cell atrophy. The mechanisms underlying neuronal atrophy and loss in normal and pathological aging are still unclear [39]. There is a hypothesis that the changes in different neuronal phenotypes in normal aging and in age-related diseases have a common denominator. This denominator could be the gradual agerelated loss of specific trophic factors and receptors [20]. There are some reports on the changes in FGF- and FGF-receptor-immunoreactivity in age-related diseases [21-241, while the changes in normal aging have been described only in rat cortex and hippocampus [25,26], and in human SN [17]. In general, these studies reveal reduction or retention of bFGF-immunoreactivity in normal aging. The present results are at odds with these observations indicating a significant increase in the staining intensity of bFGF-ir neurons in both pars compacta and pars reticulata in aging. It is interesting that Tooyama et al. [23] have found a strong bFGF-immunostaining of dopaminergic SN neurons in Parkinson’s disease, but not in control aged humans [17]. Intensification of bFGF-immunostaining has also been observed in other neurodegenerative diseases and regions of neuronal damage under experimental conditions [21,40]. It is suggested that this upregulation of bFGF in surviving cells may indicate a compensatory or unsuccessful defense response to the cell loss [16,24]. The present study demonstrates that the age-related changes in bFGF-immunoreactivity begin earlier and they are more pronounced in pars compacta than in pars reticulata. The two parts of SN differ in neuronal transmitter types, glial cell types, content of neurotrophic factors and their receptors [5,31,41]. The neurons and glial cells in pars compacta and pars reticulata have a different sensitivity to external impetuses and neurodegenerative diseases [12,23,34]. For example, GABA-ergic neurons in pars reticulata are less sensitive to the effects of bFGF and plasminogen than the dopaminergic neurons in pars compacta [11,42]. Stress hormones increase differently bFGF immunoreactivity of the neurons and astrocytes in pars compacta and pars reticulata [32]. Furthermore, the choleratoxin binding sites that reflect the presence of endogenous ganglioside GM1 are localized in all bFGF-ir neurons of pars compacta, but are not associated with bFGF-ir neurons of pars reticulata [5]. This suggests the different mechanism of the bFGF trophic effect on pars compacta and pars reticulata including in aging. In conclusion, age-related changes in bFGF-immunoreactivity occur in rat SN. The reduction of the number and size of bFGF-ir neurons is accompanied by an increase in the staining intensity with advancing age. This intensification of immunostaining may be due to a compensatory increase in the bFGF content in the remaining cells. The neuronal atrophy and loss in rat SN underlie the deficits in motor functions estimated in advancing age.
IS. L&ova, S.R. Ldov / Mech. Ageing Dev. 82 (1995) 73-89
87
References [1] A.I. Bean, R. Elde, Y. Cao, C. Oellig, C. Tamminga, M. Goldstein, R.F. Pettersson and T. Hokfelt, ExIpression of acidic and basic fibroblast growth factors in the substance nigra of rat, monkey, and human. Proc. Natl. Acad. Sci. USA, 88 (1991) 10237-10241. [2] A. Cintra, Y. Cao, C. Oellig, B. Tinner, F. Bortolotti, M. Goldstein, R.F. Pettersson and K. Fuxe, Basic FGF is present in dopaminergic neurons of the ventral midbrain of the rat. NeuroReport, 2 (1991) 597-600. [3] IA. Ferguson and E.M. Johnson, Fibroblast growth factor receptor-bearing neurons in the CNS: identification by receptor-mediated retrograde transport. J. Comp. Neurol., 313 (1991) 693-706. [4] I. Tooyama, D. Walker, T. Yamada, K. Hanai, H. Kimura, E.G. McGeer and P.L. McGeer, High molecular weight basic fibroblast growth factor-like protein is localized to a subpopulation of mesencephalic dopaminergic neurons in the rat brain. Brain Res., 593 (1992) 274-280. [5] K. Fuxe, B. Tinner, A.-M. Janson and L.F. Agnati, Codistribution of choleratoxin binding sites and tyrosine hydroxylase/FGF-2 immunoreactive nigral nerve cells. NeuroReport, 4 (1993) 857-860. [6] A. Matsuo, I. Tooyama, S. Isobe, Y. Oomura, I. Akiguchi, K. Hanai, J. Kimura and H. Kimura, Imununohistochemical localization in the rat brain of an epitope corresponding to the fibroblast growth factor receptor-I. Neuroscience, 60 (1994) 49-66. [7] G. Ferrari, M. Minozzi, G. Toffano, A. Leon and SD. Skaper, Basic fibroblast growth factor pmmotes the survival and development of mesencephalic neurons in culture. Deu. Biol., 133 (1989) 140-147. [8] B. Knusel, P.P. Michel, J.S. Schwaber and F. Hefti, Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidetmal growth factor, insulin and insulin-like growth factors I and II. J. Neurosci., 20 (1990) 5.58-570. [9] K.D. Beck, B. Knusel and F. Hefti, The nature of the trophic action of brain-derived neurotrophic fa’ctor, des(l-3)-insulin-like growth factor-I, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture. Neuroscience, 52 (1993) 855-866. [lo] S. Leonard, D. Luthman, J. Logel, J. Luthman, C. Antle, R. Freedman and B. Hoffer, Acidic and basic fibroblast growth factor messenger RNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion-assay by quantitative PCR. Mol. Brain Res., 18 (1993) 275-284. [11] D. Otto and K. Unsicker, FGF-2 modulates dopamine and dopamine related striatal transmitter systems in the intact and MPTP lesioned mouse. Eur. J. Neurosci., 5 (1993) 927-932. [12] G. Chadi, Y. Cao, R.F. Pettersson and K. Fuxe, Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience, 61 (1994) 891-910. [13] C.E. Finch, Neuron atrophy during aging: programmed or sporadic., TINS, 16 (1993) 104-110. [14] P.L. McGeer, E.G. McGeer and J.S. Suzuki, Aging and extrapyramidal function. Arch. Neurol., 34 (1977) 33-35. [15] P.L. McGeer, S. Itagaki, H. Akiyama and E.G. McGeer, Rate of cell death in parkinsonism
indicates active neuropathological process. Ann. Neurol., 24 (1988) 574-576. [16] W.G. Tatton, C.E. Greenwood, N.A. Seniuk and P.T. Salo, Interactions between MPTP-induced and age-related neuronal death in a murine model of Parkinson’s disease. Can. J. Neural. Sci., 19 (1992) 124-133. [17] I. Tooyama, E.G. McGeer, T. Kawamata, H. Kimura and P.L. McGeer, Retention of basic fibroblast growth factor immunoreactivity in dopaminergic neurons of the substantia nigra during normal aging in humans contrast with loss in Parkinson’s disease. Brain Res., 656 (1994) 165-168. [18] D.F. Emerich, P. McDermott, P. Krueger, M. Banks, J. Zhao, J. Marszalkowski, B. Frydel, S.R. Winn and P.R. Sanberg, Locomotion of aged rats - relationship to neurochemical but not morphological changes in nigro-striatal dopaminergic neurons. Brain Res. Bull., 32 (1993) 477-486. [19] R.. Schuligoi, J. Femandez, P.R. Heavens and D.J.S. Sirinathsinghji, Decreased tyrosine hydroxylase messenger RNA but not cholecystokinin messenger RNA in the pars compacta of the substantia nigra and ventral tegmental area of aged rats. Mol. Brain Res., 19 (1993) 333-338.
88
IS.
Lolova, S.R. Lolov / Mech. Age&g Dev. 82 (199.5) 73-89
[20] S.H. Appel, A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism and Alzheimer’s disease. Ann. Neural., 10 (1981) 499-505.
[21] E.G. Stopa, A. Gonzalez, R. Chorsky, R.J. Corona, J. Alvarez, E.D. Bird and A. Baird, Basic fibroblast growth factor in Alzheimer’s disease. Biochem. Biophys. Res. Commun., I71 (1990) 690-696. [22] S.L. Siedlak, P. Cras, M. Kawai, P. Richey and G. Perry, Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease. J. Histochem. Cytochem., 39 (1991) 899-904. [23] I. Tooyama, T. Kawamata, D. Walker, T. Yamada, K. Hanai, H. Kimura, M. Iwane, K. Igarashi,
[24]
[25] [26]
[27]
E.G. McGeer and P.L. McGeer, Loss of basic fibroblast growth factor in substantia nigra neurons in Parkinson’s disease. Neurology, 43 (1993) 372-376. I. Tooyama, H.P.H. Kremer, M.R. Hayden, H. Kimura, E.G. McGeer and P.L McGeer, Acidic and basic fibroblast growth factor-like immunoreactivity in the striatum and midbrain in Huntington’s disease. Brain Res., 610 (1993) l-7. T. Janet, M. Miehe, B. Pettmann, G. Labourdette and M. Sensenbrenner, Ultrastructural localization of fibroblast growth factor in neurons of rat brain. Neurosci. Lett., 80 (1987) 153-157. A. Cintra, J. Lindberg, G. Chadi, B. Tinner, A. Moller, J.A. Gustafsson, E.R. Dekloet, M. Oitzl, K. Nishikawa, L.F. Agnati and K. Fuxe, Basic fibroblast growth factor and steroid receptors in the aging hippocampus of the Brown Norway rat: immunohistochemical analysis in combination with stereotogy. Neurochem. Int., 25 (1994) 39-45. L.L. Vacca, S.J. Abrahams and N.E. Naftchi, A modified peroxidase-antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord. J. Histochem.
Cytochem., 28 (1980) 297-307. [28] J. Royet, Stereology: a method for analyzing images. Prog. Neurobiol., 37 (1991) 433-474. [29] L.J. Pellegrino, A.S. Pellegrino and A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, Plenum,
New York, 1979. [30] T. Hokfelt, R. Martensson, A. Bjorklund, S. Kleinau, and M. Goldstein, Distribution maps of tyrosine-hydroxylase immunoreactive neurons in the rat brain. In A. Bjorklund and T. Hoffelt (eds.), Handbook of Chemical Neuroanatomy, Vol. II: Classical Neurotransmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 277-379. [31] W.H. Oertel, M.L. Tappaz, A. Berod and E. Mugnaini, Two-colour immunohistochemistry for dopamine and GABA neurons in rat substantia nigra and zona incerta. Brain Res. Bull., 9 (1982) 463-474. [32] G. Chadi, L. Rosen, A. Cintra, B. Tinner, M. Zoli, R.F. Pettersson and K. Fuxe, Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. NeuroReport, 4 (1993) 783-786. [33] W.G. Tatton, C.E. Greenwood, P.T. Salo and N.A. Seniuk, Transmitter synthesis increases in substantia nigra neurons of the aged mouse. Neurosci. Lett., 132 (1991) 179-182. [34] A.M. Palmer and S.T. Dekosky, Monoamine neurons in aging and Alzheimer’s disease. J. Neural Transm. Gen. Sect., 91 (1993) 2-3. [35] F.F. Cruzsanchez, A. Cardozo and E. Tolosa, Neuronal changes in the substantia nigra with aging: a Golgi study. J. Neuropathol. Exp. Neural., 54 (1995) 74-81. [36] T.H. McNeil1 and L. Koek, Differential effects of advancing age on neurotransmitter cell loss of the substantia nigra and striatum of C57BL/6N mice. Brain Res., 521 (1990) 107-l 17. [37] L.J. Poirier, M. Giguere and R. Marchand, Comparative morphology of the substantia nigra and ventral tegmental area in the monkey, cat and rat. Brain Res. Bull., II (1983) 371-397. [38] R.L. Gulley and R.L. Wood, The fine structure of the neurons in the rat substantia nigra. Tissue Cell, 3 (1971) 675-690. [39] EC. Hirsch, Does oxidative stress participate in nerve cell death in Parkinson’s disease. Eur. Neural., 33, suppl. 1 (1993) 52-59. [40] F. Gomez-Pinilla, J.W.-K. Lee and C.W. Cotwan, Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection. J. Neurosci., 12 (1992) 345-355.
I.S. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
89
[41] L.J. Lawson, V.H. Peny, P. Dri and S. Gordon, Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience, 39 (1990) U-170. [42] K. Nagata, K. Nakajima and S. Kohsaka, Plasminogen promotes the development of rat mesencephalic dopaminergic neurons in vitro. Dev. Brain Res., 75 (1993) 31-37.
ELSEVIER
andllBwlqnmnt
A.ge-related changes in basic fibroblast growth factor-immunoreactive cells of rat substantia nigra Ivanka S. Lolova*a, Stephan R. Lolovb aInstitute of Physiology, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., bl. 23, 11 I3 Sofia, Bulgaria bInstitute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., bl. 23, 1113 Sofia, Bulgaria
Received 3 March 1995; accepted 9 May 1995
Abstract
Immunohistochemistry and computer-assisted image analysis were used to examine the age-related changes in bFGF-immunoreactivity in rat substantia nigra (SN). Distribution pattern, number, size and staining intensity of bFGF-immunoreactive (bFGF-ir) cells in pars compacta and pars reticulata of 3-, 12- and 26-month-old rats were compared. The overall distribution of bFGF-immunoreactivity was similar in the three age groups, but changes in the morphological appearance of bFGF-ir somata and processes occurred in aging. The results demonstrated a significantly reduced number of bFGF-ir cells in pars compacta (by 56.87%) and pars reticulata (by 30.4%) in 26-month-old rats compared to 3-month-old rats. The reduction of the cell number did not occur smoothly and equally in the two parts of SN. The quantitative analysis clearly indicated a significant decrease in the size of bFGF-ir neurons in pars compacta (by 18.1%) and pars reticulata (by 14.15%) of 26-month-old rats compared to 3-month-old rats. Compared to 3-month-old rats, a 19.77% and 17.83% increase in the staining intensity was observed in the remaining bFGF-ir neurons of pars compacta and pars reticulata, respectively in 26-month-old rats. Since there was no correlation between the decreased size and increased staining, it is most probable that the intensification of the staining intensity of bFGF-ir neurons was a compensatory response to the cell death. Keywords: Aging; bFGF-immunoreactivity; ysis
Substantia nigra; Computer-assisted
* Corresponding Author, e-mail: [email protected]. 0047-6374/95/$09.50
Q 1995
SSDlOO47-6374(95)01599-U
Elsevier Science Ireland Ltd. All rights reserved
image anal-
14
IS.
Lolova, S.R. Lolov 1 Mech. Ageing Dev. 82 (1995) 73-89
1. Introdbction Acidic and basic fibroblast growth-factors (aFGF, bFGF) are the most thoroughly studied members of the fibroblast growth factor family. bFGF and FGF receptors are widely distributed in various brain regions, including substantia nigra (SN) [l-6]. bFGF exerts neutrophic effects on the neurons of SN. It stimulates the histogenesis, survival and differentiation of the mesencephalic dopaminergic neurons in vitro and in vivo [7-91. bFGF protects and increases the repair of MPTP- and 6-hydroxydopamine-lesioned nigrostriatal neurons [lo- 121. Neuronal atrophy and loss occur in many brain regions during normal aging. The extent of neuronal loss in SN is unclear [13]. Some authors have reported an age-related loss of neurons in SN of rodents and humans [14-171. According to other authors, there is no reduction in the number and size of dopaminergic neurons in SN of aged animals [l&19]. There is a hypothesis that an inadequate trophic support could be a contributing factor for the neuronal atrophy and loss in normal and pathological aging [20]. Changes in bFGF immunoreactivity have been observed in the brains of patients with Alzheimer’s, Parkinson’s and Huntington’s diseases [21-241. However, little is known of bFGF-immunoreactive (bFGF-ir) neurons during normal aging [17,25,26]. We failed to find any quantitative analysis of bFGF-immunostaining of rat SN cells in advanced age. In the present study, we compared the distribution pattern of bFGF-immunoreactivity and the number, size and staining intensity of bFGF-ir neurons in SN of 3-, 12- and 26-month-old rats using a computerized image analysis system. The results showed a reduction in the number and size, but an increase in the staining intensity of bFGF-ir neurons of SN in aging. 2. Materials and methods 2.1. Immunohistochemistry Thirty-three male Wistar rats aged 3 months (n = 1l), 12 months (n = 11) and 26 months (n = 11) were used. The mean life span of the rats was 22-24 months. Under deep ether narcosis, the rats were perfused transcardially with phosphate buffered saline (PBS) followed by a fixative containing 4% paraformaldehyde, 0.08% glutaraldehyde and 0.15% picric acid in 0.1 M phosphate buffer (PB), pH 7.4. Midbrains were postfixed in the same fixative without glutaraldehyde for one night at 4°C and then soaked in 0.1 M PB containing 20% sucrose for 2 days at 4°C. Coronal sections, 30 pm thick, through the rostrocaudal extent of SN were cut on a freezing microtome (Reichert-Jung, Austria) and washed in 0.1 M PBS with 0.3% Triton X-100 (PBST) overnight at 4°C. Free-floating sections were incubated with rabbit-bFGF-antiserum (Sigma, St. Louis, MO) diluted 1:400 in PBST containing 1% bovine serum albumin (PBST-BSA) for 48 h at 4°C. Then the sections were incubated in swine anti-rabbit IgG (DAKO) diluted 1:20 and in rabbit peroxidase-
I.S. Lolova, S.R. Lolov) Mech. Ageing Dev. 82 (1995) 73-89
15
antiperoxidase complex (PAP, DAKO) diluted 1:200 according to the double amplification procedure of the PAP technique [27]. Prior to each incubation the sections were washed in PBST. The peroxidase activity was revealed with 0.02% 3-3’, 5-S’-diaminobenzidine and 0.05% hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.6. Control sections were incubated with rabbit IgG (0.1 pg/ml) or normal rabbit serum (1:lOOO) instead of the primary antibody. The sections were mounted on chrome-gelatin-coated slides and air-dried for 24 h. The immunostaining was intensified in 0.005% osmium tetroxide for 30 s. The sections were dehydrated and embedded in Entellan. In leach experiment, sections from one animal of each age group were processed simultaneously. 2.2. Quantitative analysis To assess the effects of aging on bFGF-immunoreactivity in SN, we compared the following parameters in 3-, 12- and 26-month-old rats: the number of bFGFir and Nissl-stained cells and their relationship; the rostrocaudal distribution of bFGF-ir and Nissl-stained cells; and the size and staining intensity (optical density, OD) of bFGF-ir cells. The parameters were examined in pars compacta and pars reticulata of SN. All quantitative analyses were performed on an image analysis system consisting of an Jenaval photomicroscope, a Hitachi videocamera, an IBM computer and morphometry software developed in the Biophysical Institute, Bulgarian Academy of Sciences (Dr B. Balev). 2.2. I. Determination of the volume of pars compacta and pars reticulata, the density and the true number of the N&l-stained and bFGF-ir cells For estimation of the true number of Nissl-stained and bFGF-ir cells, it was necessary to measure the volume of pars compacta and pars reticulata of SN as well as the cell density. Volume. Substantia nigra of nine rats (three in each age group) was cut serially from the most rostra1 to the most caudal levels. All sections were collected in subsets with five sections. Every first section of the subsets was stained by Nissl and every second section was immunostained for bFGF. The cross-sectional areas of both pars compacta and pars reticulata were traced bilaterally by a pointing device on all Nissl-stained sections sequentially starting with the most rostra1 and ending with the most caudal section with a x 3.2 objective lens. These areas and the section thickness (30 pm) were used for volume estimation by a formula based on the Simpson rule for integration [281. Cell density. To count the cell density, an image analyzer was used with a x 25 objective lens. With a threshold value determined empirically, the image was converted from tonal to binary form. Structures with areas less than 50 pm2 were automatically erased to avoid counting positive glial cells. Two sections at rostral, middle and caudal levels stained by Nissl and two adjacent bFGF-immunostained
76
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
sections at the same levels of pars compacta and pars reticulata of each rat were measured. The levels along the rostrocaudal axis of SN were determined according to the atlas of Pellegrino et al. [29]. The averaged values from the measurements of the two sections at each level were used. The number of cells per unit volume was counted according to the equations for a polydispersed system described by Royet LW. True cell number. Estimates of the true number of Nissl-stained and bFGF-ir cells in each pars compacta and pars reticulata were calculated by multiplying the number of cells per unit volume by the volume of the individual pars. On the basis of the cell sectional areas, the numbers of small (50- 150 pm’), medium (150-300 pm2) and large (300-600 pm2) size groups of Nissl-stained and bFGF-ir cells were automatically determined. 2.2.2. Rostrocaudal distribution of N&l-stained pacta and pars reticulata
and bFGF-ir cells in pars com-
To assess whether the distribution of Nissl-stained and bFGF-ir cells along the rostrocaudal axis of SN was changed in aging, we compared the cell density at rostral, middle and caudal levels in the three age groups. 2.2.3. Determination of the size and optical density of bFGF-ir neurons The size and OD of bFGF-ir neurons were measured at the middle level of SN with a x 40 objective. Pilot measurements revealed that there were no significant differences in size and OD of bFGF-ir neurons at the rostral, middle and caudal levels of pars compacta and pars reticulata in 3-month-old rats. Therefore, the measurements at the middle level, where the oculomotor nerve emerges, reflected accurately the size and OD of bFGF-ir neurons in the overall SN. Twenty randomly selected neurons of pars compacta and 20 neurons in the lateral subdivision of pars reticulata in every rat (n = 33) were measured. Neurons cut through the level of the nucleus or the stem of the processes were traced by a pointing device. Every cell was measured three times and the averaged values of the size and OD were used. The OD of bFGF-ir cells was expressed on an arbitrary gray scale ranging from 0 to 1 (0, OD of the glass slide; 1, maximal staining intensity). Recordings were also made of the adjacent background in the cerebral peduncle and the values were subtracted from the cellular values. To establish if there was a correlation between the size and optical density of bFGF-ir neurons, we determined the correlation coefficients. The accuracy of the estimates measured as mean coefficient of variation was 6.1% and 10.96%, respectively for neuronal size and OD determination. 2.2.4. Statistical analysis
All calculations were made for each animal before the means and standard errors of the means were determined for an age group. The statistical differences were analyzed by ANOVA test followed by Newman-Keuls test.
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
71
3. Results 3.1. Distribution of bFGF-immunoreactivity
in substantia nigra
The overall distribution of bFGF-immunoreactivity in SN was similar in the three age groups. bFGF reaction material was observed as a diffuse brown staining within the somata and processes of pars compacta and pars reticulata (Fig. 1). No reaction product was found in the neuronal nuclei. The SN cell somata in 3- and 12-monthold rats were relatively uniformly immunostained. In contrast, some somata in 26-month-old rats displayed an intensification of the immunostaining in comparison with the adjacent somata and the somata of SN in the younger rats. While an even distribution of the reaction product throughout the cell somata in pars compacta and pars reticulata was found in 3- and lZmonth-old rats, a perinuclear or spot-like accumulation of this product was seen in 26-month-old rats. The cell processes became short, thin and sparse in 26-month-old rats. The immunostaining of the neuropil of 3-month-old rats was punctiform and at higher magnification varicose fibers were seen around the cell somata and blood vessels. With advancing age, varicose fibers became more and more dark, swollen and wrinkled. 3.2. Volume of substantia nigra No significant differences in the volume of the left and right pars compacta and pars reticulata in 3-month-old rats were found. Thus, the results included the measurements of the volume of both sides. ANOVA demonstrated a significant influence of age on the volume of SN. The volume of whole SN in 26-month-old rats was significantly decreased [F(2,15) = 5.46, P < 0.051 compared to 3-month-old (by 26.7%) and 12-month-old (by 22.05%) rats. Table 1 shows that this decrease was mainly due to a significant reduction [F(2,15) = 12.21, P < O.OOl] of the volume of pars compacta in 26month-old rats compared to 3-month-old (by 51.49%) and 12-month-old (by 38.75%) rats. A 17.13% reduction in the volume of pars reticulata in 26-month-old rats as compared to 3-month-old rats was not significant. 3.3. Number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata Pars compacta. A significant effect of age on the number of Nissl-stained [F(2,6) = 7.80, P < 0.051 and bFGF-ir [F(2,6) = 8.58, P c 0.051 cells was found by ANOVA. The total number of Nissl-stained cells in 26-month-old rats was significantly decreased compared to 3-month-old (by 51%) and 12-month-old (by 41%) rats (Table 2). An even more pronounced reduction in the total number of bFGF-ir cells was observed in 26-month-old rats compared to 3-month-old (by 56.87%) and lZmonth-old (by 48.49%) rats. The decrease in the total number of Nissl-stained (by 16.7%) and bFGF-ir (by 15.78%) cells in lZmonth-old rats as compared to 3-month-old rats was not significant. As shown in Table 2, the number
78
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
of NM-stained cells in small, medium and large size groups was significantly decreased (by 45.31%, 61% and 67.14%, respectively) in 26-month-old rats compared to 3-month-old rats. Also, a significant reduction in the number of bFGF-ir cells in small, medium and large size groups (by 55.36%, 56.42% and 71.08%,
Fig. 1. bFGF-immunoreactivity in pars compacta (A$) and pars reticulata (3,D) of SN in 3-month-old (A,B) and 26-month-old (C,D) rats. The arrows show few short, thin and poorly visible processes. The arrowheads point to dark swollen fibers and varicosities in C and D in contrast to the fine fibers in B. Magnification: x 400.
IS.
Ldova,
S.R. Ldov 1 Mech. Ageing Dev. 82 (1995) 73-89
Table 1 Volume (mm3) of the whole SN, pars compacta and pars reticulata of the three age groups Age (months)
Side”
Whole SN
Pars compacta
Pars reticulata
3
L R L R L R
3.119 4.089 2.160 3.009 4.064 3.395
1.147 1.104 0.834 0.677 1.334 0.944
2.632 2.985 1.926 2.332 2.730 2.451
Mean S.E.M.
3.516 0.227
1.007 0.096
2.509 0.149
L R L R L R
3.919 4.027 2.705 3.062 2.906 3.171
1.037 1.005 0.720 0.675 0.667 0.716
2.942 3.022 1.985 2.381 2.239 2.455
Mean S.E.M.
3.308 0.229
0.803 0.069
2.505 0.165
L R L R L R
2.801 2.171 2.109 1.908 3.007 2.864
0.530 0.545 0.306 0.414 0.647 0.519
2.211 2.232 1.803 1.494 2.360 2.345
Mean S.E.M.
2.578 0.185
0.494 0.048
2.084 0.145
12
26
“L, left side; R, right side.
respect:ively) was established in 26-month-old rats compared to 3-month-old rats. In 3- and lZmonth-old rats, bFGF-ir cells accounted for 91% of the Nissl-stained cells. In 26-month-old rats, this percentage was slightly (by 9%) reduced. Pars reticufata. A decrease in the total number of Nissl-stained (by 21.3%) and bFGF-ir (by 30.4%) cells of 26-month-old rats compared to 3-month-old rats was observed. The number of Nissl-stained cells in small, medium and large size groups was also reduced (by 12.2%, 40.35% and 49.3%, respectively) compared to 3-monthold rats, but the difference was significant only in the medium size group [F(2,6) = 5.82, P < 0.051. The number of bFGF-ir cells in small, medium and large size groups of 26-month-old rats was decreased by 31.56%, 23.38% and 43%, respectively compared to 3-month-old rats. In 3-month-old rats, the vast majority of Nissl-stained cells exhibited bFGF-immunoreactivity. In 12- and 26-month-old rats, bFGF-ir cells accounted for 89% of the Nissl-stained cells. It should be noted that some cells in the medium and large size groups were not bFGF-ir. However, their amount was relatively small and obviously did not influence the percentage of bFGF-ir of the Nissl-stained cells.
I.S. Lo/ova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
80
Table 2 shows that the decrease in the number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata in aging was more pronounced in medium and large than in small size groups. To estimate whether the decrease in the number of medium and large sized cells was accompanied by a relative increase in the number of small sized cells, the ratios of small to medium and large sized cells in each population were calculated. In fact, during aging this ratio of Nissl-stained cells
Table 2 Number of Nissl-stained and bFGF-ir cells in pars compacta and pars reticulata of rat SN of three age groups
Age
Nissl-stained cells (size group)
bFGF-ir cells (size group)
(months) Total
Small
Medium
Large
Total
Small
Medium
Large
34 105 23 182 33 988
22 857 16449 23 084
8312 5458 8861
2111 1045 1492
29 457 21 280 32 083
24 970 14 667 26 475
3953 5431 4819
484 975 740
Mean S.E.M.
30 425 3622
20 797 2175
7544 1055
1549 309
27607 3253
22038 3711
4734 429
733 142
12
30 665 22731 22 632
19 901 19 568 15 184
8244 2842 6401
1755 322 953
29 168 19 705 20 881
24 618 15 987 16 324
4175 3284 3980
325 383 380
Mean S.E.M.
25343 2661
18218 1520
5829 1585
1010 415
23 251 2978
18 976 2823
3813 270
362 19
26
17 333 11 125 16 388
12 632 8945 12 532
3693 1905 3232
729 224 575
11 577 8835 15 305
10 025 7057 12 430
2194 1497 2499
113 201 323
Mean S.E.M.
14 948 1931
11 370 1213
2943 536
509 150
11 906 1875
9838 1554
2063 296
212 61
3
78 427 56 355 70 135
56 650 39 825 47 227
16 930 13 598 19 514
4477 2981 2912
79615 56712 69 858
61467 40747 55 548
14 660 14 094 12 585
2876 1480 1668
Mean S.E.M.
68 306 6437
47 901 4869
16 681 1712
3456 511
68 729 6635
52 587 6162
13 780 619
2008 437
12
78 468 62649 63 120
55 242 45215 44 443
17 015 14 174 16 239
5436 2859 2014
69 728 51907 63 012
55 620 41 567 46 670
12 563 9428 13 298
1476 772 2307
Mean S.E.M.
68079 5196
48 300 3478
15 809 848
3436 1029
61 549 5196
47 952 4107
11 763 1187
1518 444
26
65 113 46 029 50 136
48349 38 809 38 033
12 557 6484 10 810
3742 435 1082
48 686 41930 52938
39 334 31 775 36 864
8594 9220 13 861
608 781 2049
Mean S.E.M.
53 760 5799
41 730 3317
9950 1805
1753 1012
47 851 3205
35 991 2225
10 558 1661
1146 454
3
Pars compacta
Pars reticulata
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
81
Table 3 Percentage of the mean values of small, medium and large size cells of the total number in the three age groups and their ratios
Age
bFGF-ir cells (size group)
Nissl-stained cells (size group)
(months) Small
Medium
Large
Ratio
Small
Medium
Large
Ratio
Pars compacta
3 12 26
68.4 71.9 76.1
24.8 23.0 19.7
5.1 4.0 3.4
2.32 3.41 3.45
79.8 81.6 82.6
17.1 16.4 17.3
2.7 1.6 1.8
4.23 4.53 4.30
Pars reticulata
3 12 26
70.1 70.9 77.6
24.4 23.2 18.5
5.1 5.0 3.3
2.38 2.52 3.92
76.5 77.9 75.2
20.0 19.1 22.1
2.9 2.5 2.4
3.34 3.68 3.26
These ‘data were derived from the data presented in Table 2.
increased in the two parts of SN, being significant in pars reticulata [F(2,15) = 6.01, P < 0.011 (Table 3). However, the ratio of small to medium and large sized bFGF-ir cells remained unchanged with aging. 3.4. Rostrocaudal distribution of NM-stained and par’s reticulata
and bFGF-ir cells in pars compacta
The quantitative analysis showed that in 3-month-old rats, Nissl-stained and bFGF-ir cells were distributed evenly in the rostrocaudal extent of pars compacta and pars reticulata (data not shown). In 26-month-old rats, the density of Nisslstained cells was decreased only at the caudal level of pars compacta and pars reticulata compared to the same level in 3- and lZmonth-old rats. Compared to 3and 12,-month-old rats, a reduction of the density of bFGF-ir cells in pars compacta and pars reticulata in 26-month-old rats was observed at the three levels, being more pronounced at the middle level than at the rostra1 and caudal levels. 3.5. Size and staining intensity of bFGF-ir neurons in substantia nigra In contrast to automatic binary processing of the cell number, the discrimination between bFGF-ir neurons and bFGF-ir glial cells by manual tracing in the determination of the cell size and OD was much more precise. Age had a significant influence on the size and OD of bFGF-ir neurons in SN (Table 4). The size of bFGF-ir neurons was significantly decreased in both pars compacta [F(2,30) = 9.56, P < 0.001, ANOVA] and in pars reticulata [F(2,30) = 8.43, 1’ < 0.01, ANOVA] of 26-month-old rats compared to 3-month-old rats (by 18.1% and 14.15%, respectively). No differences in the size of bFGF-ir neurons between 3- and lZmonth-old rats were found. A significant increase in the OD of bFGF-ir neurons was observed in pars compacta [F(2,30) = 4.18, P c 0.05, ANOVA] and pars reticulata [F(2,30) = 4.64, P c 0.05, ANOVA] of 26-monthold rats (by 19.77% and 17.83%, respectively) compared to 3-month-old rats. The OD of bFGF-ir neurons in 3- and lZmonth-old rats did not differ significantly.
82
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
Table 4 Area (pm2) and optical density (arbitrary units) of bFGF-ir reticulata in the three age groups
neurons in pars compacta and pars
Age (months)
Pars reticulata
Pars compacta
3
Mean S.E.M. 12
Mean S.E.M. 24
Mean S.E.M.
Area
OD
Area
OD
158.3 162.7 174.9 186.1 154.3 184.4 162.6 160.3 205.5 187.2 193.0
0.23 0.40 0.26 0.26 0.36 0.39 0.31 0.36 0.24 0.33 0.27
201.6 162.4 179.2 213.1 187.0 205.7 182.4 178.9 201.6 227.0 221.1
0.24 0.38 0.21 0.26 0.34 0.39 0.33 0.28 0.25 0.33 0.34
175.4 5.1
0.31 0.02
196.4 6.0
0.30 0.02
180.4 186.1 141.5 184.1 154.6 189.2 169.1 144.9 154.3 213.2 202.5
0.29 0.31 0.31 0.33 0.29 0.38 0.37 0.34 0.34 0.36 0.21
176.2 199.7 169.4 215.0 193.1 223.1 207.0 194.6 184.9 225.8 228.1
0.29 0.31 0.27 0.33 0.23 0.35 0.31 0.30 0.32 0.33 0.24
174.5 7.1
0.32 0.01
201.5 6.1
0.30 0.01
146.8 132.1 137.0 121.0 137.9 171.7 139.3 121.0 168.7 147.1 157.6
0.30 0.40 0.41 0.37 0.34 0.40 0.35 0.34 0.43 0.45 0.31
164.4 176.6 157.5 189.2 151.8 173.1 175.5 132.8 187.7 144.8 201.0
0.29 0.36 0.42 0.29 0.32 0.38 0.40 0.33 0.46 0.37 0.33
143.7 5.1
0.37 0.01
168.6 6.2
0.36 0.02
Each figure indicates mean value per animal from the measurement of 20 cells (n = 660 in pars compacta and n = 660 in pars reticulata for the three age groups).
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
pars
compacta 40
03
months
al2
months
a26
months
30 g ._ z ‘; g20 % sp 10
ZOO-250
250-300
300-350
350-400 sq.pm
pars
reticulata 40
30 0” ._ 2 ‘; g20 B S?
10
0
50-100
100-150
150-200
200-250250-300
300-350350-400 sq.pm
area
of
IleUrOns
Fig. 2. A histogram of the size distribution of bFGF-ir neurons in pars compacta and pars reticulata of SN in three age groups.
The histogram of bFGF-ir neurons showed a shift towards smaller surfaces in pars compacta and pars reticulata of 26-month-old rats compared to 3- and 12-month-old rats (Fig. 2).
84
IS. L&ova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
There was no correlation between the size and OD of bFGF-ir neurons in pars compacta (r = - 0.16, r = - 0.16, r = - 0.08, respectively) and pars reticulata (r = - 0.13, r = - 0.0004, r = - 0.025, respectively) in 3-, 12- and 26-month-old rats. Therefore, the increase in the OD was not due to the decrease in the size of bFGF-ir neurons. 4. Discussion The present results demonstrate an extensive distribution of bFGF-immunoreactivity in the cell bodies and neuropil of rat SN. It is well known that pars compacta consists mainly of dopaminergic cells [30] while pars reticulata contains predominantly GABA-ergic neurons [31]. Although a double immunostaining was not performed in this study, the large percentage of bFGF-ir of the Nissl-stained cells in the two parts of SN indicates that bFGF is localized in the dopaminergic and GABA-ergic cells. Our results are consistent with the data that the majority of dopaminergic neurons of the ventral midbrain of rats and humans are bFGF-positive [1,2,4,32]. It is suggested that scattered bFGF-ir cells in pars reticulata are GABA-ergic ones [2,5]. The percentage, herein described, of bFGF-ir of the Nissl-stained cells shows that bFGF-immunoreactivity is localized in small, medium and some large sized GABA-ergic cells of pars reticulata. This finding is in agreement with studies reporting a trophic action of bFGF not only on dopaminergic, but also on GABA-ergic neurons of rat SN [7,11]. Neuronal atrophy and loss are considered to be the main characteristics of the aging brain, but opinions still differ concerning their extent in SN of rodents [13]. One of the main findings of our quantitative analysis is the significant reduction in the number of Nissl-stained and bFGF-ir cells in rat SN during normal aging. This age-related decrease in the number of cells per nucleus is due not only to the volume reduction of SN. We estimate also a significant reduction in the density of bFGF-ir cells at the rostral, middle and caudal levels, and of Nissl-stained cells at the caudal level of SN. The quantitative analysis pointed out that the number of Nissl-stained and bFGF-ir cells in SN did not decrease smoothly with aging. The reduction was pronounced and significant between 12 and 26 months of age, but slight and insignificant between 3 and 12 months. Also, the reduction in the number of Nissl-stained and bFGF-ir cells was not the same in the two parts of SN. In pars compacta, the decrease in the number of both Nissl-stained and bFGF-ir cells began earlier and it was more pronounced than that in pars reticulata. These results are in accordance with data about a slow and gradual age-related loss of neurons in SN of rats, mice and humans demonstrated by different methods of staining and counting [14-17,33-351. Some authors, however, have not found any significant loss of dopaminergic neurons in SN of aged rats and mice [l&19,36]. When considering these controversies, it should be born in mind that species- and genotype-specific factors are also responsible for age-related neuronal atrophy and loss [13]. The reduction of the number of bFGF-ir cells could be explained by a true loss of these cells, by a decrease in bFGF content or by both. Some of the results
IS. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
85
support the first explanation. The reduction of bFGF-ir cells in pars compacta is parallel in time and extent to the reduction of the number of Nissl-stained cells. Furthermore, the morphological appearance and quantitative analysis demonstrated a significant increase in the staining intensity of bFGF-ir neurons in pars compacta and pars reticulata in 26-month-old rats. These findings are in keeping with olbservations about age-related changes in the number of bFGF-ir and tyrosine hydroxylase-ir neurons of pars compacta of humans and mice SN [17,33]. The possib:ility that other nigral cells lose bFGF before their death should also be considered. Thus, we could explain why the decrease in the number of bFGF-ir cells in pars reticulata precedes the reduction of Nissl-stained cells. Moreover, the percentage of bFGF-ir of the Nissl-stained cells should not alter in aging if a part of the cells dies without loss of bFGF content. The quantitative analysis showed that this percentage decreased, though slightly, in pars compacta and pars reticulata. Therefore, our studies provided evidence for the presence of a true cell loss and aldditional loss of bFGF content in some cells of pars compacta and pars reticulata of rat SN with advancing age. To Idetermine whether the estimated cell loss was accompanied by an actual cell atrophy, we had to analyze size groups in the total cell population and their ratios. In the: current literature there are much discrepancies concerning the size of the neurons in rat SN (see [37] for review). Also, the discrimination of the small neurons and large glial cells on the basis of the cytological characteristics is impossible by computer-assisted image analysis. The cell sizes have been used as criteria, but the threshold for discrimination is different in the published reports. In human and mouse SN, the cells with an area smaller than 134 pm* and 100 pm*, respectively have been determined as glial cells [23,33]. However, in rat SN, Chadi et al. [32] have identified the neuronal profiles with an area larger and the glial profiles with an area smaller than 30 pm* and this threshold has been validated by computerized automatic and manual counts of both cell types. We divided the Nissl-stained and bFGF-ir cells into three size groups based on the studies of Nissl and immunohistochemical preparations from rat SN [32,38]. Similar to the criteria used lbyabove-cited authors, we discarded cells with an area smaller than 50 pm* as glial cells. Yet, it was possible that the small size group included both small neurons and large glial cells. We found that the Nissl-stained and bFGF-ir cells in the small size group represented the largest percentage in the total population, followed by the cells in the medium size group, and the cells in the large one. If there is a neuronal atrophy in aging, the ratio of the cell number in small to the cell number in the medium and large size groups should be increased. We observed that such an increase in Nissl-stained cells occurred in pars compacta of lZmonth-old rats a.nd in pars reticulata of 26-month-old rats. In contrast, this ratio of bFGF-ir cell population remained unchanged in the two parts of SN with aging. It is difficult to explain this discrepancy in the observations of Nissl-stained and bFGF-ir cells, but participation of hyperplastic glial cells exhibiting bFGF immunoreactivity cannot be excluded. Further studies by a double immunostaining and computer-assisted[ image analysis will contribute to the elucidation of this issue. Using a more precise manual discrimination between neurons and glial cells, we established a shift
86
IS.
Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
of the size histogram of bFGF-ir neurons to smaller surfaces indicating cell atrophy. Finally, some morphological features of bFGF-ir cells in 26-month-old rats such as shrinkage of the somata and processes, and uneven distribution of the reaction product provide further evidence for a cell atrophy. The mechanisms underlying neuronal atrophy and loss in normal and pathological aging are still unclear [39]. There is a hypothesis that the changes in different neuronal phenotypes in normal aging and in age-related diseases have a common denominator. This denominator could be the gradual agerelated loss of specific trophic factors and receptors [20]. There are some reports on the changes in FGF- and FGF-receptor-immunoreactivity in age-related diseases [21-241, while the changes in normal aging have been described only in rat cortex and hippocampus [25,26], and in human SN [17]. In general, these studies reveal reduction or retention of bFGF-immunoreactivity in normal aging. The present results are at odds with these observations indicating a significant increase in the staining intensity of bFGF-ir neurons in both pars compacta and pars reticulata in aging. It is interesting that Tooyama et al. [23] have found a strong bFGF-immunostaining of dopaminergic SN neurons in Parkinson’s disease, but not in control aged humans [17]. Intensification of bFGF-immunostaining has also been observed in other neurodegenerative diseases and regions of neuronal damage under experimental conditions [21,40]. It is suggested that this upregulation of bFGF in surviving cells may indicate a compensatory or unsuccessful defense response to the cell loss [16,24]. The present study demonstrates that the age-related changes in bFGF-immunoreactivity begin earlier and they are more pronounced in pars compacta than in pars reticulata. The two parts of SN differ in neuronal transmitter types, glial cell types, content of neurotrophic factors and their receptors [5,31,41]. The neurons and glial cells in pars compacta and pars reticulata have a different sensitivity to external impetuses and neurodegenerative diseases [12,23,34]. For example, GABA-ergic neurons in pars reticulata are less sensitive to the effects of bFGF and plasminogen than the dopaminergic neurons in pars compacta [11,42]. Stress hormones increase differently bFGF immunoreactivity of the neurons and astrocytes in pars compacta and pars reticulata [32]. Furthermore, the choleratoxin binding sites that reflect the presence of endogenous ganglioside GM1 are localized in all bFGF-ir neurons of pars compacta, but are not associated with bFGF-ir neurons of pars reticulata [5]. This suggests the different mechanism of the bFGF trophic effect on pars compacta and pars reticulata including in aging. In conclusion, age-related changes in bFGF-immunoreactivity occur in rat SN. The reduction of the number and size of bFGF-ir neurons is accompanied by an increase in the staining intensity with advancing age. This intensification of immunostaining may be due to a compensatory increase in the bFGF content in the remaining cells. The neuronal atrophy and loss in rat SN underlie the deficits in motor functions estimated in advancing age.
IS. L&ova, S.R. Ldov / Mech. Ageing Dev. 82 (1995) 73-89
87
References [1] A.I. Bean, R. Elde, Y. Cao, C. Oellig, C. Tamminga, M. Goldstein, R.F. Pettersson and T. Hokfelt, ExIpression of acidic and basic fibroblast growth factors in the substance nigra of rat, monkey, and human. Proc. Natl. Acad. Sci. USA, 88 (1991) 10237-10241. [2] A. Cintra, Y. Cao, C. Oellig, B. Tinner, F. Bortolotti, M. Goldstein, R.F. Pettersson and K. Fuxe, Basic FGF is present in dopaminergic neurons of the ventral midbrain of the rat. NeuroReport, 2 (1991) 597-600. [3] IA. Ferguson and E.M. Johnson, Fibroblast growth factor receptor-bearing neurons in the CNS: identification by receptor-mediated retrograde transport. J. Comp. Neurol., 313 (1991) 693-706. [4] I. Tooyama, D. Walker, T. Yamada, K. Hanai, H. Kimura, E.G. McGeer and P.L. McGeer, High molecular weight basic fibroblast growth factor-like protein is localized to a subpopulation of mesencephalic dopaminergic neurons in the rat brain. Brain Res., 593 (1992) 274-280. [5] K. Fuxe, B. Tinner, A.-M. Janson and L.F. Agnati, Codistribution of choleratoxin binding sites and tyrosine hydroxylase/FGF-2 immunoreactive nigral nerve cells. NeuroReport, 4 (1993) 857-860. [6] A. Matsuo, I. Tooyama, S. Isobe, Y. Oomura, I. Akiguchi, K. Hanai, J. Kimura and H. Kimura, Imununohistochemical localization in the rat brain of an epitope corresponding to the fibroblast growth factor receptor-I. Neuroscience, 60 (1994) 49-66. [7] G. Ferrari, M. Minozzi, G. Toffano, A. Leon and SD. Skaper, Basic fibroblast growth factor pmmotes the survival and development of mesencephalic neurons in culture. Deu. Biol., 133 (1989) 140-147. [8] B. Knusel, P.P. Michel, J.S. Schwaber and F. Hefti, Selective and nonselective stimulation of central cholinergic and dopaminergic development in vitro by nerve growth factor, basic fibroblast growth factor, epidetmal growth factor, insulin and insulin-like growth factors I and II. J. Neurosci., 20 (1990) 5.58-570. [9] K.D. Beck, B. Knusel and F. Hefti, The nature of the trophic action of brain-derived neurotrophic fa’ctor, des(l-3)-insulin-like growth factor-I, and basic fibroblast growth factor on mesencephalic dopaminergic neurons developing in culture. Neuroscience, 52 (1993) 855-866. [lo] S. Leonard, D. Luthman, J. Logel, J. Luthman, C. Antle, R. Freedman and B. Hoffer, Acidic and basic fibroblast growth factor messenger RNAs are increased in striatum following MPTP-induced dopamine neurofiber lesion-assay by quantitative PCR. Mol. Brain Res., 18 (1993) 275-284. [11] D. Otto and K. Unsicker, FGF-2 modulates dopamine and dopamine related striatal transmitter systems in the intact and MPTP lesioned mouse. Eur. J. Neurosci., 5 (1993) 927-932. [12] G. Chadi, Y. Cao, R.F. Pettersson and K. Fuxe, Temporal and spatial increase of astroglial basic fibroblast growth factor synthesis after 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine neurons. Neuroscience, 61 (1994) 891-910. [13] C.E. Finch, Neuron atrophy during aging: programmed or sporadic., TINS, 16 (1993) 104-110. [14] P.L. McGeer, E.G. McGeer and J.S. Suzuki, Aging and extrapyramidal function. Arch. Neurol., 34 (1977) 33-35. [15] P.L. McGeer, S. Itagaki, H. Akiyama and E.G. McGeer, Rate of cell death in parkinsonism
indicates active neuropathological process. Ann. Neurol., 24 (1988) 574-576. [16] W.G. Tatton, C.E. Greenwood, N.A. Seniuk and P.T. Salo, Interactions between MPTP-induced and age-related neuronal death in a murine model of Parkinson’s disease. Can. J. Neural. Sci., 19 (1992) 124-133. [17] I. Tooyama, E.G. McGeer, T. Kawamata, H. Kimura and P.L. McGeer, Retention of basic fibroblast growth factor immunoreactivity in dopaminergic neurons of the substantia nigra during normal aging in humans contrast with loss in Parkinson’s disease. Brain Res., 656 (1994) 165-168. [18] D.F. Emerich, P. McDermott, P. Krueger, M. Banks, J. Zhao, J. Marszalkowski, B. Frydel, S.R. Winn and P.R. Sanberg, Locomotion of aged rats - relationship to neurochemical but not morphological changes in nigro-striatal dopaminergic neurons. Brain Res. Bull., 32 (1993) 477-486. [19] R.. Schuligoi, J. Femandez, P.R. Heavens and D.J.S. Sirinathsinghji, Decreased tyrosine hydroxylase messenger RNA but not cholecystokinin messenger RNA in the pars compacta of the substantia nigra and ventral tegmental area of aged rats. Mol. Brain Res., 19 (1993) 333-338.
88
IS.
Lolova, S.R. Lolov / Mech. Age&g Dev. 82 (199.5) 73-89
[20] S.H. Appel, A unifying hypothesis for the cause of amyotrophic lateral sclerosis, parkinsonism and Alzheimer’s disease. Ann. Neural., 10 (1981) 499-505.
[21] E.G. Stopa, A. Gonzalez, R. Chorsky, R.J. Corona, J. Alvarez, E.D. Bird and A. Baird, Basic fibroblast growth factor in Alzheimer’s disease. Biochem. Biophys. Res. Commun., I71 (1990) 690-696. [22] S.L. Siedlak, P. Cras, M. Kawai, P. Richey and G. Perry, Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease. J. Histochem. Cytochem., 39 (1991) 899-904. [23] I. Tooyama, T. Kawamata, D. Walker, T. Yamada, K. Hanai, H. Kimura, M. Iwane, K. Igarashi,
[24]
[25] [26]
[27]
E.G. McGeer and P.L. McGeer, Loss of basic fibroblast growth factor in substantia nigra neurons in Parkinson’s disease. Neurology, 43 (1993) 372-376. I. Tooyama, H.P.H. Kremer, M.R. Hayden, H. Kimura, E.G. McGeer and P.L McGeer, Acidic and basic fibroblast growth factor-like immunoreactivity in the striatum and midbrain in Huntington’s disease. Brain Res., 610 (1993) l-7. T. Janet, M. Miehe, B. Pettmann, G. Labourdette and M. Sensenbrenner, Ultrastructural localization of fibroblast growth factor in neurons of rat brain. Neurosci. Lett., 80 (1987) 153-157. A. Cintra, J. Lindberg, G. Chadi, B. Tinner, A. Moller, J.A. Gustafsson, E.R. Dekloet, M. Oitzl, K. Nishikawa, L.F. Agnati and K. Fuxe, Basic fibroblast growth factor and steroid receptors in the aging hippocampus of the Brown Norway rat: immunohistochemical analysis in combination with stereotogy. Neurochem. Int., 25 (1994) 39-45. L.L. Vacca, S.J. Abrahams and N.E. Naftchi, A modified peroxidase-antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord. J. Histochem.
Cytochem., 28 (1980) 297-307. [28] J. Royet, Stereology: a method for analyzing images. Prog. Neurobiol., 37 (1991) 433-474. [29] L.J. Pellegrino, A.S. Pellegrino and A.J. Cushman, A Stereotaxic Atlas of the Rat Brain, Plenum,
New York, 1979. [30] T. Hokfelt, R. Martensson, A. Bjorklund, S. Kleinau, and M. Goldstein, Distribution maps of tyrosine-hydroxylase immunoreactive neurons in the rat brain. In A. Bjorklund and T. Hoffelt (eds.), Handbook of Chemical Neuroanatomy, Vol. II: Classical Neurotransmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 277-379. [31] W.H. Oertel, M.L. Tappaz, A. Berod and E. Mugnaini, Two-colour immunohistochemistry for dopamine and GABA neurons in rat substantia nigra and zona incerta. Brain Res. Bull., 9 (1982) 463-474. [32] G. Chadi, L. Rosen, A. Cintra, B. Tinner, M. Zoli, R.F. Pettersson and K. Fuxe, Corticosterone increases FGF-2 (bFGF) immunoreactivity in the substantia nigra of the rat. NeuroReport, 4 (1993) 783-786. [33] W.G. Tatton, C.E. Greenwood, P.T. Salo and N.A. Seniuk, Transmitter synthesis increases in substantia nigra neurons of the aged mouse. Neurosci. Lett., 132 (1991) 179-182. [34] A.M. Palmer and S.T. Dekosky, Monoamine neurons in aging and Alzheimer’s disease. J. Neural Transm. Gen. Sect., 91 (1993) 2-3. [35] F.F. Cruzsanchez, A. Cardozo and E. Tolosa, Neuronal changes in the substantia nigra with aging: a Golgi study. J. Neuropathol. Exp. Neural., 54 (1995) 74-81. [36] T.H. McNeil1 and L. Koek, Differential effects of advancing age on neurotransmitter cell loss of the substantia nigra and striatum of C57BL/6N mice. Brain Res., 521 (1990) 107-l 17. [37] L.J. Poirier, M. Giguere and R. Marchand, Comparative morphology of the substantia nigra and ventral tegmental area in the monkey, cat and rat. Brain Res. Bull., II (1983) 371-397. [38] R.L. Gulley and R.L. Wood, The fine structure of the neurons in the rat substantia nigra. Tissue Cell, 3 (1971) 675-690. [39] EC. Hirsch, Does oxidative stress participate in nerve cell death in Parkinson’s disease. Eur. Neural., 33, suppl. 1 (1993) 52-59. [40] F. Gomez-Pinilla, J.W.-K. Lee and C.W. Cotwan, Basic FGF in adult rat brain: cellular distribution and response to entorhinal lesion and fimbria-fornix transection. J. Neurosci., 12 (1992) 345-355.
I.S. Lolova, S.R. Lolov / Mech. Ageing Dev. 82 (1995) 73-89
89
[41] L.J. Lawson, V.H. Peny, P. Dri and S. Gordon, Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience, 39 (1990) U-170. [42] K. Nagata, K. Nakajima and S. Kohsaka, Plasminogen promotes the development of rat mesencephalic dopaminergic neurons in vitro. Dev. Brain Res., 75 (1993) 31-37.