Biomedical subjects

S Carbajo

Publications and source records attributed to S Carbajo.

At least 19 recordsLinked to original sources

PubMed1998

Severe experimental uraemia does not decrease the population of rat pituitary somatotrophs.

BACKGROUND: Growth hormone (GH) secretion by the anterior pituitary has been shown to be depressed in severely uraemic rats. Changes in the population of pituitary somatotrophs might be partially responsible for this decrease. METHODS: To analyse the population of pituitary somatotrophs in severe uraemia, immunocytochemical detection and quantification of GH-producing cells were carried out on paraffin sections from young rats either 5/6 nephrectomized, sham-operated fed ad libitum or sham-operated pair-fed with the nephrectomized animals. RESULTS: Nephrectomized rats were severely uraemic and growth retarded. The overall cell density (total pituitary cells/mm2) was higher in 5/6 nephrectomized animals in comparison with the two sham-operated groups. Thus, although the percentage of GH cells was slightly lower in nephrectomized than in control rats, no difference in either the density (cells/mm2) or the cross-sectional area of GH cells was found among groups. CONCLUSIONS: These results suggest that severe experimental uraemia interferes with the maturation process of the pituitary gland and support the contention that differences in either the number or the size of pituitary somatotrophs cannot explain the reduced GH secretion previously reported in severely uraemic rats.

Animals↗

PubMed1998

Cellular proliferation in the rat pineal gland during postnatal development.

To establish a possible correlation between the rate of cellular proliferation and already documented functional and morphological characteristics of the rat pineal gland during postnatal development, the bromodeoxyuridine labelling method was used to evaluate the fraction of cells at the S phase of the cell cycle in paraffin sections from 1-, 7-, 14- and 28-day-old rats. Numerical density, taken as an indirect measure of cell hypertrophy, was also evaluated. During the first week after birth the percentage of S phase-cells in the rat pineal gland sharply decreased from around 9% to 1.3%. A smaller but also significant decrease was found from the 7th to the 14th postnatal day where S phase cells were less than 0.5% of all pineal cells. A very low percentage was also seen in samples from 28-day-old rats. Numerical density, namely, the total number of cells per surface unit of pineal section, decreased from birth to the end of the first month. This decrease was also steeper from birth to the 7th postnatal day than at any other period of the study. These results support the idea that a strong expansion of the cellular population of the rat pineal gland precedes morphological and functional maturation and opens the way to further exploration of the relationship between functional and proliferative responses of the pineal gland.

Animals↗

PubMed1997

Expression of argyrophilic nucleolar organizer regions (AgNORs) in the harderian gland of male and female hamsters during postnatal development.

The expression of argyrophilic nucleolar organizer regions (AgNORs) was studied in the different secretory cell types of the Harderian gland of male and female Syrian hamsters during postnatal development. Mean AgNOR area was calculated for each cell type in paraffin sections from 7-, 14-, 21-, 28-, 45- and 90-day-old animals. AgNOR content was similar in male type I-cells and in female cells, decreasing in both cell types from the 7th to the 14th day, increasing afterwards at the 21st day, and remaining at relatively stable levels from that point to the end of the study. AgNOR content of male type II-cells was greater than in other cell type studied, and was greater in 45- and 90-day-old animals than in 28-day-olds. Changes of AgNOR content in type I-cells of male and female hamsters during the first two weeks seem to be related to changes in proliferative activity while metabolic activity might be responsible for changes taking place later on. Our results also support that male type I- and type II-cells have a different biological behaviour and that type II-cells are far from being degenerating cells.

Age Factors↗

PubMed1996

Comparison of Vindelov et al. and bromodeoxyuridine/DNA double-staining flow cytometry methods for analysis of cell cycle distribution in rat thymocytes.

This study compares the cell cycle distribution in rat thymocytes obtained by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the technique of Vindelov et al. (Cytometry 3:332-338, 1983). The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adult rat thymocytes after in vivo injection of BrdUrd and the results then compared with those obtained after measuring the cell DNA contents according to the Vindelov et al. method. The percentage of BrdUrd-positive cells was greater than the S-phase fraction obtained using the Vindelov et al. technique. By contrast, no major differences were observed between the percentage of BrdUrd-positive cells and the S-phase fraction obtained after analyzing the DNA histograms of the same data files with the RFIT mathematical model. The elimination of trypsin treatment used in the Vindelov et al. method did not alter the results, whereas the use of DNA denaturation with 2N HCl was shown to increase the percentage of S-phase rat thymocytes (calculated from DNA histograms) independently of whether trypsin treatment was used or not. However, the value of the S-phase fraction was not as great as that obtained after BrdUrd labeling. Thus when comparing BrdUrd-labeling and the Vindelov et al. technique, important differences in the percentage of S-phase adult rat thymocytes were observed. Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences.

Animals↗

PubMed1996

New insights into the cytodynamics of the hamster Harderian gland as provided by the bromodeoxyuridine-labelling method.

The fourth week of postnatal life is a critical point in the development of the hamster Harderian gland. During this week, cells with large lipid vacuoles (type-II cells) appear in the male gland, marking a morphological sex difference that is notorious in adult animals. The origin and fate of type-II cells are controversial. To gain insight into the mechanisms by which type-II cells become a major cell type in the gland of adult male hamsters, bromodeoxyuridine (BrdU) labelling was used to assess the proliferative activity of both types of glandular cells in 28-day-old animals. To search for possible sex differences in the proliferative activity of this gland, female animals of the same age as the males were also studied. No difference was found in the overall labelling index (BrdU-labelled cells/100 cells) between males (1.8 +/- 0.1%) and females (1.5 +/- 0.1%). In the gland of the males, the specific labelling index of type-II cells (3.4 +/- 0.4%) was significantly higher than that of type-I cells (0.9 +/- 0.2%). Interestingly, the proportion of type-II cells present in the male glands at this age (36.6%) was significantly lower than that of type-I cells. Our results strongly suggest that the proliferation of type-II cells, rather than a continuous differentiation of these cells from preexisting type-I cells, is a major event in the achievement of the mature form of this gland. The results reported here counsel a reappraisal of current theories about the cytodynamics of the hamster Harderian gland.

Animals↗

PubMed1995

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage.

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4 degrees C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling. Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4 degrees C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriously compromises the results of this technique.

Animals↗

PubMed1995

In vivo bromodeoxyuridine (BrdU)-labelling index of rat thymus: influence of different BrdU doses and exposure times as analyzed both in tissue sections and in single cell suspensions.

Tissue sections and single cell suspensions of rat thymus were analyzed by flow and image cytometry to study the effects of different doses of bromodeoxyuridine (BrdU) (10, 50 and 100 mg/kg and different labelling periods (15, 30 and 60 min) on the BrdU-labelling index. The proportions of BrdU-labelled cells, as analyzed by flow cytometry, were similar in the different groups of our study. However, the quality of the results was not exactly the same, since when the BrdU dose decreased, the separation between BrdU-positive S-phase cells and both the G0/G1 and the G2/M BrdU-negative cells on a DNA/BrdU histogram became less clear. On the other hand, the proportion of BrdU-labelled cells in tissue sections was greater in the groups of animals that received doses of 100 mg/kg and were killed 15, 30 or 60 min later than in the sections obtained from the remaining groups. Our results show that both dose and exposure time to BrdU may influence the final results when cell proliferation is assessed, the variations obtained clearly depending on the technique used for the immunological detection of BrdU-positive S-phase cells.

Animals↗

PubMed1994

Influence of luteinizing hormone-releasing hormone (LHRH) treatment on cellular proliferation in the rat anterior pituitary.

This study was designed to gain insight into the action of LHRH on the control of cellular proliferation in the anterior pituitary. The fraction of cells labelled with bromodeoxyuridine (S-phase cells) was studied in cytospin preparations of anterior pituitary cells taken from control male and female rats and from rats treated with daily doses of 2 micrograms/100 g body weight of LHRH (7 days), with doses of 40 micrograms of LHRH given on alternate days for 14 days (7 doses) or, finally, treated with daily doses of 50 ng of busereline acetate (14 days). Treatment with LHRH for 14 days resulted in a significant increase in the fraction of S-phase cells. However, neither the blockade of gonadotrophin secretion with busereline acetate nor its stimulation with LHRH for seven days resulted in a significant change in the proliferative activity of anterior pituitary cells. This action was independent of sex. No significant changes were seen in the proportions of pituitary gonadotrops of the different study groups. Regardless of the treatment-group very few cells doubly-immunostained for BrdU and LH were found. It is concluded that LHRH may stimulate cellular proliferation in the anterior pituitary, but further studies are needed to define which cells are involved in this action.

Animals↗

PubMed1994

Evaluation of cellular proliferation in human biopsy samples of lymphoid tissue according to the expression of AgNORs.

The proportions of cells with a high expression of AgNORs, as directly assessed under the light microscope, and those of Ki-67-immunoreactive cells were divided into three categories (low, < 25%; intermediate, 25-50%; high, > 50%) and compared to a series of 41 human biopsy samples of lymphoid tissue. Irrespective of histological features a strong correlation was found between both variables (contingency coefficient = 0.8; P < 0.001). It is concluded that direct evaluation of cells with a high expression of AgNORs may serve as an index of proliferative activity in biopsy samples of lymphoid tissue.

Biopsy↗

PubMed1993

Expression of silver-stained nucleolar organizer regions is coupled to cell cycle in rat thymic cells.

To directly analyze the relationship between the expression of silver-stained nucleolar organizer regions (AgNORs) and cell proliferation, thymic cells from newborn rats were separately sorted at the G0-G1-phase and early-mid and late-mid S-phase of the cell cycle according to their DNA content. Different AgNOR-derived parameters (mean area and numbers of AgNORs per cell and mean AgNOR-particle area) were evaluated after silver staining of cytospins. A linear correlation was observed between the mean area and numbers of AgNORs per cell, both parameters increasing progressively from G0-G1-phase to early-mid and late-mid S-phase. An increase of the mean AgNOR-particle area was also seen between G0-G1 and S-phase but this was not significant along the S-phase. A bias on the selection of S-phase cells linked to intrathymic maturation can be ruled out as S-phase cells labeled with bromodeoxyuridine were found throughout the thymus, and, moreover, the analysis of the frequency distribution of nuclear area did not show a bimodal pattern. It is concluded that the expression of AgNORs--evaluated as AgNOR area/cell or AgNOR numbers/cell--is causally or indirectly coupled to DNA synthesis and, thus, AgNORs can be considered as a cell proliferation marker.

Animals↗

PubMed1993

Expression of argyrophilic nucleolar organizer regions (AgNORs) can be used to assess cellular proliferation as shown in rat thymic sections.

Silver-stained nucleolar organizer regions (AgNORs) were studied in thymic sections from 4- and 30-day-old rats. By direct examination under the light microscope cells with a low or high content of AgNORs (type I and type II cells, respectively) were identified and their relative numbers calculated. The mean area of AgNORs per cell was calculated for each type of cell and age group. Additionally, the proportion of cells labelled with bromodeoxyuridine was calculated in sections from the same animals. Visual identification of type I and type II cells was confirmed by a significant (p < 0.01) difference in the mean AgNOR area in both types of cell. Both the proportion of cells with a high expression of AgNORs (type II cells) and that of bromodeoxyuridine-labelled cells were significantly greater (p < 0.01) in 4-day-old rats than in 30-day-old rats. A significant correlation was found between both variables (R2 = 0.45; p = 0.002), the relation being best between both variables and age (R2 = 0.91; p = < 0.001). These data offer support for an easy interpretation of the AgNOR reaction in which the proportion of cells with a high expression of AgNORs can be used as an index of proliferative activity in a tissue sample.

Animals↗

PubMed1993

Cellular proliferation and/or differentiation can condition modifications in the expression of AgNORs in rat anterior pituitary cells during growth.

The expression of argyrophilic nucleolar organizer regions (AgNORs) was studied in the anterior lobe of the hypophysis of 1-, 7-, 14-, 28- and 80-day-old rats. Mean area and numbers of AgNORs per nucleus decreased during this period with a simultaneous increase of both nuclear area and mean AgNOR-particle area. Comparison of our data on AgNOR expression with previously reported data on the proliferative activity and cell maturation and differentiation of anterior pituitary cells during a similar period led us to conclude that, at least in our experimental model, variations in the expression of AgNORs are more related to phenomena of cellular maturation than to proliferative activity.

Aging↗

PubMed1992

Quantification of the cellular proliferation on freshly dispersed cells from rat anterior pituitaries after in vivo and in vitro labelling with bromodeoxyuridine.

The labelling index i.e., the proportion of cells in S phase of the cell cycle, has been calculated in cytospin preparations of rat anterior pituitary cells after labelling either in vivo or in vitro with the thymidine analogue bromodeoxyuridine (BrdU). The aims of this work were (1) to check whether enzymatic digestion interferes with the incorporation of BrdU into S phase cells and/or whether it has any deletereous effect on the immunohistochemical detection of cells that have already incorporated BrdU, and (2) to check the viability of simultaneous staining for BrdU and markers for the different types of pituitary cells in the cytospins. No statistical difference was found between the labelling index after in vivo or in vitro labelling with BrdU. Identification of doubly-immunostained cells was straightforward and up to 40% of BrdU-labelled cells were immunopositive for pituitary hormones. It is suggested that cytospin preparations from biopsy samples may be used to study cellular proliferation without exposing the patient to the hazardous effects of BrdU infusion and without the interference of cell culture methods.

Animals↗

PubMed1992

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.

Animals↗

PubMed1991

Circadian variation in the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male rats as analysed by flow cytometry.

Flow cytometric analysis of nuclei stained with propidium iodide (PI) has been used to study the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male Sprague-Dawley rats at different times of the day. According to PI fluorescence intensity the relative numbers of cells in S phase (cells with a DNA content between that of somatic cells in interphase (2n) and that of somatic cells after duplication of the DNA prior to cell division (4n] and G2/M phase (4n) were calculated. A significant circadian rhythm was found for cells in both the S phase (P less than 0.05) and the G2/M phase (P less than 0.01). The wave of cells in S phase with a peak at the middle of the light period (14.00 h) precedes by about 6 h the wave of cells in G2/M phase (peak at 20.00 h). Most of the DNA-replicating cells were found during the early S phase at 11.00 h, advancing further up to the middle of this phase at 14.00 h. Cells were distributed homogeneously throughout the S phase at 17.00 h. These data strongly suggest that the beginning of the light period triggers a wave of cells to leave G0/G1 into S phase.

Animals↗

PubMed1991

Immunocytochemical study of the epithelial lining of naturally occurring cysts in the rat intermediate lobe.

An immunocytochemical study of the epithelial lining of naturally occurring cysts in the rat intermediate lobe (IL) has been carried out. Paraffin-embedded sections, in which cysts were identifiable, were treated either with anti-serotonin or anti-S-100 protein sera. S-100-positive cells were intermingled with glandular cells surrounding the cyst lumen. These S-100-positive cells sent slender cytoplasmic processes as if to cover the apical surface of neighbouring cells. Rarely were 5-HT-immunopositive cells seen in the cyst epithelial lining. Most cells of the marginal layer of the IL were found reactive either to an S-100 or a-5-HT serum. The presence of an epithelial lining positive to S-100 protein sera is in keeping with the notion that cysts in the IL might form as evaginations of the epithelial lining of the pituitary cleft. The lack of correspondence between 5-HT-positive cells in the marginal layer and the cyst lining is controversial. A peculiar spatial relationship of 5-HT cells with the vascular network of the IL is suggested.

Animals↗

PubMed1991

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.

Animals↗

PubMed1989

[Distribution of the vascular network of the adenohypophysis of the rat].

From various experiments it has been suggested that the blood flow to the different parts of the adenohypophysis is not the same. By developing the peroxidase activity of the red and white blood cells we have obtained a satisfactory image of the distribution of the intrahypophysial vessels, which indicate that the organization and distribution of these vessels varies from one portion to another in the pars distalis. On the lateral portion of the gland vascular routes are irregular, whilst in the media zone they run in a sagittal plane. Between the above-mentioned portions, on either side of the edge of the pars intermedia, a rich vascular network extending along the dorsal limit, which is fed by the long portal vessels and by numerous smaller vessels from the ventral vascular chain of the pars intermedia and neural lobe, is observed.

Animals↗