ELSEVIER DOMESTIC ANIMAL ENDOCRINOLOGY

Vol. 14(2):81-97, 1997

A S S E S S M E N T O F P I T U I T A R Y F U N C T I O N A F T E R T R A N S S P H E N O I D A L H Y P O P H Y S E C T O M Y IN B E A G L E D O G S

B.P. Meij, *'1 J.A. Mol,* T.S.G.AM. van den Ingh,** M.M. Bevers,*** H.A.W. Hazewinkel,* and A. Rijnberk*

*Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.154, NL-3508 TD

Utrecht, The Netherlands, **Department of Pathology, Faculty of Veterinary Medicine, Utrecht University,

PO Box 80.158, NL-3508 TD Utrecht, The Netherlands, and

***Department of Herd Health and Reproduction, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.151, NL-3508 TD

Utrecht, The Netherlands

Received June 21, 1996

Pituitary function was assessed in healthy adult beagle dogs before and after hypophysectomy. Anterior pituitary function was tested by use of the combined anterior pituitary (CAP) function test, which consisted of sequential 30-sec intravenous injections of four hypothalamic releasing hor- mones, in the following order and doses: 1 p,g of corticotropin-releasing hormone (CRH)/kg, 1 p~g of growth hormone-releasing hormone (GHRH)/kg, 10 ~g of gonadotropin-releasing hormone (GnRH)/kg, and 10 ~g of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin (ACTH), cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 rain after injection. Pars intermedia function was assessed by the c~-mela- notropin (~-MSH) response to the intravenous injection of the dopamine antagonist haloperidol in a dosage of 0.2 mg/kg. Posterior pituitary function was assessed by the plasma vasopressin (AVP) response to the intravenous infusion of 20% saline. Basal plasma ACTH, cortisol, thyroxine, LH, PRL, and AVP concentrations were significantly lower at 10 wk after hypophysectomy than before hypophysectomy. In the CAP test and the haloperidol test, the peaks for the plasma concentrations of ACTH, cortisol, GH, LH, PRL, and a-MSH occurred within 45 min after injection. At 2 and 10 wk after hypophysectomy, there were no responses of plasma GH, LH, PRL, and c~-MSH to stimulation. In four of eight hypophysectomized dogs, there were also no plasma ACTH and cortisol responses, whereas in the other four dogs, plasma ACTH and cortisol responses were significantly attenuated. The basal plasma ACTH and cortisol concentrations were significantly lower in the corticotropic nonresponders than in the responders. Plasma AVP responses were completely abol- ished by hypophysectomy, although water intake by the dogs was normal. Histopathological ex- aminations at 10 wk after hypophysectomy revealed that adrenocortical atrophy was much more pronounced in the corticotropic nonresponders than in the responders. No residual pituitary tissue was found along the ventral hypothalamic diencephalon. However, in all hypophysectomized dogs that were investigated, islets of pituitary cells were found embedded in fibrous tissue in the sella turcica. A significant positive correlation was found between the number of ACTH-immunopositive cells and the ACTH increment in the CAP test at 10 wk after hypophysectomy. It is concluded that [) stimulation of the anterior pituitary with multiple hypophysiotropic hormones, stimulation of the pars intermedia with a dopamine antagonist, and stimulation of the neurohypophysis with hypertonic saline do not cause side effects that would prohibit routine use, 2) in the routine stimulation of the anterior pituitary and the pars intermedia, blood sampling can be confined to the first 45 rain, 3) the ACTH and cortisol responses to hypophysiotropic stimulation are the most sensitive indicators for

© Elsevier Science Inc. 1997 0739-7240/97/$17.00 655 Avenue of the Americas, New York, NY 10010 PII S0739-7240(96)00118-X

82 MEIJ ET AL.

residual pituitary function after hypophysectomy, 4) small islets of pituitary cells in the sella turcica, containing corticotropic cells, are the most likely source of the attenuated corticotropic response that may occur after hypophysectomy, and 5) residual AVP release from the hypothalamus after hy- pophysectomy is sufficient to prevent diabetes insipidus, despite the fact that the AVP response to hypertonic saline infusion is completely abolished. © Elsevier Science Inc. 1997

INTRODUCTION

The efficacy of hypophysectomy can be determined by dynamic tests with hypophys- iotropic hormones. Over the past decade, there have been numerous reports of the combined stimulation of the anterior pituitary by the simultaneous administration of corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), and thyrotropin-releasing hormone (TRH). A study performed in male rats revealed no significant in vitro or in vivo interactions among any of four synthetic hypothalamic releasing hormones (luteinizing hormone (LH)RH, CRH, GHRH,and TRH) on anterior pituitary hormone secretions (1). The combined anterior pituitary (CAP) function test, using the simultaneous administration of CRH, GHRH, TRH, and GnRH with consecutive measurement of adrenocorticotropin (ACTH), cortisol, GH, LH, follicle-stimulating hormone (FSH), thyroid-stimulating hormone, and prolactin (PRL), has been studied thoroughly in normal human subjects (2-8). In those reports, it was concluded that the combined administration of hypophysiotropic hormones is a rapid, safe, and useful test of anterior pituitary function. The CAP test has also been described as a useful dynamic test of anterior pituitary function in patients with suspected pituitary dysfunction and in patients who have undergone pituitary surgery (5,7-9). The patients' tolerance of the CAP test has been reported to be better than that for the insulin hypoglycemia test (5). The CAP test has recently been used in dogs as well. Studies in healthy dogs have demonstrated that it allows good assessment of anterior pituitary function (I0).

Total hypophysectomy may also have consequences for neurohypopbyseal function. Immediately after surgery, there is often impaired release of vasopressin (AVP). The associated decrease in renal concentrating ability usually disappears spontaneously after days to months (11). Although a test for AVP secretory capacity has been introduced in the dog (12), the AVP response to osmotic stimulation after hypophysectomy has not yet been documented.

The human hypophysis lacks a histologically distinct pars intermedia. In contrast, the dog's posterior lobe is surrounded by a histologically heterogenous zone (pars intermedia) in which o~-melanotropin (a-MSH)-containing cells are the most abundant and ACTH- containing cells are less numerous (13). Between the pars distalis and the pars intermedia, there is an exceptionally well-developed transitional zone (13). This transitional zone and the pars tuberalis contain clusters of corticotropic cells (14) that stain similarly with anti-ACTH and only occasionally with anti-a-MSH, i.e., similar to corticotropic cells in the pars distalis proper (13,14). The pars intermedia, which is under dopaminergic inhi- bition in the dog, can be assessed by the administration of a dopamine antagonist and the measurement of ot-MSH (15).

Transsphenoidal hypophysectomy has been performed in the dog for experimental purposes (16,17) and has been carried out in both healthy dogs (11,18) and dogs with pituitary-dependent hyperadrenocorticism (PDH) (19,20). However, in those studies, there has been no systematic assessment by function tests of the postoperative residual function of the cells of the anterior pituitary, pars intermedia, or posterior pituitary.

The purpose of this study was to assess the function of the anterior pituitary, the pars intermedia, and the posterior pituitary in healthy dogs, before and after transsphenoidal

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 83

hypophysectomy, and to relate the results to histopathological findings. Pituitary function was assessed by measuring the responses of pituitary hormones to the combined admin- istration of four hypothalamic releasing hormones (anterior pituitary), to the administra- tion of a dopamine antagonist (pars intermedia), and to hypertonic saline infusion (pos- terior pituitary). The study was performed to serve as a reference for future clinical studies, including the application in dogs with PDH treated by pituitary surgery.

MATERIALS AND METHODS

Animals. Eight healthy adult male beagle dogs (median age, 2 yr; range, 1-6 yr), weighing 13-16 kg, were used. The dogs were housed individually in indoor kennels under a normal daylight regimen, with access to separate outdoor runs for about 2 hr/d. They were fed a commercial diet, and they had free access to water. The animals were adapted to these conditions for 2 wk before the start of the experiment. There was an interval of at least 1 wk between the tests, and they were performed after an overnight fast. Protocols for all experiments involving the use of dogs were approved by the Ethical Committee of the Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Neth- erlands.

Hypophysectomy. After intramuscular premedication with acepromazine (Vetranquil; Sanofi, Maassluis, The Netherlands) (0.1 mg/kg) methadone (Symoron; Gist-Brocades, Delft, The Netherlands) (1 mg/kg), and atropine (Atropini Sulfas; Pharmachemie, Haar- lem, The Netherlands) (0.07 mg/kg), anesthesia was induced by the intravenous admin- istration of sodium pentothal (10 mg/kg; Nesdonal; Specia, Amstelveen, The Nether- lands). The trachea was intubated, and inhalation anesthesia was maintained in a semiclosed system with a mixture of halothane (Halothane; Albic, Maassluis, The Neth- erlands), nitrous oxide, and oxygen.

Total hypophysectomy was performed by the transsphenoidal approach described by Markowitz et al. (16), with slight modifications. The dog was positioned in sternal re- cumbency, and the upper jaw was fixed to a metal bar connected to the operating table. The head was taped to the metal bar, and the mouth was held open by an adjustable cord passed around the lower jaw. After cleavage of the soft palate and the mucoperiosteum covering the sphenoid bone, access to the sella turcica was gained by use of an air- powered burr and bone punches. Pituitary tissue was removed by traction with fine neurosurgical grasping instruments and suction, with an operating loupe with 3.3-x ocular magnification (Carl Zeiss BV, Weesp, The Netherlands).

Postoperative analgesia was provided by buprenorphine (Temgesic; Schering-Plough, Amstelveen, The Netherlands), 0.3 mg/kg thrice daily (TID), subcutaneously for 3 d. Oral hormone substitution therapy after hypophysectomy consisted of cortisone acetate (0.25 mg/kg twice daily [BID]; Cortisoni acetas; Genfarma, Maarssen, The Netherlands) and L-thyroxine (10 txg/kg BID; Aesculaap, Boxtel, The Netherlands). Desmopressin (0.01~; Minrin; Ferring, Hoofddorp, The Netherlands) was administered in a dose of one drop in the conjunctival sac TID and was discontinued after 1 wk. Drinking behavior was moni- tored by daily recording of water intake.

Anterior Pituitary Function. Anterior pituitary function was investigated before and at 2 and 10 wk after hypophysectomy by the rapid sequential intravenous administration of four hypothalamic releasing hormones, as described by Meij et al. (10). Ovine CRH (Peninsula Laboratories Inc., Belmont, CA) and human GHRH (Peninsula Laboratories Inc.) were kept frozen at -25°C and thawed at room temperature immediately before use. A GnRH analogue, gonadorelin (Fertagyl; Intervet, Boxmeer, The Netherlands), and TRH (Hoffman-La Roche, Basel, Switzerland) were kept at 4°C. An intravenous catheter was placed in the cephalic vein of each dog to facilitate rapid injections. In the CAP test,

84 MEIJ ET AL.

all four releasing hormones were injected intravenously within 30 sec, immediately after the collection of the zero blood sample from the jugular vein, in the following order and doses: 1 ixg of CRH/kg, 1 Ixg of GHRH/kg, 10 p.g of GnRH/kg, and 10 p.g of TRH/kg. The clock for blood sampling started immediately after the administration of the last releasing hormone. Blood samples were collected at -30, -15, 0, 5, 10, 20, 30, 45, 60, 90, and 120 min from the jugular vein and transferred to ice-chilled EDTA-coated tubes. Samples were centrifuged at 4°C for 10 min. Plasma was stored at -25°C until assayed for ACTH, cortisol, GH, LH, and PRL. Substitution therapy with cortisone was continued for I0 wk after surgery, until euthanasia, and was interrupted for 48 hr before the CAP tests at 2 and 10 wk after hypophysectomy. Substitution therapy with thyroxine was continued for 8 wk after surgery and was stopped 2 wk before euthanasia. Blood samples for basal plasma T 4 measurements were collected before and at 10 wk after hypophysec- tomy.

Pars lntermedia Function. Pars intermedia function was tested before and at 2 and 10 wk after hypophysectomy by the intravenous injection of haloperidol (Haldol; Janssen Pharmaceutica BV, Tilburg, The Netherlands) in a dose of 0.2 mg/kg. Blood sampling and sample handling were as described for the CAP test. Plasma was stored at -25°C until assayed for a-MSH.

Posterior Pituitary Function. The AVP response to hypertonic saline was investigated by the intravenous infusion of 20% NaC1 for 2 hr at a rate of 0.03 ml/kg per min (12). Posterior pituitary function was tested before and 10 wk after hypophysectomy. Blood samples were collected at 0, 20, 40, 60, 80, 100, and 120 min from the jugular vein and transferred to ice-chilled EDTA-coated tubes. Samples were centrifuged at 4°C for 10 min. Plasma was stored at -25°C until assayed for AVP and osmolality.

Hormone Determinations, Plasma ACTH was measured by radioimmunoassay (RIA) without extraction, according to the procedure described by Arts et al. (21), and validated for the dog. Antiserum was obtained from IgG Corp. (Nashville, TN). The tracer was purchased from International CIS (St. Quentin-Yvelines, France), and the standard was obtained from the NIH (Bethesda, MD). The intra-assay and interassay coefficients of variation were 8 and 12%, respectively, and the detection limit was 10 ng/1.

Plasma cortisol was measured by RIA (22). The detection limit for cortisol was 1 nmol/l, and the intra-assay and interassay coefficients of variation were 6 and 8%, re- spectively.

Plasma GH was determined in a homologous RIA as described previously (23). The intra-assay and interassay coefficients of variation were 3.8 and 7.2%, respectively, and the sensitivity of the assay was 0.4 txg/1 of plasma. The degree of cross-reaction of canine PRL was 2%.

Plasma LH concentrations were assessed by a heterologous RIA, as described by Nett et al. (24). A rabbit antiserum raised against ovine LH (CSU-204), radio-iodinated NIAMDD-bLH-4, and canine pituitary standard LER 1685-1 were used in this assay. The detection limit for LH was 0.31 p~g/1, and the intra-assay and interassay coefficients of variation were 2.3 and 10.5%, respectively.

Plasma PRL concentrations were measured by a heterologous RIA, as described pre- viously (25). The detection limit was 0.8 p~g/1 of plasma, and the intra-assay and interassay coefficients of variation were 3.5 and 11.8%, respectively. The degree of cross-reaction of canine GH was 0.08%.

Plasma total thyroxine was measured by RIA as described previously (26). The detec- tion limit was 1.6 nmol/l, and the intra-assay and interassay coefficients of variation were 5.6 and 10.2%, respectively.

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 85

Plasma oL-MSH was measured by RIA with antiserum to synthetic human c~-MSH, as described by Rijnberk et al. (22). This antiserum reacted equally with a-MSH and des- acetyl-e~-MSH. Synthetic human o~-MSH was used as a standard. The detection limit was 10 ng/l, and the intra-assay and interassay coefficients of variation were 10 and 23%, respectively.

Plasma AVP was measured with the antibody against AVP (lot no. 2727) and the method of the RIA kit as purchased from Nichols Institute (Wijchen, The Netherlands), as validated for the dog (27). The detection limit was 0.25 ng/l, and the intra-assay and interassay coefficients of variation were 6 and 10%, respectively.

Pathology. Pituitary tissue collected during transsphenoidal hypophysectomy was placed in 10% neutral buffered formalin. Ten weeks after hypophysectomy, each dog was euthanized by the intravenous injection of a concentrated pentobarbital solution. The adrenals and both lobes of the thyroid gland were carefully dissected and weighed before being placed in 10% buffered formalin. Each adrenal gland was cut twice transversely, midway between the indentation of the phrenicoabdominal vein and each pole. Special attention was paid to the brain and the sella turcica. After the removal of the scalp, the brain was carefully lifted from the neurocranium and the sella turcica and hypothalamic surface were inspected macroscopically. The base of the brain, including the hypothalamic region and the sella turcica, were collected for microscopic examination.

Samples for histological examination were dehydrated and embedded in paraffin. Sec- tions were cut at 5 Ixm and stained with hematoxylin-eosin (HE). For the sella turcica, sections were made through the middle in a transverse plane. One section was stained with HE for histological examination, and adjacent sections were used for immunohistochem- ical staining by the avidin-biotin technique, with polyclonal rabbit antibodies to synthetic ACTH ~-24 (28), polyclonal rabbit antibodies to synthetic o~-MSH (PU060-UP; Biogenex Laboratories, San Ramon, CA), and rabbit antibodies to porcine GH (29). The numbers of ACTH-, GH-, and o~-MSH-immunopositive cells in sections of the sella turcica were counted.

Adrenal morphometry was performed with a TEA image manager (TIM). In each dog, the width of the zona glomerulosa (ZG) and the combined width of the zona fasciculata (ZF) and zona reticularis (ZR) were determined by the average of measurements at five different places; and the data were used to calculate the ratios [ZG/(ZF + ZR)].

Statistical Analysis. The data were normally distributed, and therefore, the results are expressed as means + SE. For statistical comparisons, the less sensitive nonparametric statistical tests were used because of the limited number of cases (n = 8). Mean basal plasma levels were calculated from the -30-, -15-, 0-rain values for ACTH, cortisol, GH, LH, PRL, T4, and a-MSH and from the 0-min values for AVP. Increments were calculated as the differences between peak levels and levels at 0 min. The significance of differences in basal plasma levels and increments before and after hypophysectomy was tested by the nonparametric Wilcoxon matched-pairs signed-ranks test. The dogs were subdivided, according to their ACTH increments at 10 wk after hypophysectomy, into ACTH re- sponders (increment > 0; n = 4) and nonresponders (increment = 0; n = 4). Statistical testing of the differences in ACTH increments between ACTH responders and nonre- sponders was considered methodically incorrect and was not performed. The significance of the differences between the responders and the nonresponders in basal plasma levels (ACTH, cortisol) and increments (cortisol), and of the differences in the numbers of ACTH-, GH-, and a-MSH-immunopositive cells in sections of the sella turcica, was tested by the nonparametric Mann-Whitney U test. Coefficients of correlation between the numbers of ACTH-, GH-, and a-MSH-immunopositive cells in sections of the sella

86 MEIJ ET AL.

turcica were calculated by use of Pearson's method (two-tailed). We also analyzed the correlat ion at 10 wk after hypophysec tomy between the number of ACTH- immunopositive cells and the ACTH increment in the CAP test, between the number of GH-immunopositive cells and the GH increment in the CAP test, and between the number of eL-MSH immunopositive cells and the cx-MSH increment in the haloperidol stimulation test. The significance of the differences between mean adrenal and thyroidal weights, and the mean ratios [ZG/(ZF + ZR)] of the responders and the nonresponders, was tested by the nonparametric Mann-Whitney U test. P < 0.05 was considered significant.

RESULTS

Pituitary Function. The combined administration of releasing hormones and the ad- ministration of haloperidol and of hypertonic saline caused no serious side effects, either before or after hypophysectomy. In some dogs, hypersalivation and restlessness occurred immediately after the combined administration of releasing hormones, but ceased within 5 min.

Hypophysectomy caused a pronounced reduction of the plasma ACTH and cortisol responses to the combined stimulation with the four hypophysiotropic hormones (Figures 1 and 2). Basal plasma ACTH concentrations were significantly lower at 2 and 10 wk after hypophysectomy than before hypophysectomy (Table 1, Wilcoxon, P < 0.05). Basal plasma cortisol concentrations were significantly lower at 10 wk after hypophysectomy than preoperatively (Table 1). There were no significant differences in basal plasma ACTH and cortisol concentrations between 2 and 10 wk after hypophysectomy (Table 1). Increments in plasma ACTH and cortisol concentrations were significantly lower at 2 and 10 wk after hypophysectomy than preoperatively (Table 2). Increments in plasma ACTH and cortisol concentrations were lower 2 wk after hypophysectomy than at 10 wk after hypophysectomy, although the differences were not significant (Table 2).

The dogs could be subdivided into two groups of four each (responders vs. nonre- sponders), according to their ACTH responses in the CAP test at 10 wk after hypophy- sectomy (decreased vs. zero ACTH increments, respectively) (Table 3). The responders

-i- I-- 0 <

400

300

200

100

l

I ~ o T r r ~ i _ _ i / m / O - O - - - - O - - O - - o - ~ _ _ _ ~ _ _ - - o 6 ~ 0

| I I I I I I i i I

-30 -15 0 15 30 45 S0 75 90 105 120

t Time (rain)

Figure 1. Plasma ACTH response (mean + SE) in eight healthy male beagle dogs after the rapid (30-sec) intravenous injection (arrow) of a combination of four hypothalamic releasing hormones before (O) and at 10 wk after (O) transsphenoidal hypophysectomy. The releasing hormones were injected in the following order and doses: 1 p,g of CRH/kg, 1 p,g of GHRH/kg, 10 p,g of GnRH/kg, and 10 p~g of TRH/kg. Basal level and increment in plasma ACTH concentration were significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 87

500

400 T T / "

OO oo /-/ l oo I " ~ o T o T T T T T T ~ 6 ~ 6 ~ . . 5 . , o ~ ~ o ~ O o-- o o

L 0 I I 1 I I I I , 1 I I I

-30 15 0 15 30 45 60 75 gO 105 120

t Time (min)

Figure 2. Plasma cortisol response (mean + SE) in eight healthy male beagle dogs after the rapid (30-sec) intravenous injection (arrow) of a combination of four hypothalamic releasing hormones before ( 0 ) and at 10 wk after (O) transsphenoidal hypophysectomy. See also legend to Figure l. Basal level and increment in plasma cortisol concentration were significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

(Dog Nos. 3, 6, 7, and 8) had posthypophysectomy plasma ACTH and cortisol responses lower than their responses before hypophysectomy but greater than those of the nonre- sponders (Nos. 1, 2, 4, and 5) after hypophysectomy (Table 3). At 10 wk after hypophy- sectomy, the basal plasma ACTH concentrations in the nonresponders were close to the lower limit of detection of the ACTH assay (10 ng/1, and were significantly lower (Mann- Whitney U, P < 0.05) than the corresponding values in the responders (Table 3). The basal plasma cortisol concentrations in the nonresponders at 10 wk after hypophysectomy did not change with stimulation and were significantly lower than the corresponding values in the responders (Table 3). The increments in plasma cortisol concentrations in the respoad- ers and the nonresponders were not significantly different before hypophysectomy, whereas at l0 wk after hypophysectomy, they were significantly lower in the nonre- sponders than in the responders (Table 3).

Plasma GH, LH, and PRL responses to stimulation were absent in all eight dogs at both 2 and l0 wk after hypophysectomy (Figures 3-5, Table 2). Basal plasma LH and PRL concentrations were significantly lower at 2 and l 0 wk after hypophysectomy than before hypophysectomy (Table 1). In three dogs (Nos. 1, 2, and 4), plasma GH levels at 10 wk after hypophysectomy had been reduced to the lower limit of detection of the assay (0.4

TABLE I. PITUITARY FUNCTION IN BEAGLE DOGS ASSESSED BY BASAL PLASMA ACTH, CORTISOL, OH, LH, PRL,

c~-MSH, AND AVP CONCENTRATIONS BEFORE AND AFTER HYPOPHYSECTOMY ( n x ) . a

2 wk I0 wk Hormone Before HX after HX after HX

ACTH (rig/l) 58.9 -+ 5.1 23.8 _+ 1.9 b 34.6 _+ 6.4 ~ Cortisol (nmol/l) 104.6 _+ 19.8 64.9 + 5.1 38.2 _+ 8.9 L" GH (Ixg/1) 1.7 + 0.3 1.7 _+ 0.2 1.4 _+ 0.4 LH (ixg/l) 5 . 3 4 + 0 . 8 2 2.25 _+-0.13 b 2.51 +0 .19 h PRL (~g/l) 1.38 -+ 0.08 0.73 + 0.04 h 0.72 -+ 0.04 t' ~ -MSH (ng/l) 14.3 + 3.1 19.7 _+ 2.4 13.8 + 2.3 AVP (ng/l) 3.7 + 0.6 1.6 _+ 0.4 h

" Values represent the means + SE in eight healthy male beagle dogs. h p < 0.05 (Wilcoxon matched-pairs signed-ranks test) vs. basal plasma hormone levels before hypophysecton b .

88 MEIJ ET AL.

TABLE 2. PITUITARY FUNCTION IN BEAGLE DOGS ASSESSED BY INCREMENTS IN PLASMA HORMONE LEVELS AFrER

STIMULATION TESTS BEFORE AND AFTER HYPOPHYSECTOMY (HX). a

2 wk 10 wk Hormone Before HX after HX after HX

ACTH (ng/1) 234.4 +_ 35.9 16.8 5:8.4 b 42.1 5:21.6 b Cortisol (nmol/l) 312.8 ± 22.0 13.3 + 14.2 b 49,6 5:28.9 b GH (Ixgfl) 13.1 5:2.4 0.2 5:0.3 b 0.5 + 0.2 b LH (Ixg/1) 19.07 5:2.77 0.04 5:0.05 b 0.53 5:0.24 b PRL (ixg/l) 28.48 + 6.77 0.02 5:0.02 b 0.10 5:0.03 b a - M S H (ng/1) 445.5 + 73.6 2.4 + 1.6 b 3.6 5:2.3 b AVP (ng/1) 56.5 5:9.0 2.2 +_ 0.5 b

"Anter ior pituitary: increment (mean + SE) for p lasma ACTH, cortisol, GH, LH, and PRL concentrations in eight healthy male beagle dogs after the intravenous rapid (30-sec) combined administrat ion of four hypotha- lamic releasing hormones. The releasing hormones were injected in the following order and doses: 1 ixg of CRH/kg, 1 Ixg of GHRH/kg, 10 ixg of GnRH/kg, and 10 ixg of TRH/kg. Pars intermedia: increment for plasma et-MSH concentrations after intravenous administration of 0.2 mg of haloperidol/kg. Posterior pituitary: incre- ment for plasma AVP concentrations after intravenous infusion of 20% NaC1 for 2 hr at a rate of 0.03 ml/kg per rain. b p < 0.05 (Wilcoxon matched-pairs signed-ranks test) vs. the increments in plasma hormone concentrations before hypophysectomy.

txg/1) at all times in the CAP test. The increments in plasma GH, LH, and PRL concen- trations were reduced to near-zero values at 2 and 10 wk after hypophysectomy (Table 2). The mean + SE plasma thyroxine concentration was 30.3 5:3.3 nmol/1 before hypophy- sectomy and 2.8 + 0.5 nmol/1 at 10 wk after hypophysectomy, which was near the detection limit of the assay (1.6 nmol/l).

There was no plasma a-MSH response after haloperidol injection at 2 and 10 wk after hypophysectomy in any of the eight dogs (Figure 6, Table 2). At 2 and 10 wk after hypophysectomy, the basal plasma ot-MSH concentrations were not significantly different from preoperative values (Table 1). In three dogs (Nos. 3, 4, and 7), plasma oL-MSH concentrations were reduced to the lower limit of detection of the assay (10 rig/l) at 10 wk after hypophysectomy, both before and after haloperidol injection. The increments in plasma o~-MSH concentration were significantly lower at 2 and 10 wk after hypophysec- tomy than preoperatively (Table 2).

During the first week after surgery, seven dogs drank about 1 1/d each, whereas one dog (No. 4) drank considerably more (2 l/d). After the discontinuation of desmopressin, water intake remained unchanged, and by the end of the second week after hypophysectomy, water intake in Dog No. 4 spontaneously decreased to about 1 1/d. The basal plasma AVP

TABLE 3. BASAL LEVELS AND INCREMENTS FOR PLASMA ACTH AND CORTISOL IN BEAGLE DOGS DIVIDED INTO

ACTH RESPONDERS (R; n = 4) AND NONRESPONDERS (NR; n = 4) AFTER HYPOPI-IYSECTOMY ( H X ) . a

Before HX 10 wk after HX

Hormone R NR R NR

ACTH (ng/1) Basal p lasma level 61.8 + 7.4 56,1 +_ 7.7 50.3 + 5.5 19.0 ± 0.0 b'c Increment 247.5 + 75.0 221,3 + 33.6 84.3 + 36.1 d 0.0 5:0.0 e

Cortisol (nmol/1) Basal plasma level 99.7 +- 32.7 109.6 5:27.4 53.6 + 14.4 22.8 5:1.1 b'c Increment 350.0 + 21.1 275.5 5:34.9 94.5 5:55.8 d 4.8 5:1.8 b'c

a Values represent the means + SE. See also footnote to Table 2 for details of the CAP test. b p < 0.05 (Mann-Whitney U test) vs. the nonresponders before HX.

P < 0.05 (Mann-Whitney U test) vs. the responders at 10 wk after HX. a p < 0.05 (Mann-Whitney U test) vs. the responders before HX.

Criterion for designation of the dogs as ACTH nonresponders in the CAP test.

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 89

20

15

~o -r C9

T

i i L i i i i I i i

-30 -15 0 15 30 45 60 75 90 105 120

t T ime (min)

Figure 3. Plasma GH response (mean + SE) in eight healthy male beagle dogs after the rapid (30-sec) intra- venous injection (arrow) of a combination of four hypothalamic releasing hormones before (O) and at 10 wk after (O) transsphenoidal hypopbysectomy. See also legend to Figure 1. Increment in plasma GH concentration was significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

concentrations were significantly lower at lO wk after hypophysectomy than before hy- pophysectomy (Table 1), whereas basal plasma osmolalities did not differ significantly. Before hypophysectomy, hypertonic saline infusion caused a rise in plasma AVP con- centrations as a linear function of plasma osmolality (Figure 7). At l0 wk after hypophy- sectomy, there was virtually no rise in plasma AVP concentrations (Figure 7, Table 2).

Pathology. The weight (mean + SE) of the lobes of the thyroid glands was 0.39 + 0.02 g, and that of the adrenals was 0.59 + 0.04 g. In all dogs, histological examination revealed inactivity of the thyroid glands and adrenocortical atrophy with regressive transformation of both the ZF and the ZR. Inactivity of the thyroid glands was characterized by follicles with flat epithelium, absence of resorption vacuoles, and a relative increase in the number of C-cells. Regressive transformation of the ZF and the ZR was evidenced by smaller cells

30

v T J

20

10

T

0 O' 0 - 0 - 0 ~ 0 - - 0 ~ 0 0 0 , 0

I I I I I I I I I I I

-30 -15 0 15 30 45 60 75 g0 105 120

t T ime (rain)

Figure 4. Plasma LH response (mean + SE) in eight healthy male beagle dogs alter the rapid (30-sec) intra- venous injection (arrow) of a combination of four hypothalamic releasing hormones before (0) and at t0 wk after (O) transsphenoidal hypophysectomy. See also legend to Figure 1. Basal level and increment in plasma LH concentration were significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

90 MEIJ ET A L

40

::L v .J rr" 13.

30

20

10

!

8 ~ 8 - o o ~ o ~ o o o I I I I I I

-30 15 0 15 30 45 60

t Iqrne (min)

- - Q o 8

I I I I

75 90 105 120

Figure 5. Plasma PRL response (mean + SE) in eight healthy male beagle dogs after the rapid (30-sec) intravenous injection (arrow) of a combination of four hypothalamic releasing hormones before (O) and at 10 wk after (O) transsphenoidal hypophysectomy. See also legend to Figure 1. Basal level and increment in plasma PRL concentration were significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

with loss of vacuolization. The ratios [ZG/(ZF + ZR)] (mean + SE; 1.2 + 0.1) in four dogs (Nos. 1, 2, 4, and 5) were significantly higher than the ratios (0.6 + 0.1) in the other four dogs (Nos. 3, 6, 7, and 8) (Mann-Whitney U test, P < 0.05). This subdivision corresponded to the above-mentioned division of the dogs according to their ACTH responses after hypophysectomy (nonresponders and responders, respectively). The mean adrenal weight (0.51 + 0.03 g) in the dogs with ratios >11 (nonresponders) was significantly lower than that (0.68 + 0.05 g) in the dogs with ratios <1 (responders).

The surgical specimens obtained by hypophysectomy contained normal pituitary tissue of the pars distalis, the pars intermedia, and the neurohypophysis. Macroscopic exami- nation of the ventral hypothalamic diencephalon revealed that all pituitary stalks had been removed completely and without damage to the hypothalamus. Histological examination

=.E. - r co

~5

500

400

300

200

100

i 1 ~ 1 1 0

t I

30 -15

I t - 0 - 0 - - " - 0 " - " " 0 0 0 0 0

I I I I , I I I I I

0 15 30 45 60 75 90 105 120

t T ime (rain)

Figure 6. Plasma a-MSH response (mean + SE) in eight healthy male beagle dogs after the intravenous injection (arrow) of 0.2 mg of haloperidol/kg before (O) and at 10 wk after (O) transsphenoidal hypophysectomy. Increment in plasma a-MSH concentration was significantly lower after hypophysectomy (Wilcoxon, P < 0.05).

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 91

80

60

c- 40

n

20

O

J / / 0 _ _ 0 ~ 0 ~ 0 - - 0 ~ 0 -0

u_-./l, , , . A , , ,

300 310 320 330 340 350 360

OSM (mOsm/I)

Figure 7. Relation between plasma AVP and plasma osmolality (OSM) (mean + SE) in eight healthy male beagle dogs during a 2-hr hypertonic saline infusion before (O) and at 10 wk after (O) transsphenoidal hypophysectomy. Basal level and increment in plasma AVP concentration were significantly lower after hy- pophysectomy (Wilcoxon, P < 0.05).

of the ventral hypothalamic diencephalon revealed no inflammatory changes, no damage to the hypothalamus, and no remnants of pituitary tissue. In one dog (No. 5), the sella turcica was lost during processing. In the sella turcica of seven dogs, microscopic islets of pituitary cells were found embedded in fibrous tissue. In five dogs (Nos. 1, 3, 4, 6, and 8) GH-, ACTH-, and e~-MSH-immunopositive cells were identified in the islets of pituitary cells in the sella turcica; in one dog (No. 7), ACTH- and c~-MSH-immunopositive cells were found, and in one dog (No. 2), only GH-immunopositive cells were found. The median (range; n = 7) number of immunopositive cells found in sections through the middle of the sella turcica was 50 (0-450) for GH, 113 (0-650) for ACTH, and 113 (0-550) for o~-MSH. There was a significant positive correlation between the number of ACTH- and the number of o~-MSH-immunopositive cells (r = 0.98, P < 0.0005), between the number of ACTH- and the number of GH-immunopositive cells (r = 0.80, P < 0.05), and between the number of GH- and the number of e~-MSH-immunopositive cells (r = 0.86, P < 0.05). The median (range) number of immunopositive cells in the nonresponders (n = 3) was 35 (5-70) for GH, 50 (0-138) for ACTH, and 50 (0-138) for ec-MSH. The median (range) number of immunopositive cells in the responders (n --- 4) was 113 (0-450) for GH. 219 (35-650) for ACTH, and 144 (50-550) for e~-MSH. The numbers of ACTH-, GH-, and ot-MSH-immunopositive cells found in sections of the sella turcica were higher in the responders (n = 4) than in the nonresponders (n = 3), but the differences were not significant. The correlation between the number of immunopositive cells and the respective increment in the CAP test at 10 wk after hypophysectomy was significant (r = 0.93, P < 0.005) for ACTH but not significant for GH (r = 0.37, P = 0.42). The correlation between the number of o~-MSH-immunopositive cells and the oL-MSH increment in the haloperidol stimulation test at 10 wk after hypophysectomy was not significant (1- -- -0.03, P -- 0.95).

92 MEIJ ET AL.

DISCUSSION

Adenohypophyseal responses to the rapid sequential administration of four hypotha- lamic releasing hormones (CRH, GHRH, GnRH, and TRH) are similar to those after the separate administration of these secretagogues. This is with the exception of the LH response, which was lower in the CAP test than after GnRH administration alone. Despite this minor interaction, the CAP test provides information on all four cell populations of the anterior lobe of the pituitary (10). In this study neither TSH nor FSH measurements were included because neither was available when the study was begun. PRL was included because TRH has distinct lactotropic activity in the dog. The doses used in the CAP test were based on the results of single stimulation tests and have been discussed previously (10). The timing of the postoperative experiments (2 and 10 wk) was chosen to allow comparison with other studies using the same postoperative intervals (11,18).

In this study, the CAP test proved to be useful for the assessment of pituitary function after hypophysectomy. The importance of testing several pituitary cell populations after hypophysectomy is illustrated by comparing our results with those of others who per- formed less comprehensive postoperative testing. In a report on the effect of graded hypophysectomy, it was concluded that only the removal of 97-99% of the anterior lobe resulted in adrenocortical, thyroidal, and gonadal atrophy (30). It was stated that there was a reasonably good correlation between the amount of pituitary tissue left in situ and the observed endocrine effects and that in clinically complete hypophysectomies, minute amounts of pituitary tissue (1-3%) may still be found (30). The latter was also seen in this study in three nonresponders. Other studies have assessed the completeness of hypophy- sectomy by GH measurements only (18) or the absence of responses in TRH (11) and GHRH (11,31) stimulation tests. However, unaffected ACTH production and normal cortisol responses to ACTH stimulation indicated that complete hypophysectomy was not accomplished, which was confirmed by the lack of adrenocortical atrophy at necropsy (11) or by the finding of remnants of adenohypophyseal tissue in the sellae turcica (18). The results of this study indicate that the corticotropic response in the CAP test is the most sensitive criterion to assess the completeness of hypophysectomy in dogs. The absence of corticotropic, somatotropic, gonadotropic, lactotropic, melanotropic, and posterior pitu- itary responses to stimulation in four dogs at 10 wk after hypophysectomy indicated complete removal of the pituitary. In the other four dogs, a small but significant cortico- tropic response in the CAP test was found, most likely originating from loose pituitary fragments.

The weights of the thyroid glands and adrenals of the hypophysectomized dogs were lower than the reported weights of thyroid glands (about 1 g) and adrenals (about 1.5 g) in normal adult beagle dogs (32,33). Low plasma thyroxine concentrations, near the lower limit of detection of the assay, were in good agreement with the finding of a 60% reduction in the postmortem weight of the thyroid glands in all hypophysectomized dogs. Inactivity of the thyroid glands was confirmed microscopically. Apart from the effect of hypophysectomy on the thyroid glands, substitution therapy with thyroxine may have caused inactivity of the remaining thyrotropic cells (and, consequently, thyroidal cells) (34). However, in the study of Panciera et al. (34), the dose rate of thyroxine was about 20 p~g/kg BID, whereas our dogs received 10 p~g/kg BID. The latter dose is somewhat low, when results of a recent pharmacokinetic study are considered (35). Thus, suppression by exogenous thyroxine is an unlikely explanation for the observed inactivity of the thyroid glands. It is rather the lack of thyrotropic hormone due to the surgery that caused the thyroidal inactivity.

Adrenocortical suppression has been reported in healthy dogs on daily prednisone

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 93

administration (36). However, it should be realized that the oral substitution dose for cortisone (0.25 mg/kg BID) in our hypophysectomized dogs was a physiological dose based on the cortisol production rate of healthy dogs (37), thereby taking into account incomplete absorption from the gut. The daily dose was equipotent to 0.1 mg of predni- sone/kg per d. This is much less than the lowest dose (0,22 mg/kg) used in the study of Chastain and Graham (36). Therefore, it is unlikely that the exogenous cortisone caused the suppression of corticotropic cells. Moreover, all dogs received cortisone according to the same protocol, so that the division in ACTH responders and nonresponders cannot be ascribed to differences in substitution therapy. The mean adrenal weights and mean [ZG/(ZF + ZR)] ratios of the ACTH responders and the nonresponders in the CAP test were significantly different. The corticotropic response in the CAP test in four dogs 10 wk after hypophysectomy led us to conclude that some functioning pituitary corticotropic cells had remained and were capable of reduced but measurable adrenocortica[ activity.

There are several possible explanations for a residual corticotropic response after by_ pophysectomy. It may be related to the persistence of corticotropic cells from the pars tuberalis of the adenohypophysis, which extends as a collar around the proximal part of the neurohypophysis (infundibulum) and envelops parts of the median eminence (13,14,38). It is easy to imagine that the pituitary stalk can be torn at different levels when the gland is removed by traction or suction during the surgical procedure, leaving variable numbers of cells of the pars tuberalis in place along the ventral 'hypothalamic diencepha- Ion. However, no pituitary remnants, either pars tuberalis or transitional zone, were found by histological examination of the ventral diencephalon in any of our hypophysectomized dogs. The corticotropic response in four hypophysectomized dogs may also be related to functional pituitary remnants in the sella turcica after hypophysectomy, as was reported previously (18). Indeed, in this study, islets of pituitary cells were found in the sella turcica with GH, ACTH, and a -MSH immunopositivity. The higher number of ACTH- immunopositive ceils found in the responders than in the nonresponders indicated that the residual responsiveness depended on the amount of tissue left in the sella turcica. Indeed, a significant posit ive correlation was found between the number of ACTH- immunopositive cells and the ACTH increment in the CAP test at 10 wk after hypophy- sectomy. Despite the absence of a GH and an c~-MSH response, GH- and c~-MSH- immunopositive cells were also found in the sella turcica after hypophysectomy; their numbers were probably too low for a measurable response to the stimulation used. The difference in the corticotropic response between the responders and nonresponders only became apparent at 10 wk after hypophysectomy. Apparently some time was required for the pituitary remnants to regain some responsiveness to blood-borne stimuli.

In humans, the CRH test has been used to evaluate selective transsphenoidal adeno- mectomy in the treatment of Cushing's disease (39-42). If surgery is successful, i.e., the ACTH-secreting pituitary adenoma is removed without affecting pituitary tissue, basal levels of all anterior pituitary hormones other than ACTH remain normal. The lack of ACTH and cortisol responses to CRH observed immediately after surgery is due to the chronic inhibition of unaffected pituitary corticotropic cells by the chronic hypercorti- solism of Cushing's disease before surgery (39,43). This suppression persists postopera- tively but gradually disappears over a period of 12 mo (43). Thus, a normal ACTH response or ACTH hyperresponsiveness to an early (1-2 wk) postoperative CRH test is a valuable criterion for the identification of patients who are at increased risk of recurrence. whereas a subnormal ACTH response or a lack of response indicates remission (40-42). The subnormal corticotropic response to CRH in the CAP test of four of our dogs at 10 wk after hypophysectomy led us to conclude that some functioning normal pituitary

94 MEIJ ET AL.

corticotropic cells had remained in the pituitary fossa, and this was confirmed at necropsy. Thus, the CAP test is also of value in the assessment of the completeness of hypophy- sectomy in dogs and may be used to assess the completeness of total hypophysectomy in dogs with PDH. The absence of a corticotropic response after total hypophysectomy in a dog with PDH could indicate complete removal of the pituitary gland, including the adenoma, but would not exclude the possibility that suppressed normal pituitary tissue was left behind in the sella turcica. A corticotropic response after total hypophysectomy in a dog with PDH would suggest incomplete removal of the pituitary adenoma or the recovery of normal pituitary corticotropic cells.

Early postoperative plasma cortisol measurements have been used recently as a pre- dictive factor for the initial cure and relapse rate after pituitary surgery in patients with Cushing's disease (44). Plasma cortisol concentrations at 6-12 wk after transsphenoidal surgery for Cushing's disease were lower than those within 2 wk after surgery and gave a better indication of permanent remission; levels less than 35 nmol/l suggested a favor- able long-term outcome (44). In this study, the basal plasma cortisol values were signifi- cantly lower at 10 wk than at 2 wk after hypophysectomy. At 10 wk after hypophysec- tomy, the ACTH nonresponders had lower basal plasma cortisol concentrations than the responders, suggesting that this can be used as a criterion for successful surgery in the normal dog. Studies after hypophysectomy in dogs with PDH are needed to determine whether a criterion for successful surgery can be found in the dog, as is used in humans.

The preoperative plasma ct-MSH responses to haloperidol administration were similar to those observed in a study by Kemppainen and Sartin (15). The absence of an e~-MSH response in all dogs at 2 and 10 wk after hypophysectomy indicated complete removal of the pars intermedia. However, six of the seven dogs investigated had pars intermedia fragments in the sella turcica with cell staining immunopositively for e~-MSH. Probably, their severance from neurogenic (dopaminergic) influences precluded responsiveness to a dopamine antagonist.

The relation between plasma AVP and plasma osmolality during hypertonic saline infusion before hypophysectomy was consistent with the reference values reported by Biewenga et al. (12). At 10 wk, hypophysectomy, hypertonic saline infusion caused virtually no AVP response. Nevertheless, the animals were not polyuric. Total hypophy- sectomy deprived the animals of the neurohypophyseal storage of AVP and the ability to release it after stimulation. However, when the pituitary stalk is sectioned low enough not to cause retrograde degeneration of supraoptic and paraventricular neurons, there is suf- ficient "leakage" of AVP to prevent diabetes insipidus (45,46). In addition, the fibers of these magnocellular neurons may regenerate to establish new neurohemal connections to form a posterior pituitary-like structure. There is evidence that the meninges play an important role in this axonal regeneration process (47). The paradox of the absence of diabetes insipidus in the hypophysectomized dogs lacking an AVP response to hypertonic saline infusion led us to conclude that sufficient AVP was released to prevent diabetes insipidus but that insufficient regrowth of posterior pituitary fibers had taken place at 10 wk after hypophysectomy to restore responsiveness to hypertonic saline infusion.

The results of this present study allow the following conclusions:

• The CAP test, stimulation with haloperidol, and hyertonic stimulation of AVP release, both before and after hypophysectomy, are associated with no serious side effects that would prohibit their routine use in the dog.

• In the hypophysiotropic stimulation of the anterior pituitary and the pars intermedia by the CAP test and haloperidol, respectively, blood sampling can be confined to 0, 5, 10, 20, 30, and 45 min; the peaks in plasma concentrations of ACTH, cortisol, GH, LH, PRL, and ct-MSH all occurred within 45 min.

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 95

• The A C T H and cor t isol r e sponses to h y p o p h y s i o t r o p i c s t imula t ion are the mos t sensi-

t ive ind ica to rs o f res idua l p i tu i ta ry func t ion af ter h y p o p h y s e c t o m y . In four o f e igh t

h y p o p h y s e c t o m i z e d dogs , some A C T H re sponse was obse rved , wi th no c o n c o m i t a n t

r e sponse by the an te r io r p i tu i ta ry somato t rop ic , gonado t rop ic , and lac to t ropic cells, no

r e sponse o f the pars i n t e rmed ia m e l a n o t r o p i c cells, and no A V P response . Cor t i co t rop ic

cel ls in islets of p i tu i tary cel ls f ound at nec ropsy in the sel la turc ica were the mos t l ikely

source o f the a t t enua ted cor t i co t rop ic r e sponse in four o f e igh t h y p o p h y s e c t o m i z e d

dogs,

• Res idua l A V P re lease f rom the h y p o t h a l a m u s is suff ic ient to p reven t d iabe tes ins ip idus

at 10 wk af ter h y p o p h y s e c t o m y , but there was no r e sponse to a hype r ton ic s t imulus .

A C K N O W L E D G M E N T S ~ F O O T N O T E S

The CSU-204 antiserum and the canine LH standard LER 1685-1 were generous gifts from Dr. G.D. Nis- wender (Fort Collins, CO) and Dr. LE. Reichert (Albany, NY), respectively. The technical assistance of Mr. H.G.H. van Engelen, Mr. J, Fama, Mr. L.C. Alflen, Mrs. J, Wolfswinkel. Mr. F. van Mil, and Mr. R. Molenbeek is gratefully acknowledged. The critical reading of the manuscript by Dr. B.E. Belshaw is highly appreciated.

r Address for correspondence and reprint requests to: Dr. B.P. Meij, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.154, NL-3508 TD Utrecht, The Netherlands.

REFERENCES

1. Wehrenberg WB, Baird A, Ying S-Y, Rivier C, Ling N, Guillemin R, Multiple stimulation of the adeno- hypophysis by combinations of hypothalamic releasing factors. Endocrinology 114:1995-2001, 1984.

2. Holl R, Fehm HL, Hetzel WD, Heinze E, Voigt KH. Globaler Hypophysen-stimulations-test mit Releasing- Hormonen. Deutsch Med Wochenschr 110:953-955, 1985.

3. Holl R, Fink P, Hetzel WD. Combined pituitary stimulation test with releasing hormones. Acta Endocrinol (Copenh) 267[Suppl 108]:18, 1985.

4. Sheldon WR Jr, DeBold CR, Evans WS, DeCheruey GS, Jackson RV, Island DP, Thorner MO, Orth DN. Rapid sequential intravenous administration of four hypothalamic releasing hormones as a combined ante- rior pituitary function test in normal subjects. J Clin Endocrinol Metab 60:623-630, 1985.

5, Cohen R, Bouquier D, Biot-Laporte S. Vermeulen E, Claustrat B, Cherpin MH, Cabrera P, Guidetti P, Ferry S, Bizollon ChA, Sassolas G. Pituitary stimulation by a combined administration of four hypothalamic releasing hormones in normal men and patients. J Clin Endocrinol Metab 62:892-898, 1986.

6. Schopohl J, Losa M, Krnig A, MUller OA, Stalla GK, Von Werder K. Combined pituitary function-test with four hypothalamic releasing hormones. Klin Wochenschr 64:314-318, 1986.

7. Kogure M. Anterior pituitary function test by combined intravenous administration of four hypothalamic releasing hormones (GRF, CRF, TRH, LH-RH). Teikyo lgaku Zasshi 10:433-441, 1987.

8. Bando H, Yamasaki R, Saito S. Evaluation of hypothalamic-pituitary function in a combination of tests with four hypothalamic releasing hormones and L-dopa in normal subjects and in patients with hypothalamic and/or pituitary disorders. Endocrinol Jpn 36:705-720, 1989.

9. Thorner MO, Vance ML, Horvath E, Kovacs K. Dynamic tests of pituitary function. In: Williams Textbook of Endocrinology, Wilson JD and Foster DW (eds). WB Saunders, Philadelphia, p. 246-260, 1992.

10. Meij BP, Mol JA, Hazewinkel HAW, Bevers MM, Rijnberk A. Assessment of a combined anterior pituitary function test in beagle dogs: Rapid sequential intravenous administration of four hypothalamic releasing hormones. Domest Anita Endocrinol 13:161-170, 1996.

11. Eantz GC, Ihle SL, Nelson RW, Carlton WW, Feldman EC, Lothrop CD, Bottoms GD. Transsphenoidal hypophysectomy in the clinically normal dog. Am J Vet Res 49:1134--1142, 1988.

12. Biewenga WJ, Van den Brom WE, Mol JA. Vasopressin in polyuric syndromes in the dog. Front Horm Res 17:139-148, 1987.

13. Halmi NS, Krieger D. Immunocytochemistry of ACTH-related peptides in the hypophysis. In: The Anterior Pituitary Gland, Bhatnagar AS (ed). Raven Press, New York, p, 1-15, 1983.

14. El Etreby MF, Dubois MP. The utility of antisera to different synthetic adrenocorticotropins (ACTIq) and melanotropins (MSH) for immunocytochemical staining of the dog pituitary gland. Histochemistry 66:245- 260, 1980.

15. Kemppainen RJ. Sartin JL. Differential regulation of peptide release by the canine pars distalis and pars intermedia. Front Horm Res 17:18-27, 1987.

16. Markowitz J, Archibald J, Downie HG, Experimental Surgery. Williams & Wilkins, Baltimore, 1964.

96 MEIJ ET AL.

17. Selman PJ, Mol JA, Rutteman GR, Van Garderen E, Rijnberk A. Progestin-induced growth hormone excess in the dog originates in the mammary gland. Endocrinology 134:287-292, 1994.

18. Niebauer GW, Eigenmann JE, Van Winkle TJ. Study of long-term survival after transsphenoidal hypophy- sectomy in clinically normal dogs, Am J Vet Res 51:677-681, 1990.

19. Rijnberk A, Der Kinderen PJ, Thijssen JHH. Spontaneous hyperadrenocorticism in the dog. J Endocrinol 41:397--406, 1968.

20. Lubberink AAME. In: Diagnosis and treatment of canine Cushing's syndrome, Thesis. Drukkerij Elinkwijk, Utrecht, p. 45-74, 1977.

21. Arts CJM, Koppeschaar HPE, Veeman W, Thijssen JHH. A direct radioimmunoassay for the determination of adrenocorticotropic hormone (ACTH) and a clinical evaluation. Ann Clin Biochem 22:247-256, 1985.

22. Rijnberk A, Mol JA, Kwant MM, Croughs RJM. Effects of bromocriptine on corticotrophin, melanotrophin, and corticosteroid secretion in dogs with pituitary-dependent hyperadrenocorticism. J Endocrinol l 18:271- 277, 1988.

23. Eigenmann JE, Eigenmarm RY. Radioimmunoassay of canine growth hormone. Acta Endocrinol (Copenh) 98:514-520, 1981.

24. Nett TM, Akbar AM, Phemister RD, Hoist PA, Reichert LE Jr, Niswender GD. Levels of luteinizing hormone, estradiol and progesterone in serum during the estrous cycle and pregnancy in the beagle bitch. Proc Soc Exp Biol Med 148:134-139, 1975.

25. Stolp R, Bevers MM, Rijnberk A, Croughs RJM, Rutteman GR. Regulation of prolactin secretion in canine pituitary-dependent hyperadrenocorticism. Horm Metab Res 18:595-598, 1986.

26. Belshaw BE, Rijnberk A. Radioimmunoassay of plasma T4 and T3 in the diagnosis of primary hypothy- roidism in dogs. J Am Anita Hosp Assoc 15:17-23, 1979.

27. Hellebrekers LJ, Mol JA, Van den Brom WE, van Wimersma Greidanus TjB. Effect of methadone on plasma arginine vasopressin level and urine production in conscious dogs. Eur J Pharmacol 136:279-286, 1987.

28. Middleton DJ, Rijnberk A, Bevers MM, Goos HJTh, Beeftink EA, Thijssen JHH, Croughs RJM. Some functional aspects of canine corticotrophs. Horm Metab Res 19:632-635, 1987.

29. Spencer GSG, Garssen GJ, Colenbrander B, Macdonald AA, Bevers MM. Glucose, growth hormone, somatomedin, cortisol, and ACTH changes in the plasma of unanaesthetised pig foetuses following intra- venous insulin administration in utero. Acta Endocrinol (Copenh) 104:240--245, 1983.

30. Ganong WF, Hume DM. The effect of graded hypophysectomy on thyroid, gonadal, and adrenocortical function in the dog. Endocrinology 59:293-301, 1956.

31. Niebauer GW. Hypophysectomy. In: Textbook of Small Animal Surgery, Slatter D (ed). WB Saunders, Philadelphia, p. 1496-1510, 1993.

32. Lane N, De Bodo RC. Generalized adrenocortical atrophy in hypophysectomized dogs and correlated functional studies. Am J Physiol 168:1-19, 1952.

33. Andersen AC, Goldman M. Growth and development. In: The Beagle as an Experimental Dog, Andersen AC and Good LS (eds). The Iowa State University Press, Ames, p. 43-105, 1970.

34. Panciera DL, Atkins CE, Bosu WTK, MacEwen EG. Quantitative morphologic study of the pituitary and thyroid glands of dogs administered L-thyroxine. Am J Vet Res 51:27-31, 1990.

35. Nachreiner RF, Refsal KR, Ravis WR, Hauptman J, Rosser EJ, Pedersoli WM. Pharmacokinetics of L-thyroxine after its oral administration in dogs. Am J Vet Res 54:2091-2098, 1993.

36. Chastain CB, Graham CL. Adrenocortical suppression in dogs on daily and alternate-day prednisone administration. Am J Vet Res 40:936-941, 1979.

37. Rijnberk A, Der Kinderen PJ, Thijssen JHH. Investigations on the adrenocortical function of normal dogs. J Endocrinol 41:387-395, 1968.

38. Hullinger RL. The hypophysis. In: Miller's Anatomy of the Dog, Evans HE (ed). WB Saunders, Philadel- phia, p. 560--567, 1993.

39. Orth DN, Debold CR, DeCherney GS, Jackson RV, Alexander AN, Rivier J, Rivier C, Spiess J, Vale W. Pituitary microadenomas causing Cushing's disease respond to corticotropin-releasing factor. J Clin Endo- crinol Metab 55:1017-1019, 1982.

40. Avgerinos PC, Chrousos GP, Nieman LK, Oldfield EH, Loriaux DL, Cutler GB. The corticotropin-releasing hormone test in the postoperative evaluation of patients with Cushing's syndrome. J Clin Endocrinol Metab 65:906-913, 1987.

4l. Schrell U, Fahlbusch R, Buchfelder M, Riedl S, Stalla GK, Miiller OA. Corticotropin-releasing hormone stimulation test before and after transsphenoidal selective microadenomectomy in 30 patients with Cush- ing's disease. J Clin Endocrinol Metab 64:1150-1159, 1987.

42. Vignati F, Berselli ME, Loli P. Early postoperative evaluation in patients with Cushing's disease: usefulness

PITUITARY FUNCTION IN HYPOPHYSECTOMIZED DOGS 97

of ovine corticotropin-releasing hormone test in the prediction of recurrence of disease, Eur J Endocrinol 130:235-241, 1994.

43. Fitzgerald PA, Aron DC, Findling JW, Brooks RM, Wilson CB, Forsham PH, Tyrrell JB. Cushing's disease: transient secondary adrenal insufficiency after selective removal of pituitary microadenornas; evidence for a pituitary origin. J Clin Endocrinol Metab 54:413-422, 1982.

44. Toms GC, McCarthy MI, Niven MJ, Orteu CH, King q-T, Monson JP. Predicting relapse after transsphe- noidal surgery tbr Cushing's disease. J Clin Endocrinol Metab 76:291-294, 1993.

45. Fisher C, Ingrain WR. The effect of interruption of the supraoptico-hypophyseal tracts on the antidiuretic, pressor and oxytocic activity of the posterior lobe of the hypophysis. Endocrinology 20:762-768, 1936.

46. Heinbecker P, White HL. Hypothalamic-hypophysial system and its relation to water balance in the dog. Am J Physiol 133:582-593, 1944.

47. lshikawa K, Kabeya K, Shinoda M, Katakai K, Mori M, Tatemoto K. Meninges play a neurotrophic role in the regeneration of vasopressin nerves after hypophysectomy. Brain Res 677:20-28, 1995.