Immunohistochemical and radioimmunological study of pituitary gonadotrophs during aging in male rats.

The impact of aging on pituitary gonadotrophs in rats is little known. We therefore undertook a quantitative immunohistochemical assessment of the follicle-stimulating hormone (FSH) and luteinizing hormone (LH) immunoreactive cell population in the pituitaries of young (4 months), old (20 months) and senescent (29 months) male rats. In addition, an attempt was made to correlate morphometric parameters with serum levels of FSH and LH. Gonadotrophs were immunostained by the peroxidase-antiperoxidase method using anti-rat LH and anti-rat FSH rabbit sera as primary antibodies. Hormones were measured in serum by specific radioimmunoassays. Analysis of morphological parameters revealed, for both types of gonadotrophs, a marked age-related reduction in cell density, volume density and surface density. Gonadotroph cell perimeter and area showed a trend towards an increase from 5 to 20 months of age but appeared drastically reduced in the 29-month-old rats. Basal serum levels of LH and FSH showed a significant increase from 4 to 20 months of age but remained at the same level in the senescent rats. The correlation between serum levels of gonadotropins and gonadotroph morphometric parameters was low in all cases. We conclude that in male rats aging brings about a significant reduction in both the number and size of pituitary gonadotrophs which, nevertheless, seem to be still capable of keeping appropriate basal levels of circulating LH and FSH.

Impact of aging on the morphology and function of the somatotroph cell population in rats.

There is little information regarding the impact of aging on pituitary somatotroph (STH) cell population in rats. We therefore undertook a quantitative immunocytochemical assessment of this cell type in young (3 months), old (20 months) and senescent (29 months) male rats. An attempt was also made to correlate morphological parameters with serum levels of growth hormone (GH). Since thyroid status is highly influential on somatotropic function, serum levels of thyroxine (T4) and triiodothyronine (T3) were also measured in the three age groups. We found a marked age-related reduction in STH cell number, volume density, and surface density, as well as a milder but significant decline in STH cell area and perimeter. Basal serum levels of GH remained unchanged with age, whereas the estimated number and amplitude of GH pulses declined from young to old animals. Thyroxine but not T3 levels also declined with age. We conclude that in rats, aging causes a marked reduction in somatotroph number and, to a lesser extent, cell size. These alterations do not affect trough levels of circulating GH. Our data also suggest that the progressive hypothyroidism associated with aging in this species may contribute to the promotion of the above changes. The present study emphasizes the convenience of combining hormone measurements with quantitative morphological analysis of cell populations for the study of pituitary function during aging.

Sexual dimorphism in the age changes of the pituitary lactotrophs in rats.

It is known that aging is associated with alterations in hypothalamic and pituitary functions. In the present study, we have undertaken a quantitative immunohistochemical assessment of the lactotroph cell population as well as prolactin (PRL) secretion, in male and female rats of different ages. Pituitaries from young (3 months), old (20 months) and senescent (29 months) male and female Sprague-Dawley rats were processed for the immunohistochemical detection of lactotrophs. Serum PRL was measured by a homologous RIA. Additionally, the in vitro PRL secretory activity was estimated by perifusion of pituitary cells from senescent animals. Analysis of morphometric parameters revealed age-related changes of PRL cell population in animals of both sexes. The cell density (CD), surface density (SD) and volume density (VD) decreased with age in both male and female rats. However, CD as well as SD appeared to have increased in females when compared to males, either in young or old animals, while VD was higher only in old females. The pituitaries of senescent females displayed chromophobic microadenomas on a background of diffuse PRL cell hyperplasia. Prolactin serum levels showed a marked increase with age in females, but only a modest elevation in males. In senescent females, PRL production per cell was reduced. We conclude that in rats, there exists a clear sexual dimorphism in the age-related changes of pituitary PRL cells.

Mechanisms involved in the beta-cell mass increase induced by chronic sucrose feeding to normal rats.

The aim of the present study was to clarify the mechanisms by which a sucrose-rich diet (SRD) produces an increase in the pancreatic beta-cell mass in the rat. Normal Wistar rats were fed for 30 weeks either an SRD (SRD rats; 63% wt/wt), or the same diet but with starch instead of sucrose in the same proportion (CD rats). We studied body weight, serum glucose and triacylglycerol levels, endocrine tissue and beta-cell mass, beta-cell replication rate (proliferating cell nuclear antigen; PCNA), islet neogenesis (cytokeratin immunostaining) and beta-cell apoptosis (propidium iodide). Body weight (g) recorded in the SRD rats was significantly (P<0.05) larger than that of the CD group (556.0+/-8.3 vs 470.0+/-13.1). Both serum glucose and triacylglycerol levels (mmol/l) were also significantly higher (P<0.05) in SRD than in CD rats (serum glucose, 8.11+/-0.14 vs 6.62+/-0.17; triacylglycerol, 1.57+/-0.18 vs 0.47+/-0.04). The number of pancreatic islets per unit area increased significantly (P<0.05) in SRD rats (3.29+/-0.1 vs 2.01+/-0.2). A significant increment (2.6 times) in the mass of endocrine tissue was detected in SRD animals, mainly due to an increase in the beta-cell mass (P=0.0025). The islet cell replication rate, measured as the percentage of PCNA-labelled beta cells increased 6.8 times in SRD rats (P<0.03). The number of apoptotic cells in the endocrine pancreas decreased significantly (three times) in the SRD animals (P=0.03). The cytokeratin-positive area did not show significant differences between CD and SRD rats. The increase of beta-cell mass induced by SRD was accomplished by an enhanced replication of beta cells together with a decrease in the rate of beta-cell apoptosis, without any evident participation of islet neogenesis. This pancreatic reaction was unable to maintain serum glucose levels of these rats at the level measured in CD animals.

Islet neogenesis: an apparent key component of long-term pancreas adaptation to increased insulin demand.

This study aimed to determine the relative importance of different functional and morphological pancreatic changes induced by the chronic administration of a sucrose-rich diet (SRD) to maintain normal glucose homeostasis. Male Wistar rats were fed either sucrose (SRD) or starch (CD) for 6 and 12 months. At both periods, serum glucose and triacylglycerol levels were significantly higher (P<0.05; paired and unpaired Student's t-test) in SRD rats. Serum insulin levels were significantly lower in SRD only at 12 months. At 6 months, the insulin secretion dose-response curve in SRD rats showed a shift to the left that was no longer observed at 12 months, when SRD islets decreased their response to 16 mM glucose. At 6 months, SRD rats showed a significant increase in beta-cell volume density (Vvi) and islet cell replication rate, together with a decrease in beta-cell apoptotic rate. Changes were not detected in the percentage of PDX-1- and islet neogenesis associated protein (INGAP)-positive cells. Conversely, at 12 months, there was a significant decrease in beta-cell Vvi and in the percentage of PDX-1-positive cells; the islet cell replication rate was not modified, and the number of apoptotic beta-cells increased significantly. No signs of increased neogenesis or INGAP-positive cells were recorded at any period in SRD rats. Our results show that SRD rats are unable to develop functional and morphological pancreatic reactive changes sufficient to maintain normal glucose and triacylglycerol levels for a long period. Such failure could be ascribed to their inability to increase the rate of neogenesis and of INGAP production.

Epinephrine effect on arachidonic acid biosynthesis in isolated adrenocortical cells.

The in vivo and in vitro effect of epinephrine on the incorporation and desaturation of [1-14C]eicosa-8,11,14-trienoic acid was studied in isolated adrenocortical cells of rats. Control cells incubated at different substrate concentrations and for different periods of time were able to incorporate eicosatrienoic acid and to desaturate it to arachidonic acid. The ultrastructural study demonstrated that most of the cells belonged to the zona fasciculata and presented a good preservation. When the cells were isolated from rats killed 7 and 12 h after the administration of a single dose of epinephrine, a decrease in the incorporation and desaturation of 20:3n6 was observed. The effect on the desaturation was independent from the incorporation of the acid, and it was also observed in the microsomes of the decapsulated adrenal gland from rats treated with epinephrine. The fine structure of adrenocortical cells did not show changes after the treatment with epinephrine. The addition of the hormone to the incubation medium containing cells isolated from untreated rats produced no effect on arachidonic acid biosynthesis, indicating that the effect of epinephrine would be indirect, through either a metabolic or a hormone mediator.

Quantitative immunocytochemical study of islet cell populations in diabetic calmodulin-transgenic mice.

The present study describes the changes in the endocrine pancreas of severely diabetic calmodulin-transgenic mice using light microscopic immunocytochemical and morphometric techniques. A marked reduction in the number and volume of islets, together with distortion of their normal architecture, was found in diabetic mice. In addition, the volume density of both endocrine tissue and B-cells was decreased. An irregular distribution of non-B-cells was also observed in diabetic animals. The volume density and the percentage of A-cells appeared increased. However, when quantified per area unit, the number of all the islet cell types diminished, although only the decrease in B-cell number was statistically significant. The decrease in B-cell mass might account for the diabetic state developed in this animal model.

Long-term administration of a sucrose-rich diet to normal rats: relationship between metabolic and hormonal profiles and morphological changes in the endocrine pancreas.

The aim of the present investigation was to study normal rats fed a sucrose-rich diet (SRD) for a prolonged period (up to 30 weeks) (1) to obtain additional data on the hormonal and metabolic patterns induced by this treatment and (2) to provide information on changes taking place in the pancreatic islet cell populations. We found that long-term feeding with a SRD resulted in a steady state of hypertriglyceridemia and hyperglycemia in which insulin levels remained unchanged and unable to compensate for the increased demands of the developing metabolic changes. The endocrine pancreas showed a significant increase of both islet number and B-cell area, as well as changes in the profile of islet cell distribution. However, these changes were not accompanied by an increase in the pancreatic content of immunoreactive insulin (IRI). It may therefore be postulated that the newly emerged B-cell mass has some sort of derangement with the increased insulin demand resulting from insulin resistance induced by the long-term SRD feeding. Thus, feeding a SRD to normal rats may prove to be an attractive animal model for studying the role of environmental nutritional factors in the unsettled issue of the relationship between insulin resistance and relative insulin deficiency. The model might provide key information for understanding the pathophysiology of human diseases such as type II diabetes, dyslipidemia, and a number of entities included in so-called syndrome X.

Sequential morphological changes in pancreatic islets of spontaneously diabetic rats.

This study describes the sequential morphological changes in pancreatic islets from 1-, 6-, and 18-month-old male eSS rats, as compared to aged-matched control animals. Spontaneous diabetes mellitus was confirmed in 6- and 18-month-old eSS rats after an oral glucose tolerance test. Light microscopic immunocytochemical and morphometric techniques were used to study islet-cell populations. The pancreas was normal, and the morphometric methods did not reveal significant changes in islets from 1-month-old rats. However, 6-month-old eSS animals showed disruption of islet architecture and fibrosis in the stroma. The volume density (Vvi) of endocrine tissue and the Vvi and percentage of B cells were increased, whereas the Vvi of exocrine tissue and the Vvi and percentage of A cells were diminished. Eighteen-month-old eSS rats also exhibited conspicuous islet lesions. Nevertheless, the Vvi of endocrine tissue and the Vvi and percentage of B cells were diminished, while the Vvi of exocrine tissue and the Vvi and percentage of D cells were increased. Our results provide further quantitative evidence for the sequential morphological events occurring in the pancreatic islets of a useful animal model of diabetes mellitus.

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice.

The nonobese diabetic (NOD) mouse is an animal model that shares a number of clinical, genetic, and immunologic characteristics with human insulin-dependent diabetes mellitus. Since little is known about the morphometric cell profiles in the endocrine pancreas of these NOD animals, it was of interest to assess their changes in morphometry within the pancreatic islet cell types during two stages of this syndrome. Prediabetic (6-week-old) and diabetic (16-week-old) NOD female mice, as well as normal C57BL/6 female mice (15 weeks old), were used. Light microscopic immunocytochemical and morphometric methods were employed to study the endocrine cell populations. The immunoperoxidase technique for the identification of insulin, glucagon, somatostatin, and pancreatic polypeptide, as well as the point-counting method, was used on serial sections of pancreas tissue. Compared to those of normal and prediabetic mice, pancreata from diabetic animals showed a decrease in both the number of islets and the volume density of the endocrine component. Analysis of islet tissue revealed a significant diminution of B-cell volume density, as well as an increased A-, D-, and PP-cell volume density. A parallel variation in the number of B and non-B cells was also found. In addition, when the total pancreatic tissue surface was taken as reference, the fractional area occupied by all the different types of islet cells was seen to be diminished in a variable fashion. We conclude that the diabetic syndrome of NOD mice not only severely affects the B-cell mass, but also causes marked changes in the non-B endocrine-cell populations.

Postnatal sequential changes in islet morphology and insulin secretion of normal hamsters.

We have studied the postnatal development of the endocrine pancreas from normal female Syrian golden hamsters 1, 8, and 24 weeks of age. The observations were made by (a) analysis of insulin secretion in response to glucose using isolated pancreatic islets and (b) identification and quantitation of insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-secreting cells. Glucose-induced insulin secretion showed typical dose-response curves. However, whereas in 24-week-old animals maximal secretion was already present with 8 mM glucose, in younger hamsters such a response was attained only with 20 mM glucose. The volume density of the endocrine pancreas and the number of islets were increased in 1-week-old hamsters compared to the older animals. The islet volume average in 8-week-old hamsters was almost three times higher than that measured in 1-week-old animals. However, the proportion and size of each cell type in the islets did not present significant differences among the groups studied. Our results show that, in hamsters, the endocrine pancreas reaches the adult general characteristics late after birth. Furthermore, the definite morphological pattern is attained far earlier than the secretory response. These observations provide basic information for further studies regarding the mechanisms and factors that control both the growth and the differentiation of endocrine cell populations as well as glucose-induced insulin secretion in a simple experimental model.

Morphometric and ultrastructural analysis of different pituitary cell populations in undernourished monkeys.

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20% (control group) and a 10% (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings.

Ammonium thiocyanate for detaching antibodies from histological specimens.

When different antigens must be demonstrated in the same structure, the permanence of former antibodies can lead to false identification of another antigen. The peroxidase-antiperoxidase method was used, followed by the oxygen acceptor ethyl-carbazole. After staining the sections, they were destained with xylene and the antibodies detached with 3 M ammonium thiocyanate; then the specimens were treated for the demonstration of the other antigen. The procedure could be repeated and thus as many as four antigens could be demonstrated without damaging the tissues. Antigens participating in the immunohistochemical staining were well-preserved after destaining and detaching the antibodies as demonstrated by their ability to react again in a second staining.

Immunocytochemical detection of bromodeoxyuridine in tissues of the toad Bufo arenarum Hensel.

We present a modified protocol for the immunocytochemical identification of 5-bromo-2'-deoxyuridine (BrdU) as an indicator of cell replication in different tissues of the toad, Bufo arenarum Hensel. Animals were sacrificed 60 min after BrdU (5 mg/100 g body weight) was injected into the dorsal lymph sac. The tissues were fixed in Carnoy's fluid and stained by the immunoperoxidase method using an anti-BrdU monoclonal antibody. This protocol can be used safely for the study of cell replication in toads and other Anura amphibia.

Quantitative ultrastructural changes induced by sucrose administration in the pancreatic B cells of normal hamsters.

We have previously reported that young male Syrian hamsters receiving a sucrose-rich diet presented increased B-cell replication rate and size. The aim of the present study was to analyze, under the same experimental conditions, the ultrastructural changes in B cells. For this purpose, young male Syrian hamsters were fed with a commercial diet and 10% sucrose in their drinking water (S group) while the control group (C) received the same diet and tap water, for 5 weeks. Samples of the pancreas removed after that period were processed for the immunohistochemical identification of B cells as well as for measuring several ultrastructural parameters. S hamsters showed higher serum insulin levels, while similar serum glucose values were obtained in animals from both groups. The B cells from S group exhibited lesser number of dense secretory granules at expenses of an increase of the pale ones, increased number of both exocytosis profiles and fusion-granule images, as well as enlargement of the intercellular space and mitochondrial area. Marked expansions of this space, limited by junctional complexes, were observed between adjacent B cells. These results would indicate that sucrose administration to normal hamsters not only increases the pancreatic B-cell mass but also induces measurable subcellular changes in the individual B-cell characteristic of an enhanced secretory activity. The present model would represent a useful tool for testing strategies in preventing the damage or promoting the recovery of the pancreatic B cells.

Immunohistochemical and ultrastructural age-related changes in rat lactotroph cells.

Ageing produces alterations in some functions of the hypothalamo-pituitary axis leading to sexually dimorphic changes in the prolactin (PRL)-secreting cells. Since quantitative morphological data of these age-associated alterations are scarce, we carried out a morphometric immunohistochemical assessment as well as an ultrastructural study of the PRL cell population in male and female rats of different ages. Young (3-month-old), old (20-month-old), and senescent (31-month-old) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries immediately dissected out and processed for both immunohistochemistry and electron microscopy. Analysis of different morphometric parameters revealed that the cell density (CD) and volume density (VD) significantly decreased with age in male rats. In females, while CD showed a significant age-related diminution when young rats were compared to old ones, this parameter increased in senescent animals. The VD presented higher values in senescent rats. When the data were compared between sexes, VD was found to be higher in females if old and senescent rats were considered. Finally, CD increased significantly in females when compared to males. The ultrastructure of the PRL cells from old and senescent animals of both sexes exhibited changes suggestive of an hyperstimulation state, with some prolactotrophs having the appearance of cells undergoing an involutive process. We conclude that ageing has a differential impact on the PRL cells of male and female rats with respect to the immunohistochemical and ultrastructural features of that cell population.

Histomorphological and quantitative immunohistochemical changes in the rat pancreas during aging.

Although the endocrine pancreas is the purpose of several deep investigations, morphological data referred to the effect of aging on the gland are not homogeneous. The purpose of the current work was to analyze the changes occurring in the pancreas of aged rats, with especial reference to the islet cell populations. Six young (Y), old (O) and senescent (S) male Sprague-Dawley rats were used. The pancreas tails were processed for light microscopy and studied by means of routine stains as well as by immunohistochemical identification of insulin-, glucagon-, somatostatin-, and pancreatic polypeptide- secreting cells (Dako Envision System, DAB as chromogen). A progressive pancreatic histoarchitecture distortion was found among the aged animals. Even when the alterations were not uniformly observed, they appeared more evident and severe in the S group. The S rats showed significantly increased volume density and cell density of the B cell population, as well as larger number of islet profiles, when compared to O rats. A significant progressive increment of adipose tissue was also evident in aged animals. No abnormal changes were detected in the non-B cell populations of the different groups. The quantitative changes found in aged animals suggest a possible compensatory reaction of the B cell population in an attempt to curb the influence of diabetogenic factors mounting with advanced age.

[Structure and ultrastructure of the endocrine pancreas in diabetic transgenic mice]. / Estructura y ultraestructura del páncreas endocrino de ratones transgénicos diabéticos.

The aim of the present study was to confirm the structural changes and to establish the ultrastructural alterations that occur in the endocrine pancreas of mice with an induced insulin-dependent diabetes mellitus (IDDM) syndrome. For that purpose, we used transgenic mice (OVE 27) that overexpress a calmodulin gene in the beta cells of the endocrine pancreas. In these animals, the excess of calmodulin decreases the cytosolic calcium levels in beta cells, leading to morphological and functional alterations that produce a severe IDDM. Sections of pancreas (tail) from 4 male 5-week-old diabetic mice (glycemia: 376 +/- 2 mg/dl) and from 4 normal age-matched males (glycemia: 113 +/- 13 mg/dl) were processed. Light microscopic immunohistochemical observations confirmed a decrease in the number and size of pancreatic islets in transgenic mice, together with a disruption in their architecture, without an associated inflammatory response. The ultrastructural studies revealed diverse degrees of injury in the beta cells, such as the presence of membrane interdigitations and alterations in their organelles and secretory granules. These findings are in agreement with the quantitative and functional impairment of beta cells, coexisting with a normal appearance of non-beta cell populations within the pancreatic islets. Our results demonstrate the existence of ultrastructural changes in the pancreatic beta cells of the experimental model studied. Such changes, together with the immunohistochemical alterations previously described, contribute to explain the appearance of a diabetic syndrome in these animals.