Age-Related Effects of DexamethasoneAdministration in Adrenal ZonaReticularis

HENRIQUE ALMEIDA, LILIANA MATOS, JORGE FERREIRA,AND DELMINDA NEVES

Laboratory for Molecular Cell Biology, Faculdade de Medicina do Porto,4200-319 Porto, Portugal

Instituto de Biologia Molecular e Celular da Universidade do Porto, Rua doCampo Alegre, 4150-180 Porto, Portugal

ABSTRACT: Suppression of adrenocorticotropic hormone results in re-duced adrenal steroid output, adrenocortical cell atrophy, and apoptosisin young rats. To verify such effects during aging, dexamethasone wasinjected into rats for 3 days at five different ages; at day 4, adrenals andblood were collected for morphologic and corticosterone assay. Adrenalstructure was similar at all ages, but in dexamethasone-injected ani-mals there were ultrastructural features of apoptosis and a higher per-centage of TUNEL and caspase-3-labeled nuclei and cytoplasm; theircorticosterone decreased significantly. In both groups, there was age-related decrease in the percentage of apoptotic cells, significant only indexamethasone-injected rats. The data suggest that aged adrenocorti-cal cells are less susceptible to the lack of adrenocorticotropic hormone(ACTH), possibly as a result of their decreased functional ability.

KEYWORDS: adrenal; age-related; TUNEL; caspase-3; apoptosis; corti-costerone

INTRODUCTION

The inner zone (IZ) of the adrenal cortex (AC) comprises the zona fascicu-lata (ZF) and the zona reticularis (ZR). Under close regulation by the pituitary-originated adrenocorticotropic hormone (ACTH) the IZ cells synthesize andsecrete glucocorticoids to the circulation, mostly cortisol in humans and cor-ticosterone in rats.

The administration of ACTH is followed by enhancement of steroidogenicenzyme activity, enlargement of IZ cell volume and mitochondrial and smooth

Address for correspondence: Henrique Almeida, Laboratorio de Biologia Molecular e Celular, Fac-uldade de Medicina do Porto 4200-319 Porto, Portugal. Voice: +351 22 551 3654; fax: +351 22 5513655.

e-mail: almeidah@med.up.pt

Ann. N.Y. Acad. Sci. 1067: 354–360 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1354.050

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ALMEIDA et al.: DEXAMETHASONE IN ADRENAL ZONA RETICULARIS 355

endoplasmic reticular compartments, and an increase in steroid output.1,2 How-ever, when ACTH is suppressed, as occurs after hypophysectomy or corti-costeroid administration, those effects are reversed1,2 and notable apoptoticfeatures are observed in ZR and its transition to ZF.3,4

So far, to the best of our knowledge, all experimental studies of ACTHsuppression were done using rat fetuses or young adult animals. Thereforedata are lacking about such effects in the adrenal parenchymal cells of the agedanimals or during aging. Yet, a recent study in a setting of ACTH blockade5

indicated an age-related increase in macrophages, suggesting an increment inthe need for the clearance of parenchymal dead cell and debris.

It was thus the purpose of this structural and immunocytochemical study toobserve and quantify apoptotic age-related changes in adrenal ZR parenchymalcells.

MATERIAL AND METHODS

Wistar male rats from the colony of the Institute of Molecular and CellBiology (IBMC, Porto) were maintained with free access to water and labora-tory diet. At 2, 6, 12, 18, and 24 months, eight apparently healthy animals wererandomly selected and divided into two groups. The first group was injectedintramuscularly for three consecutive days with 4 mg/kg of dexamethasonephosphate (Decadron�, Merck, Sharp & Dohme, Portugal), and the second(control) received a similar volume of saline solution. On the fourth day, theanimals were sacrificed by decapitation. One adrenal was processed for ul-trastructural morphologic study using previously described procedures;6 theother adrenal was fixed in formalin, embedded in paraffin, and sectioned ontopoly-L-lysine–coated slides.

The sections were processed for TUNEL by means of a TdT-FragEL kit fromOncogene Research Products (now Calbiochem) using the manufacturer’s in-structions; in the immunocytochemical study, a monoclonal antibody, anti-activated caspase-3 (Cell, Signaling Technology), diluted at 1:200, was em-ployed and a streptavidin/biotin/peroxidase system (Dakocytomation) usingdiaminobenzidine (DAB) was used for the final labeling.

Morphometric study, to assess parenchymal cell volume (CV), nuclear vol-ume (VN), and numerical density (Nv), was done as previously described.7 Theapoptotic index (AI) was quantified in ZR random photographs of TUNEL sec-tions, as the percentage of apoptotic nuclei per total number of nuclei; for eachrat a total 2,500–3,000 nuclei were counted.

Trunk blood was collected for corticosterone assay by high-performanceliquid chromatography (HPLC).6

ANOVA was used to compare the means of measurements at different ages.The statistical significance of differences between individual pairs of meanswas assessed using the least-significant-difference procedure of Fisher (LSDtest). For a comparison between the two groups, the Student t-test was used.

356 ANNALS NEW YORK ACADEMY OF SCIENCES

RESULTS

Microscopic Study

Light microscopy revealed that the general structure of the AC was conservedthroughout aging, allowing the identification of the usual cortical zones andthe medulla. In the IZ of dexamethasone (DEX)-treated animals, there was anapparent decrease in cell and nuclear volumes.

The ultrastructural study did not disclose major changes in the cytoplasmand nuclei of DEX animals at any age, but, in contrast to the controls, asubstantial number of cells displayed nuclei with a wavy outline and distortedand clumped chromatin, indicating apoptotic changes. In addition, variousapoptotic bodies appearing as membrane-bound structures containing packedmitochondria, lipid droplets, and portions of chromatin (some enveloped bynuclear membrane) were observed close to, or within, the capillaries (FIG. 1A).Some of the capillaries had a disrupted structure, allowing direct contact ofparenchymal cells with the lumina and access of apoptotic bodies into thelumina.

In the TUNEL sections, several nuclei in the ZR and ZF/ZR transition wereshrunken and intensely stained with DAB (FIG. 1B). Clearly, they were morecommon in DEX animals when compared to controls. Immunocytochemicalstudy of caspase-3 some parenchymal cells of the ZR of DEX rats had a distinc-tive DAB-stained cytoplasm (FIG. 1C), but in the same zone other cells had faintlabeling. In the sections processed either for TUNEL or caspase-3 labeling, thecells from the zona glomerulosa ZG, most of the ZF and medulla presented nolabeling.

Quantitative Study

The results for CV (�m3), VN (�m3), and Nv (×103/mm3) are shown inTABLE 1. There was a statistically significant decrease in cell and nuclear vol-umes of DEX animals when assessed by the Student t-test. However, ANOVAdid not reveal any significant age-related trend.

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→FIGURE 1. (A) Ultrastructure of ZR from a 24-month-old DEX rat displaying an

apoptotic body beside a capillary lumen (black star). Mitochondria (m), lysosome (arrow),and possible fragments of the nucleus (white star) are seen; some lipid droplets are extrudingfrom the body (L). N and M: nucleus and mitochondria from a parenchymal cell. Bar:2 �m. (B) ZR from a 24-month-old DEX rat with DAB-stained nuclei (circles). M: adrenalmedulla. TUNEL. Bar: 50 �m. (C) Same material as (B). Several ZR cells have DAB-stained cytoplasm (arrows). M: adrenal medulla. Caspase-3 immunocytochemistry. Bar:50 �m.

ALMEIDA et al.: DEXAMETHASONE IN ADRENAL ZONA RETICULARIS 357

358 ANNALS NEW YORK ACADEMY OF SCIENCES

The results for the apoptotic and corticosterone assay are displayed inTABLE 2. There was an age-related decrease of AI in both groups, signifi-cant for DEX rats and marginally not significant for controls, and a reducedbasal blood corticosterone in older animals compared to young ones. In someDEX animals, the assay failed to detect any corticosterone peak.

DISCUSSION

The pituitary peptide ACTH exerts a trophic action on the IZ of the adrenalcortex. ACTH mobilizes cholesterol obtained from circulating lipoproteinsand intracellular lipid stores. Then, the steroidogenic acute regulatory enzyme(StAR) delivers the cholesterol to the mitochondria for glucocorticoid synthesisand ultimate secretion into the blood; in addition, in vivo mitogenic effectson the adrenal cortex have been attributed to the peptide. Conversely, thisbalance is changed when ACTH secretion is blocked by hypophysectomy orcorticosteroid administration: steroid output is reduced, adrenals lose weight,the cortex becomes atrophic, and IZ cells decrease in volume1,8 and displayapoptotic features.3,4

The present study confirmed a decrement of circulating corticosterone andZR cell volume, and showed that the effects on ZR cells are maintained duringaging. Either ultrastructural observations or the results of the TUNEL study andcaspase-3 labeling provided clear evidence that frequent apoptotic phenomenawere taking place at any age under these experimental conditions.

The cell migration theory of the adrenal cortex9 states that cell proliferationoccurs in the ZG, or the intermediate zone between ZF and ZG, and, as the cellsdifferentiate, they move centripetally and die at the ZR, where they are removedby the macrophages. Whether for the purpose of clearance or as on additionalmodulation effect, adrenal macrophages attain their highest numerical densityin ZR, where they increase in an age-related fashion.10 This trend is maintainedin DEX-treated animals,5 supporting their role in removing the dead cells ordebris.

This study showed significant quantitative differences between controls andDEX animals in blood corticosterone, ZR nuclear and cellular volumes, andapoptotic indexes at any age. However, in the assessment of differences duringaging, the decrease of the apoptotic index in DEX rats was the only significanttrend observed. Interestingly, higher levels of AI were observed in the 6-month-old and 12-month-old groups, whereas the lowest level was present in 24-month-old animals. At this age, the lowest levels of corticosterone, eitherbasal or in response to the ACTH challenge, were recorded in a previousstudy,6 indicating a lesser functional ability of aged cells eventually due to adecrease in ACTH receptors.11

It is conceivable that this lesser ability of aged cells to respond to ACTHenables them to better withstand the lack of ACTH, thus explaining the age-related decrease in the apoptotic index.

ALMEIDA et al.: DEXAMETHASONE IN ADRENAL ZONA RETICULARIS 359

TA

BL

E1.

Par

ench

ymal

cellu

lar

(CV

,�

m3)

and

nucl

ear

(VN

,�

m3)

volu

mes

and

Nv

(×10

3/m

m3)

ofco

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ls(C

t)an

dde

xam

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d(D

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tag

es

CV

Ct

CV

Dx

VN

Ct

VN

Dx

Nv

Ct

Nv

Dx

2m

14

19

.01

±48

.07

10

37

.05

b±6

2.7

72

10

.10

±8.0

71

49

.10

b±8

.25

54

2.5

8±1

6.8

55

97

.09

±28

.25

6m

13

64

.15

±68

.20

10

94

.35

±90

.83

18

5.3

9±1

.95

14

9.9

8a

±12

.24

58

9.8

2±2

7.1

26

91

.16

a±2

5.7

3

12

m1

50

4.8

6±1

13

.07

10

29

.37

b±5

7.4

41

84

.69

±8.6

11

34

.28

b±4

.37

52

6.8

9±2

9.1

76

96

.81

±65

.03

18

m1

55

8.7

9±1

56

.10

10

44

.73

a±9

6.3

02

02

.80

±19

.31

13

4.5

0a

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75

11

.90

±65

.72

63

0.3

2±7

1.7

02

4m

15

28

.21

±18

1.7

01

11

7.2

9a

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.68

21

4.2

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0.9

71

36

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.91

53

0.2

4±7

2.9

35

83

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P<

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360 ANNALS NEW YORK ACADEMY OF SCIENCES

TABLE 2. Apoptotic index (AI, %) in ZR and blood corticosterone (B, ng/mL) of controls(Ct) and dexamethasone-injected (Dx) rats at different ages

AI Ct AI Dx B Ct B Dx

2 m 0.39 ±0.11 3.07b ±0.42 285.5∗ ±25.4 15.1b ±9.9

6 m 1.25 ±0.43 4.80a ±0.72 236.1 ±39.7 0.0b ±0.0

12 m 0.98 ±0.16 4.41b ±0.67 305.5 ±61.8 4.0b ±4.0

18 m 0.54 ±0.12 3.98b ±0.26 175.0∗ ±2.0 0.0b ±0.024 m 0.65 ±0.10 2.28a ±0.60 143.4 ±41.6 18.4a ±10.6P< 0.051 0.05 n.s. n.s.

a and b refer to comparisons with the controls, respectively, for P < 0.05 and P < 0.01 in theStudent’s t-test. Means ± standard error, n = 4 or n = 2 (∗).

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