Giovanni L. Rossi

Ultrastructure of Somatotrophs of Rats with Median Eminence Lesions: Studies in Basal Conditions and after Thyrotropin-Releasing Hormone Stimulation

Anterior pituitaries from female Sprague-Dawley rats (160-200 g b.w) bearing electrolytic lesions of the median eminence were qualitatively and quantitatively investigated by electron microscopy and the findings compared with those from intact animals or sham-operated controls. This study was performed in basal conditions and after stimulation with thyrotropin-releasing hormone. Animals with lesions showed a reduction of both the number of somatotrophic cells and growth hormone granules. After injection of thyrotropin-releasing hormone exocytosis of growth hormone granules from somatotrophs was frequent in rats with lesions of the median eminence but rare in sham-operated animals. The differences were statistically significant.

Thyroid and pituitary secretory disorders in streptozotocin-diabetic rats are associated with severe structural changes of these glands

SummaryStreptozotocin diabetes in rats is associated with reduced function of the hypothalamopituitary-thyroid axis. The structure and hormone secretion of the thyroid and pituitary glands were studied in adult male rats 1 month after streptozotocin injection. The thyroid of diabetic rats was characterized by decreased follicle area and epithelial thickness. By electron microscopy, thyroid epithelial cells were characterized by flattened and almost empty rough endoplasmic reticulum cisternae, scanty exocytotic apical and endocytotic vesicles as well as degenerate mitochondria and rough endoplasmic reticulum. By immunohistochemistry, intracolloidal thyroglobulin and T3 as well as intraepithelial thyroglobulin were reduced. Electron microscopic and immunohistochemical analysis of pituitary glands showed that in diabetic rats thyrotrophs were mostly of type II, and the number of thyrotrophs (type I + type II) was greater than in controls. By radioimmunoassay (RIA), plasma T3, T4, and TSH levels were markedly reduced, and the TSH response to TRH was deficient in diabetic animals. The pituitary TSH concentration was increased, as expected from the morphological data. This study demonstrates severe structural changes in the thyroid and pituitary glands of diabetic rats which are accompanied by marked alterations of their secretory activity.

Continuous Subtherapeutic Insulin Counteracts Hypothalamopituitary-Gonadal Alterations in Diabetic Rats

In experimental animal models, gonadal axis lesions are probably responsible for reproductive disorders associated with diabetes mellitus. The pathogenesis of these disorders is not yet known, but it is assumed that insulin deficiency plays an important role. To check this hypothesis, we have investigated the hypothalamopituitary-gonadal axis of insulin-treated streptozocin-induced diabetic (STZ-D) rats and compared it with that of untreated diabetic and control animals. Insulin was delivered by subcutaneously implanted osmotic minipumps. Furthermore, to determine whether possible beneficial insulin effects are selectively limited to the gonadal axis or act generally, we also studied retinal microangiopathy. The hypothalamopituitary-gonadal axis of insulin-treated diabetic animals was almost unchanged. On the contrary, retinal microangiopathy was only slightly influenced by subtherapeutic insulin doses. In conclusion, continuous administration of insulin at subtherapeutic doses can successfully counteract most of the effects of diabetes on the gonadal axis. Thus, the gonadal-axis impairment in STZ-D animals appears to be related to the fall of plasma insulin below a critical level. Furthermore, the various organ systems may respond to different plasma insulin threshold levels.

Functional and Morphological Changes in Mediobasal Hypothalamus of Streptozocin-induced Diabetic Rats: In Vitro Study of LHRH Release

To investigate the role of the mediobasal hypothalamus (MBH) in diabetic gonadal axis disorders, the MBHs of adult male streptozocin-induced diabetic (STZ-D) rats were examined after incubation in basal conditions or in K+-enriched medium and compared with those of controls. Diabetes lasted 1 mo. Both luteinizing-hormone-releasing hormone (LHRH) release and MBH morphology were studied. After incubation in basal conditions, the LHRH release was unchanged. By light microscopy, the dilated-axon cross sections were more numerous (P < .01) in the basal arcuate nucleus and in the median eminence. By electron microscopy, the ratio of exocytoses to neurosecretory granules observed in the median eminence axon cross sections was smaller (P < .05). The total LHRH immunoreactivity, the number of labeled axons, and the amount of positive material in the axons were reduced (P < .05). After incubation in K+-enriched medium, the LHRH release was markedly reduced (P < .01). The number and area of dilated-axon cross sections, possibly because of the relation between exocytosis and physiological dilation, were less augmented (P < .01). Whereas the number of exocytoses and the ratio of exocytoses to neurosecretory granules were not decreased, the total LHRH immunoreactivity and the number of labeled axons were reduced (P < .05). The releasable LHRH pool therefore seems to be exhausted in control MBH because of long-term stimulation and reduced in the MBH of STZ-D rats because of diabetes. In conclusion, STZ-D causes functional and anatomical MBH lesions that should be pathogenetically relevant for the disorders of the gonadal axis documented in this animal model.

Technical Aspects in the Study of Pathologic Lesions in the Hypothalamus of the Rat

Adequate techniques are of primary importance for successful morphological studies of lesions in the central nervous system of laboratory animals For investigations of the rat hypothalamus in particular the following points deserve special attention: (a) method of fixation, (b) dissection of tissues, and (c) further processing. Over some years we have developed and employed the techniques herewith described (Bestetti and Rossi 1980, and this volume p. 331; Rossi and Bestetti 1981). They allow accurate study of lesions by combining morphological and immunohistochemical methods.

Studies on retinal microangiopathy and coronary macroangiopathy in rats with streptozotocin-induced diabetes

SummaryDiabetes mellitus was induced in male rats by streptozotocin injection. After 4, 8 and 12 months, retinal and coronary vessels were prepared and their structure was studied. An age-dependent increase in thickness of the capillary basement membrane of retinal vessels was found both in controls and diabetics. Furthermore, diabetics had an added 20%, diseaserelated thickening, present after 4 months and thereafter. No significant change in coronary arteries was found. After 8 and 12 months, plasma lipids, triiodothyronin and thyroxin were measured. In diabetics, triglycerides were higher at both time intervals, high density lipoprotein was slightly increased and thyroxin was reduced after 8 months, and triiodothyronin was reduced after both 8 and 12 months. Diabetes of 12 months duration appears insufficient to induce significant arteriosclerotic changes in rats.

Stimulation of growth hormone release by luteinizing hormone-releasing hormone and melanocyte-stimulating hormone-release inhibiting hormone in the hypophysectomized rat bearing an ectopic pituitary.

Intrajugular administration of LHRH (0.6 and 1.2 μg) in hypophysectomized rats which received renal grafts of anterior pituitary induced a small but significant rise in plasma GH 5 and 10 min post‐treatment. LHRH, at the same dose levels, was ineffective in weight‐matched intact controls. MIF, at the dose of 1.2 μg, induced a slight GH rise 5 min after treatment in hypophysectomized‐transplanted rats, while it was ineffective in intact controls. Unlike the two hypothalamic peptides, α‐MSH (0.6 and 1.2 μg) was ineffective as a GH‐releaser in both transplanted and intact rats.

Effect of growth hormone-releasing stimuli in streptozotocin diabetic rats.

The dynamics of growth hormone (GH) secretion in response to different GH secretagogues has been studied in adult freely moving male rats one month after induction of diabetes by single i.v. injection of streptozotocin (60 mg/kg). Baseline plasma GH concentrations and pituitary GH content were not different in streptozotocin-diabetic (St-D) rats and controls. Clonidine (0.15 mg/kg i.v.), an alpha 2-adrenergic agonist, failed to evoke GH release in St-D rats. Substitution therapy with insulin (1 IU/100 g b.wt.daily) delivered through subcutaneously implanted minipumps, allowed re-institution of a normal GH responsiveness to clonidine. At odds with clonidine, FK 33-824 (0.1 mg/kg i.v.), a potent analog of the opioid peptide Met-enkephalin, induced a similar rise in plasma GH levels in control and St-D rats. Finally, administration of a synthetic replicate of a GH-releasing hormone of human pancreatic origin, hpGRF-40 (2.5 micrograms/kg i.v.) elicited a higher GH response in St-D rats than in controls. These data indicate that in St-D rats: (1) an impaired function of noradrenergic pathways controlling GH release is present; (2) contrary to previous beliefs, an alpha 2-adrenergic mechanism is not involved in the GH-releasing effect of opioid peptides; and (3) pituitary GH responsiveness to hpGRF is increased.

Tuberoinfundibular dopaminergic neurons and lactotropes in young and old female rats.

Aging in female rats is accompanied by several endocrine dysfunctions, such as reproductive decline associated with characteristic hyperprolactinemia, lactotrope hyperplasia, and functional impairment of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. The aim of this morphometrical, immunocytochemical, and densitometrical study was to gain a better anatomical knowledge of TIDA neurons and axons as well as of lactotropes in old female rats with (A) or without (NA) pituitary adenomas, compared with young animals. At the hypothalamic level, we found that tyrosine hydroxylase (TH)-labeled neurons in the arcuate nucleus were comparable in young and old NA yet their size and TH-content were increased in A animals. Also the TH-labeled median eminence axons did not differ significantly between young and old NA but were more numerous in the old A rats. Independently from adenomas, both number of prolactin (PRL)-labeled structures and content of immunoreactive PRL were increased in pituitaries of old rats, the plasma PRL levels, however, were high only in A. Our findings support the documented lactotrope hypertrophy and hyperplasia in old female rats and suggest that TIDA-neuron changes only occur in hyperprolactinemic animals carrier of adenomas.

Functional and Morphological Aspects of Impaired TRH Release by Mediobasal Hypothalamus of STZ-Induced Diabetic Rats

Streptozocin-induced diabetes (STZ-D) in rats is associated with marked hypothyroidism characterized by functional impairment and structural lesions of the pituitary-thyroid axis. Degenerative axonal lesions, which can be prevented by insulin administration, have been reported in the mediobasal hypothalamus (MBH) of STZ-D rats. However, direct evidence connecting anatomic MBH lesions with functional impairment is still missing. We therefore performed a combined functional and morphological investigation in 4-mo-old STZ-D male rats (diabetes lasted 1 mo), applying an in vitro model to study in the same isolated MBH 1) the basal and depolarization-induced thyrotropin-releasing hormone (TRH) release during two successive incubations of 20 min each and 2) morphological and morphometric aspects, including distribution and amount (densitometric evaluation) of immunoreactive TRH in the incubated tissue. In basal conditions, TRH release was much lower in diabetic than control MBH during both incubations (P < .01 vs. P < .05). In depolarizing conditions, TRH release was increased during the second incubation in control (P < .05) and during both incubations in diabetic (P < .01) rats, the percentage increase of the TRH release due to ionic stimulation being much higher in diabetic than control animals (P < .01). As determined by light-microscope morphometry, the total area of dilated-axon cross sections was larger in diabetic than control MBH under basal conditions (P < .01), thus confirming degenerative axonopathy in diabetic rats. By densitometry determination, the amount of immunoreactive TRH was higher in stimulated diabetic MBH compared with both stimulated control and basal diabetic MBH (P < .01). The total area of immunoreactive TRH was increased in stimulated diabetic MBH (P < .05). Our findings show impaired in vitro TRH secretion without reduced TRH content in diabetic MBH. This should play an important role in the pathogenesis of the pituitary and thyroid lesions responsible for the hypothyroidism observed in STZ-D rats.