Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21% of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 µg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets

Aversive stimulation during the stress-hyporesponsive period does not affect the number of corticotroph cells in neonatal male rats

Immunohistochemistry was used to evaluate the effects of neonatal handling and aversive stimulation during the first 10 days of life on the number of corticotrophs in the anterior lobe of the pituitary of 11-day-old male Wistar rats. Since adult rats handled during infancy respond with reduced corticosterone secretion in response to stressors and with less behavior inhibition in novel environments, we assumed that neonatal stimulation could affect pituitary morphology during this critical period of cell differentiation. Three groups of animals were studied: intact (no manipulation, N = 5), handled (N = 5) and stimulated (submitted to 3 different aversive stimuli, N = 5). The percentage of ACTH-immunoreactive cells in the anterior lobe of the pituitary (number of ACTH-stained cells divided by total number of cells) was determined by examining three slices per pituitary in which a minimum of 200 cells were counted by two independent researchers. Although animals during the neonatal period are less reactive to stress-like stimulation in terms of ACTH and corticosterone secretion, results showed that the relative number of ACTH-stained cells of neonatal handled (0.25 ± 0.01) and aversive stimulated (0.29 ± 0.03) rats was not significantly different from intact (0.30 ± 0.03) animals. Neonatal stimulation may have a differential effect on the various subpopulations of corticotroph cells in the anterior pituitary

Veratridine increases the survival of retinal ganglion cells in vitro

Neuronal cell death is an important phenomenon involving many biochemical pathways. This degenerative event has been studied to understand how the cells activate the mechanisms that lead to self-destruction. Target cells and afferent cells play a relevant role in the regulation of natural cell death. We studied the effect of veratridine (1.5, 3.0, 4.5 and 6.0 µM) on the survival of neonatal rat retinal ganglion cells in vitro. Veratridine (3.0 µM), a well-known depolarizing agent that opens the Na+ channel, promoted a two-fold increase in the survival of retinal ganglion cells kept in culture for 48 h. This effect was dose-dependent and was blocked by 1.0 µM tetrodotoxin (a classical voltage-dependent Na+ channel blocker) and 30.0 µM flunarizine (a Na+ and Ca2+ channel blocker). These results indicate that electrical activity is also important for the maintenance of retinal ganglion cell survival in vitro

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Histopathological diagnosis of intra- and extrahepatic neonatal cholestasis

The histopathology of the liver is fundamental for the differential diagnosis between intra- and extrahepatic causes of neonatal cholestasis. However, histopathological findings may overlap and there is disagreement among authors concerning those which could discriminate between intra- and extrahepatic cholestasis. Forty-six liver biopsies (35 wedge biopsies and 11 percutaneous biopsies) and one specimen from a postmortem examination, all from patients hospitalized for neonatal cholestasis in the Pediatrics Service of Hospital de Clínicas de Porto Alegre, were prospectively studied using a specially designed histopathological protocol. At least 4 of 5 different stains were used, and 46 hepatic histopathological variables related to the differential diagnosis of neonatal cholestasis were studied. The findings were scored for severity on a scale from 0 to 4. Sections which showed less than 3 portal spaces were excluded from the study. Sections were examined by a pathologist who was unaware of the final diagnosis of each case. Bile tract permeability was defined by scintigraphy of the bile ducts and operative cholangiography. The F test and discriminant analysis were used as statistical methods for the study of the hepatic histopathological variables. The chi-square method with Yates correction was used to relate the age of the patients on the date of the histopathological study to the discriminatory variables between intra- and extrahepatic cholestasis selected by the discriminant function test. The most valuable hepatic histopathological variables for the discrimination between intra- and extrahepatic cholestasis, in decreasing order of importance, were periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, foci of myeloid metaplasia, and portal-portal bridges. The only variable which pointed to the diagnosis of intrahepatic cholestasis was myeloid metaplasia. Due to the small number of patients who were younger than 60 days on the date of the histopathological study (N = 6), no variable discriminated between intra- and extrahepatic cholestasis before the age of 2 months and all of them, except for the portal expansion, were discriminatory after this age. In infants with cholestasis, foci of myeloid metaplasia, whenever present in the liver biopsy, suggested intrahepatic cholestasis. Periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis and portal-portal bridges suggested extrahepatic obstructive cholestasis.

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

Dialysis, time and death: comparisons of two consecutive decades among patients treated at the same Brazilian dialysis center

The survival of hemodialysis patients is likely to be influenced not only by well-known risk factors like age and comorbidity, but also by changes in dialysis technology and practices accumulated along time. We compared the survival curves, dialysis routines and some risk factors of two groups of patients admitted to a Brazilian maintenance hemodialysis program during two consecutive decades: March 1977 to December 1986 (group 1, N = 162) and January 1987 to June 1997 (group 2, N = 237). The median treatment time was 22 months (range 1-198). Survival curves were constructed using the Kaplan-Meier method and compared using the log-rank method. The Cox proportional hazard regression model was used to investigate the more important variables associated with outcome. The most important changes in dialysis routine and in patient care during the total period of observation were the progressive increase in the dose of dialysis delivered, the prohibition of potassium-free dialysate, the use of bicarbonate as a buffer and the upgrading of the dialysis equipment. There were no significant differences between the survival curves of the two groups. Survival rates at 1, 5 and 10 years were 84, 53 and 29%, respectively, for group 1 and 77, 42 and 21% for group 2. Patients in group 1 were younger (45.5 ± 15.2 vs 55.2 ± 15.9 years, P<0.001) and had a lower prevalence of diabetes (11.1 vs 27.4%, P<0.001) and of cardiovascular disease (9.3 vs 20.7%, P<0.001). According to the Cox multivariate model, only age (hazard ratio (HR) 1.04, confidence interval (CI) 1.03-1.05, P<0.001) and diabetes (HR 2.55, CI 1.82-3.58, P<0.001) were independent predictors of mortality for the whole group. Patients of group 2 had a lower prevalence of sudden death (19.1 vs 9.7%, P<0.001). After adjusting for age, diabetes and other mortality risk factors, the risk of death was 17% lower in group 2, although this difference was not statistically significant. We conclude that the negative effects of advanced age and of higher frequency of comorbidity on the survival of group 2 patients were probably offset by improvements in patient care and in the quality and dose of dialysis delivered, so that the survival curves did not undergo significant changes along time.

Galectin-1, an alternative signal for T cell death, is increased in activated macrophages

Galectin-1 belongs to an evolutionarily conserved family of animal ß-galactoside-binding proteins, which exert their functions by crosslinking the oligosaccharides of specific glycoconjugate ligands. During the past decade, attempts to identify the functional role of galectin-1 suggested participation in the regulation of the immune response. Only in the last few years has the molecular mechanism involved in these properties been clearly elucidated, revealing a critical role for galectin-1 as an alternative signal in the generation of T cell death. In the present study we will discuss the latest advances in galectin research in the context of the regulation of the immune response, not only at the central level but also at the periphery. Moreover, we will review the purification, biochemical properties and functional significance of a novel galectin-1-like protein from activated rat macrophages, whose expression is differentially regulated according to the activation state of the cells. The novel role of a carbohydrate-binding protein in the regulation of apoptosis is providing a breakthrough in galectin research and extending the interface between immunology, glycobiology and clinical medicine.

The regulation of apoptotic cell death

Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment). Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement). The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution). Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial).

Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

Implications of ischemic penumbra for the diagnosis of brain death

The data reviewed here suggest the possibility that a global reduction of blood supply to the whole brain or solely to the infratentorial structures down to the range of ischemic penumbra for several hours or a few days may lead to misdiagnosis of irreversible brain or brain stem damage in a subset of deeply comatose patients with cephalic areflexia. The following proposals are advanced: 1) the lack of any set of clinically detectable brain functions does not provide a safe diagnosis of brain or brain stem death; 2) apnea testing may induce irreversible brain damage and should be abandoned; 3) moderate hypothermia, antipyresis, prevention of arterial hypotension, and occasionally intra-arterial thrombolysis may contribute to good recovery of a possibly large subset of cases of brain injury currently regarded as irreversible; 4) confirmatory tests for brain death should not replace or delay the administration of potentially effective therapeutic measures; 5) in order to validate confirmatory tests, further research is needed to relate their results to specific levels of blood supply to the brain. The current criteria for the diagnosis of brain death should be revised.

Detection of early apoptosis and cell death in T CD4+ and CD8+ cells from lesions of patients with localized cutaneous leishmaniasis

Human localized cutaneous leishmaniasis (LCL), induced by Leishmania braziliensis, ranges from a clinically mild, self-healing disease with localized cutaneous lesions to severe forms which can present secondary metastatic lesions. The T cell-mediated immune response is extremely important to define the outcome of the disease; however, the underlying mechanisms involved are not fully understood. A flow cytometric analysis of incorporation of 7-amino actinomycin D and CD4+ or CD8+ T cell surface phenotyping was used to determine whether different frequencies of early apoptosis or accidental cell death occur at different stages of LCL lesions. When all cells obtained from a biopsy sample were analyzed, larger numbers of early apoptotic and dead cells were observed in lesions from patients with active disease (mean = 39.5 ± 2.7%) as compared with lesions undergoing spontaneous healing (mean = 17.8 ± 2.2%). Cells displaying normal viability patterns obtained from active LCL lesions showed higher numbers of early apoptotic events among CD8+ than among CD4+ T cells (mean = 28.5 ± 3.8 and 15.3 ± 3.0%, respectively). The higher frequency of cell death events in CD8+ T cells from patients with LCL may be associated with an active form of the disease. In addition, low frequencies of early apoptotic events among the CD8+ T cells were observed in two patients with self-healing lesions. Although the number of patients in the latter group was small, it is possible to speculate that, during the immune response, differences in apoptotic events in CD4+ and CD8+ T cell subsets could be responsible for controlling the CD4/CD8 ratio, thus leading to healing or maintenance of disease.

Stimulatory effect of dexamethasone on angiotensin-converting enzyme in neonatal rat cardiac myocytes

Angiotensin-converting enzyme (ACE) plays a central role in cardiac remodeling associated with pathological conditions such as myocardial infarction. The existence of different cell types in the heart expressing components of the renin-angiotensin system makes it difficult to evaluate their relative role under physiological and pathological conditions. Since myocytes are the predominant cellular constituent of the heart by mass, in the present study we studied the effects of glucocorticoids on ACE activity using well-defined cultures of neonatal rat cardiac myocytes. Under steady-state conditions, ACE activity was present at very low levels, but after dexamethasone treatment ACE activity increased significantly (100 nmol/l after 24 h) in a time-dependent fashion. These results demonstrate the influence of dexamethasone on ACE activity in rat cardiac myocytes. This is consistent with the idea that ACE activation occurs under stress conditions, such as myocardial infarction, in which glucocorticoid levels may increase approximately 50-fold.

Reduction of intraspecific aggression in adult rats by neonatal treatment with a selective serotonin reuptake inhibitor

Most studies suggest that serotonin exerts an inhibitory control on the aggression process. According to experimental evidence, this amine also influences growth and development of the nervous tissue including serotoninergic neurons. Thus, the possibility exists that increased serotonin availability in young animals facilitates a long-lasting effect on aggressive responses. The present study aimed to investigate the aggressive behavior of adult rats (90-120 days) treated from the 1st to the 19th postnatal day with citalopram (CIT), a selective serotonin reuptake inhibitor (20 mg/kg, sc, every 3 days). Aggressive behavior was induced by placing a pair of rats (matched by weight) in a box (20 x 20 x 20 cm), and submitting them to a 20-min session of electric footshocks (five 1.6-mA - 2-s current pulses, separated by a 4-min intershock interval). When compared to the control group (rats treated for the same period with equivalent volumes of saline solution), the CIT group presented a 41.4% reduction in the duration of aggressive response. The results indicate that the repeated administration of CIT early in life reduces the aggressive behavior in adulthood and suggest that the increased brain serotoninergic activity could play a role in this effect.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively), paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9) or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.