This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gayrard, V. Articles by Toutain, P. L. Search for Related Content PubMed PubMed Citation Articles by Gayrard, V. Articles by Toutain, P. L.

Major Hypercorticism Is an Endocrine Feature of Ewes with Naturally Occurring Scrapie

Unité associée INRA de Physiopathologie et Toxicologie Expérimentales (V.G., N.P.-H., C.G., J.G., P.L.T.) Ecole Nationale Vétérinaire de Toulouse, 31076 Toulouse, France; Laboratoire de Neuroendocrinologie Expérimentale (M.G., N.S.), Institut National de la Santé et de la Recherche Médicale U501, 13326 Marseille, France; and Unité associée INRA de Physiopathologie Respiratoire des Ruminants (O.A., F.S.), Ecole Nationale Vétérinaire de Toulouse, 31076 Toulouse, France

Address all correspondence and requests for reprints to: P. L. Toutain, Laboratoire de Physiologie & Thérapeutique, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, 31076 Toulouse. E-mail: pl.toutain{at}envt.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of this study was to identify the origin of scrapie-induced hypercortisolism. Cortisol and ACTH kinetics and production rate were measured in 14 ewes (6 healthy and 8 scrapie-affected). It was shown that cortisol plasma clearance remained unmodified but that cortisol production rate and plasma concentrations of free cortisol were increased by a factor of 5, whereas the total cortisol plasma concentrations were only doubled. The apparent discrepancy between adrenal secretion rate and the corresponding total cortisol plasma levels was attributable to the scrapie-induced lower corticosteroid-binding globulin (CBG) binding capacity, which altered the ratio of free-to-bound cortisol. The secretion rate of ACTH from diseased ewes was increased by a factor of 1.5, in comparison with healthy ewes, and 4 of the 8 scrapie-affected ewes exhibited a decreased response to a low dexamethasone suppression test. The administration of tetracosactide induced a 2-fold increase in the cortisol production in diseased ewes, compared with that of healthy ewes, but the pituitary sensitivity to ovine CRF was not modified by the prion disease.

In conclusion, natural scrapie displays a syndrome of hypercorticism associated with increased ACTH secretion, hyperresponsiveness of the adrenals, and lower CBG binding capacity, which leads to overexposure to CBG-free cortisol.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
SCRAPIE IS AN ovine subacute transmissible spongiform encephalopathy caused by unconventional transmissible agents, which include human Creutzfeldt-Jacob disease and Bovine Spongiform Encephalopathy. We demonstrated in a previous study that this prion disease displays a syndrome of hypercortisolism (1). The mechanism by which scrapie-affected ewes maintain high plasma cortisol levels may involve a specific scrapie-induced increase in hormone production rate and/or a generally disrupted elimination process resulting in decreased cortisol clearance. Determination of the cortisol production rate requires evaluation of both the plasma cortisol clearance and a 24-h plasma cortisol profile. Because of the nonlinear binding of cortisol to corticosteroid-binding globulin (CBG), the plasma clearance of total plasma cortisol (i.e. both free and bound) is not a parameter but a variable that is subjected to both short-term and long-term fluctuations linked to ultradian and circadian rhythms of cortisol secretion, respectively. In contrast, the CBG-free plasma cortisol clearance (i.e. clearance of cortisol unbound to CBG) is a parameter and was used in the present experiment to demonstrate that the hypercortisolism of scrapie-affected ewes resulted from adrenal cortisol hypersecretion alone.

Different endocrine tests have to be carried out to distinguish the possible causes of any Cushing-like syndrome. In ewes, the principal regulator of ACTH release by pituitary corticotrophs is the hypothalamic CRH that drives the adrenal secretion of cortisol (2). Glucocorticoids provide negative feedback control for ACTH release by inhibiting anterior pituitary corticotrophs, as well as hypothalamic centers (3). Thus, scrapie-induced hypercorticism could be mediated by 1) scrapie-induced damage to the control centers of the nervous system, including the hypothalamus and/or the pituitary gland; or 2) autonomous hyperactivity of the adrenal glands. The spontaneous pattern of ACTH secretion was examined to identify the central or peripheral origin of the syndrome of hypercorticism. In addition, the responsiveness of the hypophysis to ovine CRF (oCRF) and that of adrenals to an exogenously administered 1–24 fragment of ACTH (ACTH_1–24, tetracosactide) were determined. Finally, the responsiveness of the pathways involved in the feedback inhibition of cortisol of scrapie-affected ewes was examined using a low-dose dexamethasone suppression test.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
General
Experiments were performed on six healthy and eight scrapie-affected Manech ewes. The animals were kept in a light-sealed room under an artificial short photoperiod (12 h of light, 12 h of darkness). The ages of both healthy and diseased ewes ranged from 1 yr and 6 months to 5 yr. The scrapie diagnosis, based on classical clinical signs (i.e. pruritus, behavioral changes, tremor, and locomotor incoordination) was established at least 10 days before the beginning of the experiments. The healthy ewes were included in the trial on the basis of an absence of clinical signs of scrapie. None of the classical clinical signs of scrapie were subsequently observed in these ewes during the 6 months after the sampling sessions. The ewes were individually tied in metabolism cages, where they received daily rations of concentrate plus hay ad libitum and had free access to water. The scrapie-affected ewes were killed when they manifested the final clinical stages of scrapie, i.e. when the clinical signs had progressed to irreversible recumbency.

Design
The objective of Exp 1 was to compare cortisol disposition in scrapie-affected ewes with that of healthy animals. During the first period, blood samples were collected at 1-h intervals for 24 h. The experiment was begun at 0800 h. During the second period, which took place 1 week later, cortisol (hydrocortisone, Sigma; l'Isle d’Abeau Chesnes, La Verpillière, France) was iv administered at a dosage of 1 mg/kg at 1000 h. Dexamethasone (0.1 mg/kg, Cortamethasone, Vetoquinol, Lure, France) was iv administered at 0700 h to prevent interference with endogenous cortisol secretion. Peripheral blood samples were collected at 1-h intervals for 3 h before and after cortisol administration at 1, 2, 4, 8, 15, 30, 45, and 60 min, then at 1-h intervals until 12 h post administration.

Exp 2 was performed 4 days later and was designed: 1) to test the integrity of the pathway involved in the negative feedback inhibition of cortisol; and 2) to compare both the endogenous secretion of ACTH and the adrenal gland responsiveness to stimulation with an exogenously administered 1–24 fragment of ACTH (ACTH_1–24, tetracosactide) of scrapie-affected ewes with that of healthy ewes. During the first period, blood samples were collected at 10-min intervals from 1300 h to 1730 h. The samples were chilled in wet ice. During the second period, i.e. the following day, dexamethasone (Cortamethasone, 0.01 mg/kg) and tetracosactide (Synacthene immediat, Ciba-Geigy, Rueil Malmaison, France, 5 µg/kg) were successively iv administered at a 6-h interval. Peripheral blood samples were collected before (at 0800 h) and 3, 4, and 6 h after dexamethasone administration, then after tetracosactide administration at 2, 4, 8, 15, 30, 60, 90, 120, 150, and 210 min. One scrapie-affected ewe was killed after the completion of Exp 2.

Nine days later, Exp 3 was performed to compare the pituitary and adrenal responsiveness to a stimulation with oCRF of scrapie-affected ewes with that of healthy ewes. The experiment was begun at 1300 h. Peripheral blood samples were collected 60 and 30 min before and after an iv administration of oCRF (Sigma, 0.4 µg/kg) at 4, 8, 15, 30, 45, and 60 min; then at 15-min intervals for 2 h; and finally 3.5, 4 and 5 h post administration. The samples were chilled in wet ice. The seven remaining scrapie-affected ewes were killed from 12 days to 4 months after the completion of Exp 3.

Administrations and blood sampling
All drugs were injected in the right jugular vein via an indwelling catheter that had been inserted the day preceding the experiments. Cortisol was dissolved in dimethyl sulfoxide and ethanol 50:50 (vol/vol) to produce a concentration of 50 mg/ml. oCRF was dissolved in saline containing 2% sterile ovine plasma and HCl 0.001 M 1%. Blood samples were obtained from the left jugular vein with an indwelling catheter inserted the day before the experiments. Blood samples for cortisol assay were collected in heparinized tubes and centrifuged for 10 min at 1400 x g. Blood samples for ACTH assay were collected in tubes containing EDTA/benzamidine placed in ice and centrifuged at 700 x g for 8 min within 15 min of collection. Finally, blood samples for oCRF assay were collected in tubes containing EDTA (4%) and centrifuged at 700 x g for 10 min. The plasma was separated and stored at -20 C until assayed.

Histology
After iv pentobarbital injection and exsanguination, the brain was removed and stored in a 10% formalin buffered solution for 3 weeks. Samples (obex, midpons) were dehydrated, embedded in paraffin, cut into 2-µm-thick sections, and stained using a classical Hematoxylin-eosin method. The diagnosis of scrapie was confirmed by the identification of perikaryonic and/or neuropilar vacuolisation in at least three gray matter nuclei.

Analytical methods
All hormone measurements involved radioimmunological techniques. Cortisol was assayed in duplicate using 50-µl aliquots of plasma and the RIA method adapted from Gomez Brunet and Lopez Sebastian (4). The level of quantification of the assay was 4 ng/ml. The mean intraassay coefficient of variation for three plasma levels (4, 16, and 32 ng/ml) was 13% (4 assays); the mean interassay coefficient of variation for these plasmas was 14%.

ACTH levels were measured in polyethyleneglycol plasma extracts by RIA, as previously described (5). Plasma tetracosactide concentrations were measured using the same RIA method, except that [¹²⁵I] ACTH_1–24 and ACTH_1–24 were used instead of [¹²⁵I] ACTH_1–39 and ACTH_1–39. Circulating CRF was measured in acetone plasma extracts using a previously described RIA method (6). The intraassay coefficients of variation within the measurement range of each assay were 5% for ACTH and tetracosactide and 6% for CRF. The limits of detection of the assays were 5 pg ACTH/ml, 2.5 pg tetracosactide/ml, and 10 pg CRF/ml plasma.

Kinetic analysis
Plasma cortisol (total concentrations) were analyzed using a compartmental approach: monocompartmental and bicompartmental models were constructed assuming that the cortisol not specifically bound to CBG (i.e. free cortisol and nonspecifically bound to albumin) was the sole form eliminated from the central compartment, with a rate constant of k₁₀, (min^-1) from a central compartment with a volume of V_c (liter/kg). In the central compartment, cortisol is specifically bound to CBG with B_max (nmol) being the CBG maximal binding capacity, and K_d (nmol) the cortisol dissociation constant, i.e. the free plasma cortisol corresponding to half saturation of CBG. B_max and K_d were estimated as amounts but were expressed in terms of concentration by dividing the estimated amount by the volume of distribution of the free fraction, i.e. V_c. It should be understood that in our model, the nonspecific cortisol binding was ignored, and the free cortisol corresponded to truly free cortisol plus the cortisol that was not specifically bound to albumin (see Discussion).

A fifth-order Runge-Kutta method with variable step size was used to solve the models numerically. The parameters were obtained using REVOL, a free derivative of the Monte Carlo minimizing algorithm (7). The goodness of fit of the described model was assessed using least-square criteria. The data points were weighted using 1/y_i², with y_i the i^th fitted concentration. An F test was used to select the appropriate number of compartments (1 or 2), and a bicompartmental model was selected.

Finally, the estimated parameters were k₁₀ (min^-1), k₁₂ (min^-1, first-order rate constants between central and peripheral compartments), and k₂₁ (min^-1, first-order rate constants between peripheral and central compartment), V_c (liter/kg), B_max (nmol), and K_d (nmol).

The plasma clearance of CBG-free cortisol (Cl_F, liter/kg·min) was calculated using Eq 1:

(1)

with k₁₀ and V_c as defined above.

The plasma half life for terminal phase was calculated using Eq 2:

(2)

with k₁₀ as defined above.

The steady-state volume of distribution of CBG-free cortisol was obtained from Eq 3:

(3)

with k₁₂, k₂₁, and V_c as defined above.

The nycthemeral cortisol production rate (PR_0–24h) was calculated from Eq 4:

(4)

where AUC_{free(0–24h)} is the area under the plasma CBG-free cortisol concentration-time curve calculated using the arithmetic trapezoidal rule from t = 0 to t = 24 h from data obtained during the first period of Exp 1.

The CBG-free cortisol concentrations (CBG_free) were calculated from raw data using Eq 5:

(5)

where TOT is the measured plasma cortisol concentration and B_max and K_d are the individual estimated binding parameters of cortisol to CBG. The estimated K_d is actually the product of the dissociation constant of binding with (NS+1), where NS is a dimensionless proportionality constant for the nonspecific binding of cortisol to albumin.

Tetracosactide and oCRF
The kinetic parameters for oCRF and tetracosactide were calculated from the plasma oCRF and tetracosactide concentration-time profile, respectively, according to the classical equations of Gibaldi and Perrier (8) and using a program for nonlinear regression analysis adapted from Multi (9).

The cortisol production rate induced by tetracosactide and oCRF administration was calculated from Eq 6:

(6)

where Cl_F is the plasma clearance of CBG-free cortisol, as previously defined, and AUC_{free(0-tlast)} is the area under the plasma CBG-free cortisol concentration-time curve, calculated using the arithmetic trapezoidal rule from t = 0 (time of tetracosactide or oCRF administration) to the last measurable concentration (t_last) from raw data converted to CBG-free cortisol concentrations using Eq 5 and individual values of estimated CBG binding parameters. For the determination of cortisol production rate induced by oCRF administration, AUC_{free(0-tlast)} was calculated from raw data firstly converted into CBG-free cortisol concentrations and then corrected by removing a basal CBG-free cortisol level corresponding to the mean values of CBG-free cortisol plasma levels obtained during the 1-h period preceding oCRF administration.

The ACTH production rate induced by oCRF administration was estimated from Eq 7:

(7)

where AUC_{ACTH (0-tlast)} is the area under the plasma ACTH concentration-time curve, calculated using the arithmetic trapezoidal rule from t = 0 (time of oCRF administration) to the last measurable concentration (t_last) from data corrected by removing a basal level corresponding to the mean values of ACTH plasma levels obtained during the 1-h period preceding oCRF administration and Cl_tetra is the tetracosactide plasma clearance.

Statistical analysis
Results are reported as mean ± SD. Statistics were performed using the Statgraphics program (version 5, STSC Inc., Rockville, MD, 1991). A P value lower than 0.05 was considered as significant. Corticoid concentrations below the limit of quantification of the assay were arbitrarily fixed at 2 ng/ml. The effect of scrapie on cortisol pharmacokinetics and CBG binding parameters was assessed using a one-way ANOVA. Because of the heterogeneity of variances in Exp 1, the nonparametric Kruskall-Wallis test was used to analyze the effect of scrapie on total and CBG-free cortisol plasma concentrations.

Similarly, in Exp 2, the nonparametric Kruskall-Wallis unilateral test was used to analyze the effect of scrapie on cortisol and ACTH concentrations. The effect of scrapie on tetracosactide and oCRF pharmacokinetic parameters was assessed using a one-way ANOVA. The same test was performed to examine the effect of scrapie on tetracosactide and cortisol production rates induced by the oCRF administration.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exp 1
No evidence of behavioral stress was recorded during the different sampling sessions, and scrapie-affected ewes behaved and ate normally, because they were excluded from the experiment if severe clinical signs developed. Fig. 1 indicates the nycthemeral variations in the individual and mean plasma levels of total and CBG-free cortisol in healthy and scrapie-affected ewes. The overall mean cortisol plasma levels of scrapie-affected ewes were about 2 times greater than that of healthy ewes (23.5 ± 17.2 vs. 13.9 ± 8.5 ng/ml, Kruskal-Wallis, P < 0.01). The corresponding CBG-free plasma cortisol concentrations of diseased ewes were increased by a factor of 5, when compared with healthy ewes (12.4 ± 14.8 vs. 2.6 ± 2.8 ng/ml, Kruskal-Wallis, P < 0.01).

View larger version (33K): [in this window] [in a new window]   Figure 1. Nycthemeral variations of individual (thin line) and mean (thick line) plasma total and CBG-free cortisol concentrations (ng/ml) observed in healthy and scrapie-affected ewes. hh, Hour of the day; mm, minutes.

The mean disposition parameters of cortisol obtained after a 1-mg/kg iv cortisol administration and the CBG-binding parameters are given in Table 1. The mean CBG-free cortisol plasma clearance of scrapie-affected ewes [29.7 ± 10.5 ml/(kg·min)] did not differ from that of healthy ewes [26.4 ± 4.2 ml/(kg·min)], ANOVA, P > 0.05). Similarly, the other pharmacokinetic parameters were unaffected by the disease, except for V_c, which was significantly higher in scrapie-affected ewes (ANOVA, P < 0.05). The overall mean CBG maximal binding capacity of scrapie-affected ewes (14.8 ± 6.1 ng/ml) was about two times lower than that obtained in healthy ones (34.3 ± 15.3 ng/ml, ANOVA, P < 0.05). The mean value of the apparent K_d was 2.6 ± 1.6 ng/ml in scrapie-affected ewes and 3.9 ± 2.6 ng/ml in healthy ones. This difference was not statistically significant (ANOVA, P > 0.05).

Exp 2
Fig. 2 shows the individual temporal variations in plasma ACTH and cortisol concentrations observed in healthy and scrapie-affected ewes. Visual inspection of the figure indicates that the range of mean ACTH plasma concentrations of scrapie-affected ewes was wider than that obtained in healthy ewes, extending from 68.7–335.5 pg/ml for diseased ewes vs. 85.7–137.1 pg/ml for healthy ones. Mean plasma ACTH concentrations of scrapie-affected ewes observed throughout the 4-h period (177.2 ± 123.5 pg/ml) were significantly higher than those observed in healthy ewes (116.4 ± 32.5 pg/ml, unilateral Kruskal-Wallis, P < 0.05).

View larger version (29K): [in this window] [in a new window]   Figure 2. Temporal variations in plasma ACTH and cortisol concentrations observed during a 4-h period of blood sampling in healthy (left) and scrapie-affected ewes (right).

Six hours after the administration of dexamethasone at a dosage of 0.01 mg/kg, cortisol and ACTH could not be detected in any of the healthy ewes, whereas four of the eight scrapie-affected ewes exhibited higher plasma cortisol levels than the limit of quantification of the assay.

Mean tetracosactide disposition parameters [Plasma clearance, T_1/2, V_c, V_SS, mean residence time (MRT)] of scrapie-affected ewes did not differ from values observed in healthy ewes (Table 2).

View larger version (21K): [in this window] [in a new window]   Figure 3. Effect of a 5-µg/kg tetracosactide iv administration on mean (±SD) total plasma cortisol concentrations and plasma CBG-free cortisol concentrations (ng/ml) in healthy (square) and scrapie-affected ewes (circle).

View larger version (24K): [in this window] [in a new window]   Figure 4. Temporal variations of plasma ACTH (black symbol) and cortisol (gray symbol) concentrations observed during the 1-h period before and the 5-h period after the 0.4 µg/kg iv administration of oCRF in healthy and scrapie-affected ewes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Stimulation of the adrenocortical function in scrapie-affected ewes is consistent with the increased urinary corticoid levels demonstrated in a previous study (1) and the abnormal enlargement of the adrenal glands observed in ewes naturally affected with scrapie (10) and in experimentally infected mice (11) and hamsters (12, 13).

Cortisol kinetics have been investigated in ewes using various methodological approaches (14, 15, 16). In our study, we used a modeling method based on the evaluation of CBG-free cortisol plasma clearance. To compare our results with those from the literature, the value for total cortisol plasma clearance can be approximated to 0.5 liter/kg·h by multiplying the mean value of CBG-free cortisol plasma clearance of the present experiment (1.6 liter/kg·h) by an estimated percentage of CBG-free cortisol corresponding to the physiological plasma cortisol levels (i.e. 30% for a mean plasma cortisol concentration of 15 ng/ml, assuming a B_max of 28.2 ng/ml and a K_d of 3.3 ng/ml, 17). The value of total plasma cortisol clearance generally reported or calculated from tracer or perfusion methods varied from 1–2 liter/kg·h (14, 15, 16, 18). The wide range of reported values for total plasma cortisol clearance can be explained by the fact that this variable is directly linked to the fraction of cortisol available for elimination, i.e. the CBG-free cortisol fraction that is subjected to instantaneous variations each time the plasma cortisol levels reach values that exceed the CBG binding capacity. In contrast, the plasma CBG-free cortisol clearance is a parameter that is independent of the temporal fluctuations in secretion of the hormone and reflects the real capacity of elimination. We have demonstrated that the plasma CBG-free cortisol clearance is unaffected by the prion disease. The calculated mean cortisol production rate of healthy ewes, based on individual values of this clearance, was 3.5 µg/kg·h. The cortisol secretion rate was increased by a factor of 5 in scrapie-affected ewes, whereas the plasma hormone concentrations were only doubled. The apparent discrepancy between adrenal secretion rate and plasma cortisol levels was attributable to the nonlinearity of plasma cortisol binding to CBG and to the lower CBG binding capacity of scrapie-affected ewes, which resulted in an increased CBG-free cortisol fraction. Indeed, the CBG binding capacity of healthy ewes, estimated using our pharmacokinetic model, varied within the range of physiological values previously obtained using equilibrium dialysis (17), whereas it was decreased by a factor of 2 in scrapie-affected ewes. The reduced CBG binding capacity of diseased ewes was confirmed using the reference method of equilibrium dialysis (19). The fall in plasma CBG could result from the depressive effect of hypercorticism on CBG synthesis, as previously suggested in rats (20). Whatever the origin of the scrapie-induced decrease in CBG binding capacity, it will result in an amplifying mechanism of the still stimulated adrenal function. Indeed, the total plasma cortisol concentrations in diseased ewes often attain higher levels than the reduced threshold of CBG maximal binding capacity, thereby resulting in a more-than-proportional increase in the diffusible plasma CBG-free cortisol levels.

From a mechanistic point of view, the stimulation of adrenal activity in scrapie-affected ewes could result from specific alterations of the neuronal systems involved in control of the hypothalamic-pituitary-adrenal axis (HPA) or from autonomous hyperactivity of the adrenal gland of infected animals. Our results support the hypothesis that adrenal dysfunction results from scrapie-induced cerebral damage, rather than from pathological changes in the adrenals. Indeed, we showed that the plasma ACTH concentrations of scrapie-affected ewes were about 1.5-fold greater than those observed in healthy ewes. Assuming that the kinetic disposition parameters of tetracosactide reflect the disposition parameters of natural ACTH, we can conclude that the secretion of ACTH was increased by a factor of 1.5 in diseased ewes despite the 5-fold increase in cortisol secretion rate. The ratio between cortisol and ACTH production rate increases in scrapie-affected ewes was partially explained by the increased responsiveness of the adrenal cortex. Indeed, the administration of tetracosactide induced a 2-fold increase of cortisol production in diseased ewes, compared with healthy ones. Due to the sigmoidal pattern of the dose-response relationship, the sensitivity of the response will depend on tetracosactide concentrations. Because, in our experimental conditions, the dosage of tetracosactide (5 µg/kg) was near maximal (21), it is likely that the response tended to be maximal and that a lower dose of tetracosactide could have induced a more-than-2-fold increase of adrenal response. However, we cannot rule out that extrapituitary mechanisms of adrenal regulation may assist in the maintenance of high plasma cortisol levels. Indeed, it is often reported that ACTH levels do not correspond to elevated concentrations of glucocorticoids (22). The increased adrenocortical responsiveness to tetracosactide could reflect the hypertrophy of the cells of the zona fasciculata of the adrenal cortex evidenced in diseased ewes. Alternatively, we cannot exclude the hypothesis that the sensitivity of adrenocortical cells to ACTH was increased in scrapie-affected ewes by a mechanism involving a positive effect of the hormone on its own receptors (23).

The higher secretion profiles of ACTH were observed in three of the four diseased ewes that exhibited only a partial response to the low dexamethasone suppression test. A recent study performed in knockout mice demonstrated that dexamethasone brain penetration is low, thus supporting the concept of a pituitary rather than a central site of dexamethasone action in its suppression of the HPA axis (24). According to this assumption, our results indicate that the pathways involved in cortisol feedback inhibition at the pituitary level remained functional in some diseased ewes. A decreased sensitivity of the nervous central system to cortisol feed back inhibition without functional alteration of the pituitary target could explain both the effectiveness of dexamethasone and the maintained pituitary secretion of ACTH in these ewes.

The oCRF administration stimulated ACTH secretion from the pituitary, resulting in increased cortisol production in both healthy and scrapie-affected ewes. The pituitary response to oCRF was not modified by the prion disease, whereas the stimulated adrenal secretions seemed to be greater in scrapie-affected ewes than in the healthy ones. However, the increased adrenal response to oCRF-induced ACTH secretion was not statistically evidenced in scrapie-affected ewes. As demonstrated for cortisol, the pharmacokinetic parameters of tetracosactide and oCRF were not affected by the prion disease, thus demonstrating that scrapie-induced adrenal dysfunction did not involve alteration of the elimination processes.

Our results suggest that scrapie could induce a sequence of HPA adaptations, ultimately resulting in a total or partial state of cortisol refractoriness reflected by sustained ACTH secretion and reduced suppression response to dexamethasone. Scrapie-induced cortisol resistance could result from two mechanisms: 1) increased arginine vasopressin secretion by the magnocellular neurons located in the paraventricular and supraoptic nuclei, which might permit the escape of ACTH from cortisol suppression; and/or 2) increased CRF and/or arginine vasopressin secretion by the parvocellular neurons located in the paraventricular nucleus, which could result from an impaired feedback inhibition. In agreement with the former hypothesis, a functional hypertrophy of the supraoptico-infundibular neurosecretory subsystem was evidenced in ewes naturally affected with scrapie (25). The second hypothesis is comforted by the increased number of hypothalamic CRF neurons demonstrated in the experimental model of hamsters infected with the 139H strain of scrapie (26).

Whatever the origin of scrapie-induced hypercorticism, attention should be paid to the pathophysiological meaning and consequences of chronic overexposure of the central nervous system to CBG-free cortisol. In Alzheimer’s disease, glucocorticoids have been shown to have a protective effect by slowing the rate of progression, because the activation of specific inflammatory mechanisms contributes to neurodegeneration (27). Recent studies suggest that a glial production of cytokine induced by PrPsc deposition may contribute to the development of pathological lesions in a murine scrapie model (28). The inflammatory nature of the brain lesions could explain the reduced susceptibility to scrapie when steroids are administered to experimentally infected mice (29). Thus, it can be hypothesized that the hypercorticism response observed in the present experiment is aimed to counteract a brain inflammation process. Indeed, glucocorticoids are potent inhibitors of the Nuclear factor–B family, which could be involved in the central nervous system response to pathogenic stimuli leading to neuronal death (30). However, a long-term sustained secretion of cortisol could exacerbate the slow neurodegenerative process involved in the prion disease. Indeed, excessive exposure to glucocorticoids was shown to have deleterious effects in the rodent brain (31). On the other hand, a more general inhibitory effect of cortisol overexposure on the immune system of infected animals could help to explain the absence of a detectable immune response observed in prion disease (see Ref. 32 for review).

In conclusion, the main result of the present experiment is that ewes with naturally occurring scrapie display a major syndrome of hypercorticism associated with a hyperresponsive adrenal cortex and lowered CBG binding capacity. The secretion of ACTH was increased in diseased ewes despite the high secretion rate of cortisol, this suggesting a disruption of the pathways involved in the feedback inhibition of cortisol, which could result from cerebral damage.

	Acknowledgments

 
The authors are grateful to S. Baurès and N. Gautier for their assistance and F. Lyazrhi for his critical analysis of the statistics.

Received July 1, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Schelcher F, Picard-Hagen N, Laroute V, Gayrard V, Popot MA, Andreoletti O, Toutain PL 1999 Corticoid concentrations are increased in the plasma and urine of ewes with naturally occurring scrapie. Endocrinology 140:2422–2425[Abstract/Free Full Text]
2. Guillaume V, Conte-Devolx B, Magnan E, Boudouresque F, Grino M, Cataldi M, Muret L, Priou A, Deprez P, Figaroli JC, Oliver C 1992 Effect of chronic active immunization anti-corticotropin-releasing factor on the pituitary-adrenal function in the sheep. Endocrinology 130:2291–2298[Abstract/Free Full Text]
3. Keller-Wood ME, Dallman MF 1984 Corticosteroid inhibition of ACTH secretion. Endocr Rev 5:1–24[Abstract/Free Full Text]
4. Gomez Brunet A, Lopez Sebastian A 1991 Effect of season on plasma concentrations of prolactin and cortisol in pregnant, non-pregnant and lactating ewes. Anim Reprod Sci 26:251–268[CrossRef]
5. Usategui R, Oliver C, Vaudry H, Lombardi G, Rozenberg I, Mourre AM 1976 Immunoreactive alpha-MSH and ACTH levels in rat plasma and pituitary. Endocrinology 98:189–196[Abstract/Free Full Text]
6. Caraty A, Grino M, Locatelli A, Guillaume V, Boudouresque F, Conte-Devolx B, Oliver C 1990 Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophyseal portal blood of conscious, unrestrained rams. J Clin Invest 85:1716–1721
7. Koeppe P, Hamann C 1980 A program for non-linear regression analysis to be used on desk-top computers. Comput Methods Programs Biomed 12:121–128
8. Gibaldi M, Perrier D 1982 Pharmacokinetics, ed 2. Marcel Dekker, New York
9. Yamaoka K, Tanigawara K, Nakagawa T, Uno T 1981 A pharmacokinetic analysis program (Multi) for microcomputer. J Pharmacobiodyn 4:879–889[Medline]
10. Beck E, Daniel PM, Parry HB 1964 Degeneration of the cerebellar and hypothalamo-neurohypophysial systems in sheep with scrapie and its relationship to human system degenerations. Brain 87:153–176[Free Full Text]
11. Kim YS, Carp RI, Callahan SM, Wisniewsky HM 1988 Adrenal involvement in scrapie-induced obesity. Proc Soc Exp Biol Med 189:21–27[CrossRef][Medline]
12. Carp RI, Kim YS, Callahan SM 1990 Pancreatic lesions and hypoglycemia-hyperinsulinemia in scrapie-infected hamsters. J Infect Dis 161:462–466[Medline]
13. Ye X, Carp RI 1996 Histopathological changes in the pituitary glands of female hamsters infected with the 139H strain of scrapie. J Comp Pathol 114:291–304[CrossRef][Medline]
14. Paterson JYF, Harrison FA 1967 The specific activity of plasma cortisol in sheep during continuous infusion of [1,2-³H₂]cortisol, and its relation to the rate of cortisol secretion. J Endocrinol 37:269–277[Abstract/Free Full Text]
15. Hennessy DP, Coghlan JP, Hardy KJ, Scoggins BA, Wintour EM 1982 The origin of cortisol in the blood of fetal sheep. J Endocrinol 95:71–79[Abstract/Free Full Text]
16. Soding P, Coghlan JP, Denton DA, Graham WF, Humphery TJ, Scoggins BA 1983 The effect of ACTH on the blood clearance rate of aldosterone, cortisol, 17,20-dihydroxy-4-pregnen-3-one in the sheep. J Steroid Biochem 18:173–177[CrossRef][Medline]
17. Gayrard V, Alvinerie M, Toutain PL 1996 Interspecies variations of corticosteroid-binding globulin parameters. Domest Anim Endocrinol 13:35–45[CrossRef][Medline]
18. Thompson K, Coleman ES, Hudmon A, Kemppainen RJ, Soyoola EO, Sartin JL 1995 Effects of short-term cortisol infusion on growth hormone-releasing hormone stimulation of growth hormone release in sheep. Am J Vet Res 56:1228–1231[Medline]
19. Picard-Hagen N, Gayrard V, Alvinerie M, Laroute V, Touron C, Andréoletti O, Toutain PL Naturally occurring scrapie is associated with a lower CBG binding capacity in ewes. J Endocrinol, in press
20. Fleshner M, Deak T, Spencer RL, Laudenslager ML, Watkins LR, Maier SF 1995 A long-term increase in basal levels of corticosterone and a decrease in corticosteroid-binding globulin after acute stressor exposure. Endocrinology 136:5336–5342[Abstract]
21. Fulkerson WJ, Jamieson PA 1982 Pattern of cortisol release in sheep following administration of synthetic ACTH or imposition of various stressor agents. Aust J Biol Sci 35:215–222[Medline]
22. Bornstein SR, Chrousos GP 1999 Adrenocorticotropin (ACTH)- and non-ACTH mediated regulation of the adrenal cortex: neural and immune inputs. J Clin Endocrinol Metab 84:1729–1736[Free Full Text]
23. Picard-Hagen N, Penhoat A, Hue D, Jaillard C, Durand P 1997 Glucocorticoids enhance corticotropin receptor mRNA levels in ovine adrenocortical cells. J Mol Endocrinol 19:29–36[Abstract/Free Full Text]
24. Meijer OC, Lange ECM, Breimer DD, De Boer AG, Workel JO, De Kloet ER 1998 Penetration of dexamethasone into brain glucocorticoid targets is enhanced in mdr1A P-glycoprotein knockout mice. Endocrinology 139:1789–1793[Abstract/Free Full Text]
25. Parry HB, Livett BG 1973 A new hypothalamic pathway to the median eminence containing neurophysin and its hypertrophy in sheep with natural scrapie. Nature 242:63–64[CrossRef][Medline]
26. Ye X, Carp RI, Yu Y, Kozielski R, Kozlowski P 1994 Effect of infection with the 139H scrapie strain on the number, area and/or location of hypothalamic CRF- and VP-immunostained neurons. Acta Neuropathol (Berl) 88:44–54[CrossRef][Medline]
27. Aisen PS, Pasinetti GM 1998 Glucocorticoids in Alzheimer’s disease. Drugs Aging 12:1–6
28. Williams A, Van Dam AM, Ritchie D, Eikelenboom P, Fraser H 1997 Immunocytochemical appearance of cytokines, prostaglandin E2 and lipocortin-1 in the CNS during the incubation period of murine scrapie correlates with progressive PrP accumulations. Brain Res 754:171–180[CrossRef][Medline]
29. Outram GW, Dickinson AG, Fraser H 1974 Reduced susceptibility to scrapie in mice after steroid administration. Nature 249:855–856[CrossRef][Medline]
30. Grilli M, Memo M 1999 Nuclear factor-B/Rel proteins a point of convergence of signalling pathways relevant in neuronal function and dysfunction. Biochem Pharmacol 57:1–7[CrossRef][Medline]
31. Sapolski RM 1996 Why stress is bad for your brain. Science 273:749–750[CrossRef][Medline]
32. Schreuder BEC 1994 Animal spongiform encephalopathies - an update part I. Scrapie and lesser known animal spongiform encephalopathies. Vet Q 16: 174–181

This article has been cited by other articles:

				J D Bailey, J G Berardinelli, T E Rocke, and R A Bessen Prominent pancreatic endocrinopathy and altered control of food intake disrupt energy homeostasis in prion diseases J. Endocrinol., May 1, 2008; 197(2): 251 - 263. [Abstract] [Full Text] [PDF]

				A. H. Peden, D. L. Ritchie, H. P. Uddin, A. F. Dean, K. A. F. Schiller, M. W. Head, and J. W. Ironside Abnormal prion protein in the pituitary in sporadic and variant Creutzfeldt-Jakob disease J. Gen. Virol., March 1, 2007; 88(3): 1068 - 1072. [Abstract] [Full Text] [PDF]

				C Viguie, Y Chilliard, V Gayrard, N Picard-Hagen, P Monget, A Dutour, and P-L Toutain Alterations of somatotropic function in prion disease in sheep J. Endocrinol., November 1, 2004; 183(2): 427 - 435. [Abstract] [Full Text] [PDF]

				N. Picard-Hagen, V. Gayrard, M. Alvinerie, H. Smeyers, R. Ricou, A. Bousquet-Melou, and P. L. Toutain A nonlabeled method to evaluate cortisol production rate by modeling plasma CBG-free cortisol disposition Am J Physiol Endocrinol Metab, November 1, 2001; 281(5): E946 - E956. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gayrard, V. Articles by Toutain, P. L. Search for Related Content PubMed PubMed Citation Articles by Gayrard, V. Articles by Toutain, P. L.