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.

Mecanismo de producción del aumento de la masa de células B inducido por la administración crónica de sacarosa a ratas normales / Mechanisms producing an increase in B-cell mass induced by the chronic administration of sucrose to normal rats

El objetivo del presente trabajo fue clarificar los mecanismos mediante los cuales una dieta rica en sacarosa produce, en el páncreas de la rata, un aumento de la masa celular B. Se utilizaron ratas Wistar normales, alimentadas durante 30 semanas con una dieta rica en sacarosa (DRS; 63 por ciento o bien con una dieta similar en la que la sacarosa se reemplazó por la misma proporción de almidón (DC). Se estudió el peso corporal, los niveles séricos de glucosa y triacilglicerol, el tejido endocrino, la masa celular B, el grado de replicación de las células B (antígeno de proliferación nuclear celular, PCNA), la neogénesis insular (inmunomarcación de citoqueratina, CK) y la apoptosis de células B (yoduro de propidio). El peso corporal de las ratas DRS fue significativamente mayor (p<0,05) que el registrado en el grupo DC (556,0ñ8,3 vs 470,0ñ13,1). Tanto los niveles séricos de glucosa como los de triacilglicerol (mmol/L) fueron significativamente mayores (p<0,05) en las ratas DRS que en las DC: Glucemia 8,11 ñ 0,14 vs 6,62 ñ 0,17; triacilglicerol 1,57 ñ 0,18 vs 0,47 ñ 0,04. El número de islotes pancreáticos por unidad de área aumentó significativamente (p<0,05) en las ratas DRS (3,29 ñ 0,1 vs 2,01 ñ 0,2). Además, se detectó un incremento significativo (2,6 veces) en la masa de tejido endocrino de los animales DRS, principalmente debido a un aumento en la masa celular B (p=0,0025. El porcentaje de células B insulares en replicación (PCNA positivas) aumentó 6,8 veces en las ratas DRS (p<0,03). El número de células apoptóticas del páncreas endocrino disminuyó significativamente en el grupo DRS (3 veces, p=0,03). No hubo diferencias significativas en el área positiva para CK entre los animales DRS y DC. El aumento de la masa celular B inducido por la DRS se correspondió con un aumento en la replicación de las células B, junto con una disminución en la proporción de dichas células en apoptosis. No hubo evidencias de neogénesisen los islotes. Dichos cambios pancreáticos no pudieron mantener la glucemia de estos animales en valores semejantes a los obtenidos en las ratas DC. Estos resultados muestran que en este modelo de manipulación alimentaria, aunque el aumento de replicación de las células B y la disminución de la apoptosis jugarían un rol importante, la neogénesis insular constituiría el mecanismo compensador decisivo en la adaptación a la mayor demanda de insulina

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 percent (control group) and a 10 percent (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

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

Non enzymatic liver cells isolation: citrate-perchlorate technique

In the present study we describe a non-enzymatic technique for the isolation of rat hepatocytes by perfusion of liver through portal vein. The perfusion media consist of 1 mM sodium percholorate. 5 mM sodium citrate, 10 mM glucose, 129 mM NaCl and 0.1 per cent bovine-serum albumin at pH 7.4. After purification through diatrizoate gradient, electron microscopical studies revealed that most of purified hepatocytes were well preserved and presented a normal ultrastructure, thus correlating with previous biochemical results. The present method enables the recovery of metabolically and morphologically normal hepatocytes.