Effect of acute heat stress on rat adrenal glands: a morphological and stereological study
Faculty of Biology, University of Belgrade, Serbia and Montenegro
* Author for correspondence (e-mail: vesnak{at}bf.bio.bg.ac.yu)
Accepted 7 September 2004
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Summary |
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Key words: heat stress, rat, adrenal gland, histology
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Introduction |
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Bearing in mind that ACTH is the major stimulator of adrenal cortical
function, investigation of their relationship is of great importance in both
basal and stress conditions. Short-term ACTH treatment provokes a decrease in
volume of the lipid-droplet compartments in rat zona glomeruloza (ZG) cells,
and a rise in plasma and intracellular concentrations of corticosterone and
aldosterone (Mazzocchi et al.,
1986). ZG growth (i.e. the volume of the ZG and its parenchymal
cells) is stimulated by angiotensin II, sodium deficiency, potassium loading,
ACTH and prolactin, and inhibited by somatostatin. Rat ZG hyperplasia and
mitotic activity were found to be induced by chronic ACTH and cAMP treatment
(Lewinski and Szkudlinski,
1981
; Payet and Lehoux,
1982
). It was shown that ACTH itself, or stress-induced increased
ACTH secretion, exert tropic (short-term) and trophic (long-term) effects on
the adrenocortical zona fasciculata (ZF) and zona reticularis (ZR). Tropic
effects involve an immediate increase in corticosteroid hormone secretion,
which appears about 10 min after the beginning of stress and reaches the
maximum 1530 min later (Jaanus et
al., 1970
; Normand et al.,
1982
). The trophic effect of ACTH involves an increase in adrenal
mass and in the steroidogenic capacity of adrenocortical cells
(Nussdorfer, 1986
).
Other results suggest that the HPA axis is activated in any type of stress
(acute or chronic), but the adrenal gland response varies
(Nussdorfer and Mazzocchi,
1983; Robba et al.,
1985
; Mazzocchi et al.,
1986
). Taking all this into consideration, as well as the fact
that there are very few reports on morphological and quantitative analysis of
adrenal glands after acute heat exposure, we decided to determine the effect
of high ambient temperarture on rat adrenal gland morphology.
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Materials and methods |
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Animals were weighed and decapitated. The body mass of control rats was 203±9.79 g (mean ± S.E.M.), and 190.2±7.90 g for heat stressed rats. Animals were decapitated by guillotine (Harward-Apparatus, Holliston, MA, USA). The left adrenal gland was quickly excised, freed of fat tissue (4°C) and weighed. Blood was collected from the trunk, divided into two sets of tubes, and EDTA added to obtain plasma. Serum and plasma were frozen for the ACTH and corticosterone (CORT) determination. Serum CORT was determined using a RIA kit (Biochemicals, Costa Mesa, CA, USA) and the values expressed as ng CORT ml1 serum. Plasma ACTH concentration was determined by a chemiluminescence method using an IMMULITE automatic analyzer (DPC, Los Angeles, CA, USA). The values are expressed as pg ACTH ml1 plasma. This protocol has been approved by the Canadian Council on Animal Care.
The total volume of the adrenal gland was determined according to Swinyard
(1938). Relative adrenal mass
was calculated as a % of body mass. Each left adrenal gland was fixed in
Bouin's solution and embedded in paraffin, according to standard procedures. 5
µm thick sections were stained by the AZAN trichrome technique.
Stereological analysis of every tenth section of adrenal gland and its
components (capsule, cortex and medulla) was performed using Weibel's
multipurpose test grid M42 by a point counting technique
(Weibel et al., 1966
).
The volume fractions of parenchyma cell nuclei, cytoplasm and connective
tissue together with blood vessels (stroma) were estimated and the number of
nuclear profiles of adrenocortical cells per unit area of section was counted
at a magnification of 1000 x. Ten fields of each adrenal zona (zona
glomerulosa, ZG; zona fasciculata, ZF; zona reticularis, ZR) were counted in a
single section from each adrenal gland. On the basis of earlier karyometric
studies (Malendowiez et al., 1974), the shape coefficient ß, which
relates NV to NA and
VV and depends on the axial ratio of estimated nuclei, was
assumed to be 1.382 for ZF and ZR, and 1.500 for ZG. The number of nuclei of
adrenocortical cells per mm3 was calculated according to the method
of Weibel and Gomez (see Aherne and
Dunnill, 1982). Since rat adrenocortical cells are mononucleic,
the numerical density of nuclei corresponds to the number of cells per
mm3. The average volume V of cells and nuclei in each
cortical zone was estimated from the formula:
Vcell(nuclei)=VVcell(nuclei)/NVcell(nuclei).
The average diameter D of blood vessels in ZF and ZR was estimated
from the formula: D=6VVblood
vessels/SVblood vessels, where
Sv=2I/Lt (I, number
of intersections on blood vessels; Lt, total length of
test lines). The length of the blood vessels was calculated from the formula:
L=4/
xVVblood vessels/D.
The Student t-test was employed to determine statistically significant differences between means.
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Results |
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The absolute and relative adrenal gland masses of heat-exposed rats were significantly decreased, as well as the absolute mass of adrenal cortex in comparison to controls (Table 1), probably as a consequence of the reduction of all cortical zones (ZG, ZF and ZR), especially that of ZF.
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Histological investigation of adrenal gland revealed the existence of dark and light regions in the adrenal cortex of stressed rats (Fig. 2A). The cells in the light regions were filled with large lipid droplets (Fig. 2B), but those in the dark cortical regions were deprived of them. The whole ZG area was reduced and filled with enlarged cells and nuclei. Condensed nuclei were observed in ZR cells. Interstitial fibrosis was present in all parts of the cortical zones (Fig. 2C), as well as in the medulla. There were no mitoses visible in the adrenal glands of rats exposed to heat stress. In control rats, however, all components of the adrenal glands were clearly differentiated (Fig. 2D). ZG of control rats contained many nuclei, as compared to ZG cells in stressed rats, and many mitotic figures were present in the outer cortical zones (ZF in close proximity to ZG). The cytoplasm of ZF cells contained a moderate number of lipid droplets (Fig. 2E). All the nuclei in ZR of control animals cortex were large and light.
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The volume densities of adrenal gland component and cortical zones are shown in Fig. 3. The volume density of medulla was significantly increased, and that of cortex decreased. Cortical ZG was significantly decreased, and that of ZR increased, in the rats exposed to heat stress. There were no differences in volume density of ZF between two groups of rats.
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Fig. 4 illustrates that the absolute volume of adrenal glands and that of cortex, as well as the absolute volume of ZF was significantly reduced in comparison to controls.
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The results of the stereological investigation of the cortical zones are shown in Table 2. The volumes of cells and nuclei in ZF and ZR were decreased and those in the ZG increased. There were no significant differences between the two groups of rats with respect to the volume of cells and their nuclei, except for ZR nuclei (P<0.05). The numerical density of cells showed a slight reduction in ZG, and augmentation in ZF and ZR. The mean diameter and lenght of blood vessels in ZF and ZR were similar in both groups of rats.
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Discussion |
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High ambient temperature induced a significant reduction of absolute and relative adrenal mass due to the reduction of cortical mass, especially that of ZF. This was expected because glucocorticoid synthesis is performed mostly in the ZF and this is the largest part of the cortex.
Morphological and stereological studies revealed that heat exposure
provoked a depletion of lipid droplets from the ZF cells and also a reduction
of their volumes. The volume reduction was also observed in ZR cells, which
represent the innermost layer adjacent to the medulla. Although the ZR cells
were able to secrete glucocorticoids they primarily produced and secreted weak
androgens. However, heat stress induced an increment of ZG cell volume. These
cells are responsible for production of aldosterone (the salt-retaining
hormone), whose physiological effects correspond to the hypothalamic hormone,
vasopressin (AVP). This hormone, according to Gallo-Payet and Guillon
(1998), can also stimulate
cortisol secretion, but not that of corticosterone, which is the dominant
glucocorticoid in rats. It was shown that under conditions of stress, both
ACTH and AVP secretion was increased
(Aguilera, 1996
), and when ACTH
was secreted in large amounts, such as in conditions of stress, it could also
stimulate aldosterone secretion. Thus, under the heat stress, both ACTH and
AVP stimulated enlargement of ZG cell volume, the site of aldosterone
accumulation. Bearing in mind that, under heat stress conditions the animals
were in hyperthermia, which is characterised by increased body temperature
(39.9°C vs 37.2°C in controls, P<0.001) and
accompanying salivation, resulting in water and salt loss. The increased
amount of aldosterone is necessary to re-establish osmotic homeostasis.
In the unstimulated adrenal gland, intracortical capillaries were
constricted, and after operative stress, or following a 1 h period of ACTH
perfusion, they become massively expanded
(Pudney et al., 1981). In
spite of the fact that the ACTH level in heat stressed rats was very high, the
mean diameter and length of blood vessels in the ZF and ZR regions did not
significantly change. Another type of acute stress is ethanol injection, which
also caused a significant rise in ACTH and CORT levels
(Rivier, 1996
;
Rivier and Lee, 1996
), and
induced the opposite morphological and stereological changes in adrenal glands
(Milovanovic et al., 2003
) to
those observed by us on heat stress. That is, ethanol provoked increased ZF
and ZR and decreased ZG cell volumes with dilated small blood vessels and
prominent hyperemia. These authors did not find any sign of fibrosis such as
we found after heat stress. This could support the idea that adrenal gland
responds differently to the various stressors
(Pacak and Palkovits, 2001
),
despite the same reaction in the hypothalamicpituitary part of the HPA
axis.
There are reports suggesting that the outer part of the adrenal glands,
particularly the ZG, as the source of new cortical cells, is the major site of
mitosis and that most cell deaths occur within the inner part of the cortex,
particularly in the ZR (Wright and
Voncina, 1977; Stachowiak et
al., 1990
; Miyamoto et al.,
2000
). We also observed mitoses in ZG and ZF in control rats, but
did not find any sign of mitoses in rat adrenal gland after heat stress. This
phenomenon could be explained by the great sensitivity of mitotic cells to
hyperthermia, in contrast to the interphase cells. Hyperthermia induces
disruption of the mitotic spindle, a reappearance and an extension of the
Golgi apparatus, an inactivation of microtubule nucleation, and a
disorganization of the centrosome (Debec
and Marcaillou, 1997
). Regulation of the cell cycle during thermal
stress involves many heat shock proteins. The 47 kDa heat shock protein (HSP
47) is a collagen-binding glucoprotein that is heat-inducible and sensitive to
malignant transformation (Nagata et al.,
1986
; Nagata and Yamada,
1986
). It is involved in collagen processing and/or secretion
under normal conditions. Under stress conditions, HSP 47 synthesis is
increased, which correlates with collagen synthesis in several cell lines
(Nagata and Yamada, 1986
;
Takechi et al., 1992
;
Kudo et al., 1994
).
Schiaffonati et al. (1991
)
showed that after a 50 min heatexposure (41°C), there was an immediate
increase in Hsp70 and Hsp90 levels in rat liver. Inaguma et al.
(1995
) suggested that induction
of Hsp27 and Hsp70 in various tissues of heat stressed rats (20 min exposure,
42°C), including adrenal glands, is controlled by a physiological
process(es) that is sensitive to ethanol and prazosin and is operative for a
short time during the application of heat stress. These literature reports
confirm and explain an interstitial fibrosis in all parts of the adrenal gland
in rats after heat stress, as we found in the present study.
In conclusion, acute exposure to heat significantly increased the circulating ACTH and CORT concentrations in rats. A high ambient temperature reduced the absolute and relative mass of the adrenal cortex, especially that of ZF, which is partially depleted of lipid droplets. Fibrosis was present and mitotic figures were absent in all parts of adrenal glands of rats exposed to heat stress.
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Acknowledgments |
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