Thymic
involution in Wistar and OXYS rats at different ages
The thymus of OXYS rats, like that of
Wistar rats, consists of two lobes of unequal size, which are tightly
juxtaposed and separated by a thin layer of loose connective tissue. The
anatomy of the thymus of OXYS and Wistar rats seem to be similar. However,
microscopic studies of serial sections of OXYS rat thymus at 10 days of age
revealed immature lobules which were remarkable for their lack of distinct
differentiation between cortex and medulla as well as low cell density.
The
absolute weight of the thymus in OXYS rats is significantly lower than in
Wistar rats (Figure 1A-F). This is partially due to smaller body weight of OXYS
rats. However, at age 2 and 3.5 months, not only absolute thymic weight but
also thymic weight normalized to the body weight (thymic index) of OXYS rats
was shown to be lower than that of Wistar rats (Figure 1B).
The
total volume of the thymus significantly increased in both rat strains from age
10 days to age 2 months, reached a maximum, and then declined slightly between
2 and 3.5 months of age and significantly between 3.5 and 14 months of age. The
total volume was always lower in OXYS rats, and the difference was
statistically significant at age 10 days and 2 and 14 months (Figure 1C).
The
thymic cortex volume in both strains of rats increased between 10 days and 2
months of age, reached a maximum, and then decreased (Figure 1D). At all time
points, the thymic cortex volume was lower in OXYS rats, and statistically
significant differences were observed at age 10 days and 2 and 14 months.
The
volume of the medulla also increased in both strains of rats between 10 days
and 2 months of age, reached a maximum at 3.5 months, and then declined (Figure 1E). The medulla volume was always lower in OXYS rats.
The
ratio of cortex to medulla (C/M index) changed with age in a fashion similar in
the two studied rat strains (not shown in figures).
The
volume of connective tissue stroma of the thymus, the capsule, and septa
changed as a function of age of the animals. It increased significantly between
10 days and 2 months of age, did not noticeably change between 2 and 3.5
months, and declined by 14 months in both rat strains. At all time points, the
absolute value was lower in OXYS rats due to lower total thymic size (not
shown).
One
of the characteristic signs of age-related involution of the thymus is
replacement of lymphoid tissue with adipose tissue. In the thymuses of
10-day-old Wistar and OXYS rats, adipose tissue was not detectable. At age 2
and 3.5 months, single adipocytes were observable in the capsule and septa. At
age 14 months, adipose tissue occupied measurable part of the thymus. This
volume was two-fold higher in Wistar rats compared
to OXYS rats (2.8 ± 0.3 mm³ and 1.4 ± 0.2 mm³, respectively, p =
0.0033). Percent content of adipose tissue in the thymus of OXYS and Wistar rats was
similar (12.6 ± 1.4 % vs. 13.5 ± 2.8 %, respectively).
Figure 1A. Age-related changes in the thymus of Wistar and OXYS rats.
Absolute weight of the thymus (here and below, mean ±
S.E.).
Figure 1B. Age-related changes in the thymus of Wistar and OXYS rats.
Thymic weight index.
Figure 1C. Age-related changes in the thymus of Wistar and OXYS rats.
Total thymic volume.
Figure 1D. Age-related changes in the thymus of Wistar and OXYS rats.
Volume of thymic cortex.
Figure 1E. Age-related changes in the thymus of Wistar and OXYS rats.
Volume of thymic medulla.
Figure 1F. Age-related changes in the thymus of Wistar and OXYS rats.
Total cell number (cellularity) of the thymus.
Table 1. Cellular content of the thymus in Wistar and OXYS rats at different ages (absolute numbers of various subpopulations of lymphocytes in the thymus, mean ± S.E. x10 6).
Measurements
|
Age
|
Age group, ID
(size)
|
Wistar
|
OXYS
|
Statistical significance of
interstrain differences, p |
CD3+
|
10 d.
|
1 (n=7)
|
2300±386
|
2308±17
|
not significant (>0.05)
|
2 mo.
|
2 (n=7)
|
1550±131
|
859±92
|
0.0012
|
3.5 mo.
|
3 (n=5)
|
405±35
|
168±43
|
0.0029
|
Comparison age groups, p
value
|
1-2, =0.00031
2-3, =0.000123
|
1-2, =0.0001
2-3, =0.0001
| |
CD3+hi |
10 d.
|
1 (n=7)
|
34.1±4.3
|
49.1±6.9
|
not significant
|
2 mo.
|
2 (n=7)
|
227±9.4
|
162±17.2
|
0.0062
|
3.5 mo.
|
3 (n=5)
|
90.1±13.6
|
38.9±6.8
|
0.0101
|
Comparison age groups, p
value
|
1-2, <0.0001
2-3, <0.0001
|
1-2, =0.0001
2-3, =0.0002
| |
CD4+/
CD8+
|
10 d.
|
1 (n=7)
|
267±44.5
|
291±20.2
|
not significant
|
2 mo.
|
2 (n=7)
|
1610±135.1
|
972±115.9
|
0.004
|
3.5 mo.
|
3 (n=5)
|
481±44.2
|
192±36.4
|
0.0011
|
Comparison age groups, p
value
|
1-2, <0.0001
2-3, <0.0001
|
1-2, =0.00013
2-3, =0.00013
| |
CD4+
|
10 d.
|
1 (n=7)
|
22.6±4.0
|
21.8±2.9
|
not significant
|
2 mo.
|
2 (n=7)
|
173±18.9
|
136±9.8
|
not significant
|
3.5 mo.
|
3 (n=5)
|
44.1±4.1
|
19.2±3.0
|
0.0014
|
Comparison age groups, p
value
|
1-2, <0.0002
2-3, <0.0001
|
1-2, =0.00017
2-3, =0.00013
| |
CD8+
|
10 d.
|
1 (n=7)
|
6.1±1.2
|
6.4±0.8
|
not significant
|
2 mo.
|
2 (n=7)
|
53.9±4.3
|
31.9±3.8
|
0.003
|
3.5 mo.
|
3 (n=5)
|
23.1±3.1
|
7.4±1.2
|
0.002
|
Comparison age groups, p
value
|
1-2, <0.00013
2-3, <0.0001
|
1-2, =0.00017
2-3, =0.00013
| |
CD4+/
CD8+
|
10 d.
|
1 (n=7)
|
3.8±0.1
|
3.4±0.3
|
not significant
|
2 mo.
|
2 (n=7)
|
3.2±0.3
|
4.6±0.5
|
0.038
|
3.5 mo.
|
3 (n=5)
|
1.9±0.1
|
2.6±0.2
|
0.002
|
At
age 10 days and 2 and 3.5 months, the total number of cells (cellularity) in
the thymus and immunological phenotype of thymocytes was determined using flow
cytometry. Major subpopulations of thymocytes were detected by means of surface
markers for CD3+, CD4+, and CD8+ cells. Two-way ANOVA analysis showed that the
total cell number in the thymus changed with age. In both strains, it increased
between 10 days and 2 months, reached a maximum, and then declined. No strain
difference in total cell number was found on day 10. At the age of 2 and 3.5
months, this parameter was lower in OXYS than in Wistar rats (Figure 1F).
Double-positive lymphocytes were the predominant cell type in the thymus at all
time points in both strains. Their relative number was the highest in
10-day-old animals regardless of the strain. This measure tended to decrease
with age (not shown).
The
second most numerous subpopulation among thymocytes was mature CD4+ lymphocytes
(Т-helper cells). Their number was not significantly different between
the rat strains, but changed with age. The percentage of CD4+ cells was the
lowest on day 10 in both rat strains, increased at age 2 months and decreased
at age 3.5 months (Table 1).
The
third most numerous subpopulation of thymocytes was CD8+ cells (cytotoxic T
lymphocytes and suppressor T cells). Their dynamics were similar to that of
CD4+ cells (Table 1). The relative number of all CD3+ thymocytes was dependent
on both genotype and age of the animals. This parameter lowered with age in
both rat strains, the lowering being stronger in the OXYS rats (Table 1).
Analysis of subfractions of CD3+ cells showed that the content of CD3+low+medcells was lower in OXYS rats compared to Wistar rats (not shown). As to
CD3+hi, it first increased and then decreased with age in both
strains of rats (Table 1).
An
important parameter of a population of single-positive thymocytes is the
CD4+/CD8+ ratio. In the present study, it decreased with age in both strains
(Table 1).
These results indicate that both OXYS and
Wistar rats exhibit age-dependent signs of primary hypoplasia of the thymus, as
evidenced by the lowering of average thymic weight and total volume, decrease
in volumes of cortex and medulla (epithelial compartment of the thymus), as
well as total numbers of thymic cells, and lowering of absolute numbers of
major subpopulations of thymocytes. The direction of all these changes was
similar in two strains. However, by the end of fast body growth and the
beginning of age-related thymic invo-lution (age 2 to 3.5 months), lowering of
the thymic cellularity developed more quickly in OXYS rats.
Effect
of mitochondria-targeted antioxidant SkQ1 on thymic characteristics of OXYS and
Wistar rats
In
the second part of the investigation, we studied the effect of SkQ1 on the
morphological and functional state of the thymus of Wistar and OXYS rats. The
animals received the drug with food at the dose of 250 nmol SkQ1/kg body per
day weight from the age of 1.5 to 14 months.
Two-factorial
dispersion analysis of weight measurements at age 3.5 months revealed that the
drug treatment increases the absolute weight of the thymus (not shown) and
thymic weight index (Figure 2A). In OXYS rats, the increase was stronger, being
statistically significant. Similar relationships were revealed when the total
thymic volume was measured (Figure 2B). The SkQ1 treatment also enhanced the
volumes of structural components of the thymus - cortex, medulla, and
connective tissue stroma. Again, the effect (a volume increase) was stronger in
OXYS rats (Figure 2C-E).
The
total number of cells in the thymus was affected by both drug treatment and
genotype of the animals. The thymic cellularity in OXYS rats was much lower
than in Wistar rats and was increased threefold by the SkQ1 treatment (Figure 2F).
Data
on amounts of various cell fractions in the thymus of Wistar and OXYS rats
treated and non-treated with SkQ1 are shown in Table 2. The table shows that
percentage content of various subpopulations of thymo-cytes did not undergo
significant changes as a result of the SkQ1 treatment. However, the analysis of
absolute values revealed very strong positive effect of SkQ1.
Cell cycle analysis in thymocytes did not show
significant differences between OXYS and Wistar rats. SkQ1 also had no effect
on the percentage of cells in various phases of the cell cycle. The percentage
of cells with fragmented DNA, characteristic of apoptosis (
The
number of thymocytes at the various stages of apoptosis was determined using
parallel staining of the tissue from 3.5 months rats with annexin V,
7-amino-actinomycin D (7-ААD), or TUNEL followed by flow cytometry.
No significant effects of strain or SkQ1 were revealed (not shown).
Figure 2A. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Age of the animals, 3.5 months. SkQ1 was fed during the last 2 months.
Figure 2B. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Total thymic volume.
Figure 2C. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Volume of thymic cortex.
Figure 2D. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Volume of thymic medulla.
Figure 2E. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Volume occupied by connective tissue stroma.
Figure 2F. Effect of SkQ1 on thymic involution in Wistar and OXYS rats.
Thymic cellularity.
In the last series of experiments, the effect of SkQ1 was
studied on old (14 months) Wistar and OXYS rats. The data are summarized in
Table 3. One can see pro-nounced SkQ1-induced increase in the thymic weight
index, total volume of thymus, and volumes of its cortex and medulla. Volumes of thymic stroma and adipose tissue
slightly or strongly increased, respectively. All the above changes with the
exception of the adipose tissue volume were larger in Wistar than in OXYS rats
(in con-trast to that observed in 3.5-month-old rats, see Figure 2A-F).
Table 2. Effect of SkQ1 on cellular content (mean ± S.E. X10 6 )
of the thymus in Wistar and OXYS rats. Data for age 3.5 months with SkQ1
administration started at age 1.5 months.
Strain
|
Wistar
|
OXYS
|
Comparison of groups, p
value
|
no SkQ1
(group 1)
|
SkQ1
(group 2)
|
no SkQ1
(group 3)
|
SkQ1
(group 4)
|
CD3+
(absolute)
|
405±35
|
526±73
|
168±43
|
488±66
|
1-3, =0.008
3-4, =0.002
|
CD3+hi
(absolute)
|
90.1±13.6
|
87.1±9.7
|
38.9±6.8
|
92.2±13.8
|
1-3, =0.018
3-4, =0.025
|
CD4+/CD8+(absolute)
|
481±44
|
588±85
|
191±36
|
582±72
|
1-3, =0.004
3-4, =0.001
|
CD4+(absolute)
|
44.1±4.1
|
47.9±5.6
|
19.2±3.0
|
49.3±9.5
|
1-3, =0.01
3-4, =0.017
|
CD8+(absolute)
|
23.1±3.1
|
21.6±2.9
|
7.4±1.2
|
27.4±5.8
|
1-3, =0.02
3-4, =0.008
|
CD4/CD8
|
1.9±0.1
|
2.3±0.2
|
2.7±0.2
|
1.8±0.1
|
1-2, =0.025
3-4, =0.017
|
CD3+ (%)
|
72.0±1.2
|
78.7±4.1
|
72.6±5.6
|
72.2±2.0
|
-
|
CD3+low+med
(%)
|
55.9±1.7
|
65.4±4.9
|
54.9±6.3
|
58.5±1.2
|
-
|
CD3+hi (%)
|
16.1±2.1
|
13.3±1.2
|
17.6±1.0
|
13.7±1.1
|
-
|
CD4+/CD8+ (%)
|
85.5±1.4
|
87.6±0.6
|
85.6±1.1
|
86.6±1.1
|
-
|
CD4+ (%)
|
7.9±0.7
|
7.2±0.3
|
9.0±0.8
|
7.1±10.6
|
-
|
CD8+ (%)
|
3.8±0.4
|
3.9±0.3
|
3.7±0.2
|
3.5±0.2
|
-
|
Table 3. Effect of SkQ1 on the thymus (mean ± S.E.) in 14-month-old Wistar and OXYS rats.
Strain
|
Wistar
|
OXYS
|
Comparison
of groups, p value
|
no
SkQ1
(group
1)
n=6
|
SkQ1
(group
2)
n=9
|
no
SkQ1
(group
3)
n=7
|
SkQ1
(group
4)
n=9
|
Thymic weight index (mg/100 g)
|
11.8±1.7
|
17.8±2.0
|
8.97±0.54
|
10.1±0.94
|
1-2,
=0,008
2-4,
=0,003
|
Total volume of thymus (mm3 )
|
26.9±7.1
|
44.4±4.6
|
10.7±0.6
|
13.6±2.9
|
1-2,
=0,007
1-3,
=0,03
2-4,
=0,0002
|
Volume of thymic cortex (mm3
)
|
12.4±3.9
|
23.2±3.0
|
4.2±0.3
|
6.2±1.5
|
1-2,
=0,005
2-4,
=0,0003
|
Volume of thymic medulla (mm3
)
|
6.7±2.1
|
12.7±1.4
|
2.4±0.1
|
3.6±0.9
|
1-2,
=0,003
2-4,
=0,0002
|
Volume of thymic stroma (mm3
)
|
5.0±1.2
|
6.8±0.6
|
2.8±0.2
|
3.2±0.6
|
2-4,
=0,002
|
Volume of adipose tissue (mm3
)
|
2.8±0.4
|
1.7±0.3
|
1.4±0.2
|
0.7±0.2
|
1-2,
=0,0035
1-3,
=0,0011
2-4,
=0,023
|