Cholinergic- rather than adrenergic-induced sweating play a role in developing and developed rat eccrine sweat glands.

Both cholinergic and adrenergic stimulation can induce sweat secretion in human eccrine sweat glands, but whether cholinergic and adrenergic stimulation play same roles in rat eccrine sweat glands is still controversial. To explore the innervations, and adrenergic- and cholinergic-induced secretory response in developing and developed rat eccrine sweat glands, rat hind footpads from embryonic day (E) 15.5-20.5, postanal day (P) 1-14, P21 and adult were fixed, embedded, sectioned and subjected to immunofluorescence staining for general fiber marker protein gene product 9.5 (PGP 9.5), adrenergic fiber marker tyrosine hydroxylase (TH) and cholinergic fiber marker vasoactive intestinal peptide (VIP), and cholinergic- and adrenergic-induced sweat secretion was detected at P1-P21 and adult rats by starch-iodine test. The results showed that eccrine sweat gland placodes of SD rats were first appeared at E19.5, and the expression of PGP 9.5 was detected surrounding the sweat gland placodes at E19.5, TH at P7, and VIP at P11. Pilocarpine-induced sweat secretion was first detected at P16 in hind footpads by starch-iodine test. There was no measurable sweating when stimulated by alpha- or beta-adrenergic agonists at all the examined time points. We conclude that rat eccrine sweat glands, just as human eccrine sweat glands, co-express adrenergic and cholinergic fibers, but different from human eccrine sweat glands, cholinergic- rather than adrenergic-induced sweating plays a role in the developing and developed rat eccrine sweat glands.

RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization.

Rats are effective model animals and have contributed to the development of human medicine and basic research. However, the application of reproductive engineering techniques to rats is not as advanced compared with mice, and genome editing in rats has not been achieved using embryos obtained by in vitro fertilization (IVF). In this study, we conducted superovulation, IVF, and knock out and knock in using IVF rat embryos. We found that superovulation effectively occurred in the synchronized oestrus cycle and with anti-inhibin antiserum treatment in immature rats, including the Brown Norway rat, which is a very difficult rat strain to superovulate. Next, we collected superovulated oocytes under anaesthesia, and offspring derived from IVF embryos were obtained from all of the rat strains that we examined. When the tyrosinase gene was targeted by electroporation in these embryos, both alleles were disrupted with 100% efficiency. Furthermore, we conducted long DNA fragment knock in using adeno-associated virus and found that the knock-in litter was obtained with high efficiency (33.3-47.4%). Thus, in this study, we developed methods to allow the simple and efficient production of model rats.

Expression of genome defence protein members in proliferating and quiescent rat male germ cells and the Nuage dynamics.

During epigenetic reprogramming germ cells activate alternative mechanisms to maintain the repression retrotransposons. This mechanism involves the recruitment of genome defence proteins such as MAEL, PIWIL4 and TDRD9, which associate with piRNAs and promote Line-1 silencing. MAEL, PIWIL4 and TDRD9 form the piP-bodies, which organization and dynamics vary according to the stage of germ cell epigenetic reprogramming. Although these data have been well documented in mice, it is not known how this mechanism operates in the rat. Thus, the aim of this study was to describe the distribution and interaction of MAEL, PIWIL4, TDRD9 and DAZL during rat germ cell development and check whether specific localization of these proteins is related to the distribution of Line-1 aggregates. Rat embryo gonads at 15 days post-conception (dpc), 16dpc and 19dpc were submitted to MAEL, PIWIL4, TDRD9 and DAZL immunolabelling. The gonads of 19dpc embryos were submitted to the double-labelling of MAEL/DAZL, TDRD9/MAEL and PIWIL4/MAEL. The 19dpc gonads were submitted to co-immunoprecipitation assays and fluorescent in situ hybridization for Line-1 detection. MAEL and TDRD9 showed very similar localization at all ages, whereas DAZL and PIWIL4 showed specific distribution, with PIWIL4 showing shuttling from the nucleus to the cytoplasm by the end epigenetic reprogramming. In quiescent 19dpc gonocytes all proteins colocalized in a nuage adjacent to the nucleus. DAZL interacts with PIWIL4 and MAEL, suggesting that DAZL acts with these proteins to repress Line-1. TDRD9, however, does not interact with DAZL or MAEL despite their colocalization. Line-1 aggregates were detected predominantly in the nuclear periphery, although did not show homogeneous distribution as observed for the nuage. In conclusion, the nuage in quiescent rat gonocytes show a very distinguished organization that might be related to the organization of Line-1 clusters and describe the association of DAZL with proteins responsible for Line-1 repression.

Optimization of Pulmonary Vasculature Tridimensional Phenotyping in The Rat Fetus.

Comparative, functional, developmental, and some morphological studies on animal anatomy require accurate visualization of three-dimensional structures. Nowadays, several widely applicable methods exist for non-destructive whole-mount imaging of animal tissues. The purpose of this study was to optimize specimen preparation and develop a method for quantitative analysis of the total pulmonary vasculature in fetal rats. Tissues were harvested at E21 and fetuses fixed overnight in 4% paraformaldehyde/phosphate buffered saline. They were treated with 25% Lugol solution for 72 hours to ensure perfusion. Four different methods were used for fetal specimen preparation; isolated lung, upper torso, direct right ventricle contrast injection, and whole body with partial thoracic skin excision. The microCT scan was performed, and pulmonary vasculature was segmented. Vessels were analyzed for diameter, length, and branching. Of the four preparation methods, only whole body with partial thoracic skin excision resulted in adequate reconstruction of the pulmonary vasculature. In silico generated 3D images gathered by micro CT showed pulmonary vasculature distributed throughout the lung, which was representative of the shape and structure of the lungs. The mean number of vessels segmented in the pulmonary tree was 900 ± 24 with a mean diameter of 134.13 µm (range 40.72-265.69 µm). While up to the 30th generation of vessels could be segmented, both for arteries and veins, the majority of branching was between the 21st and 30th generations. Passive diffusion of contrast material enables quantitative analysis of the fetal pulmonary vasculature. This technique is a useful tool to analyze the characteristics and quantify the fetal pulmonary vasculature.

Identification of excitatory-inhibitory links and network topology in large-scale neuronal assemblies from multi-electrode recordings.

Functional-effective connectivity and network topology are nowadays key issues for studying brain physiological functions and pathologies. Inferring neuronal connectivity from electrophysiological recordings presents open challenges and unsolved problems. In this work, we present a cross-correlation based method for reliably estimating not only excitatory but also inhibitory links, by analyzing multi-unit spike activity from large-scale neuronal networks. The method is validated by means of realistic simulations of large-scale neuronal populations. New results related to functional connectivity estimation and network topology identification obtained by experimental electrophysiological recordings from high-density and large-scale (i.e., 4096 electrodes) microtransducer arrays coupled to in vitro neural populations are presented. Specifically, we show that: (i) functional inhibitory connections are accurately identified in in vitro cortical networks, providing that a reasonable firing rate and recording length are achieved; (ii) small-world topology, with scale-free and rich-club features are reliably obtained, on condition that a minimum number of active recording sites are available. The method and procedure can be directly extended and applied to in vivo multi-units brain activity recordings.

Neuroprotective astrocyte-derived insulin/insulin-like growth factor 1 stimulates endocytic processing and extracellular release of neuron-bound Aß oligomers.

Synaptopathy underlying memory deficits in Alzheimer's disease (AD) is increasingly thought to be instigated by toxic oligomers of the amyloid beta peptide (AßOs). Given the long latency and incomplete penetrance of AD dementia with respect to Aß pathology, we hypothesized that factors present in the CNS may physiologically protect neurons from the deleterious impact of AßOs. Here we employed physically separated neuron-astrocyte cocultures to investigate potential non-cell autonomous neuroprotective factors influencing AßO toxicity. Neurons cultivated in the absence of an astrocyte feeder layer showed abundant AßO binding to dendritic processes and associated synapse deterioration. In contrast, neurons in the presence of astrocytes showed markedly reduced AßO binding and synaptopathy. Results identified the protective factors released by astrocytes as insulin and insulin-like growth factor-1 (IGF1). The protective mechanism involved release of newly bound AßOs into the extracellular medium dependent upon trafficking that was sensitive to exosome pathway inhibitors. Delaying insulin treatment led to AßO binding that was no longer releasable. The neuroprotective potential of astrocytes was itself sensitive to chronic AßO exposure, which reduced insulin/IGF1 expression. Our findings support the idea that physiological protection against synaptotoxic AßOs can be mediated by astrocyte-derived insulin/IGF1, but that this protection itself is vulnerable to AßO buildup.

Expression of components of the serotonergic system in the developing rat thymus.

The developing thymus of rat fetuses contains all components of the serotonergic system: receptors, enzymes of synthesis, and membrane transporters. The expression of receptors suggests the possibility of a direct influence of serotonin on thymic development. The presence of tryptophan hydroxylase (the key rate-limiting enzyme of serotonin synthesis) and aromatic l-amino acid decarboxylase indicates the ability of fetal thymic cells to synthesize serotonin. It was shown that the cells of a developing thymus can actively uptake extracellular monoamines. The results of this study suggest different functions of the intrathymic and circulating serotonin pools in the regulation of thymic development.

Quantitative changes in the V2 lateral visual cortex in developing rats with induced protein malnutrition

In this paper we have analyzed the quantitative changes in the lateral visual cortex V2 (V2L) from developing rats with induced isocaloric protein malnutrition that looks like Kwashiorkor condition, as well as the changes induced after refeeding. Using stereological techniques the cortical volume, numerical density and the absolute number of neurons and the average neuronal volume were estimated. The result obtained led us to the following conclusions: i) A significant enlargement in the ratio between brain weight and body weight was detected, disappearing after refeeding. ii) The volume of the cerebral cortex, and V2L was reduced. This was partially reversed by the refeeding at the level of the cerebral cortex, but there was not significant reversal in V2L. iii) The neuronal density was enlarged in V2L. Globally considered, this parameter was unchanged after refeeding. Nevertheless, in those animals refed from post-gestational age P0 the numerical density of neurons was significantly lower than in rats refed from post-gestational age P21. iv) The absolute number of neurons and their average volume was unchanged in V2L. Nevertheless, the average neuronal volume was significantly lower in rats with more than thirty days of postgestational age (>P30) in comparison with the rats with less than thirty days of post-gestational age (greater than or equal to P30). v). No differences regarding sex were observed at the level of the studied parameter

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Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are involved in vasculogenesis during rat embryonic pituitary development.

We have recently shown that cells positive for the paired-related homeobox transcription factors PRRX1 and PRRX2 occur in the rat pituitary, and that they are derived from two different origins: pituitary-derived cells positive for stem cell marker SOX2 and extra-pituitary-derived cells negative for SOX2. In this study, we have further characterized the PRRX1- and PRRX2-positive cells that originate from extra-pituitary cells. Immunohistochemical analyses were performed with specific antibodies against PRRX1 and PRRX2 in order to clarify their roles in pituitary vasculogenesis. PRRX1- and PRRX2-positive cells were found in Atwell's recess and at the periphery of the pituitary on embryonic day 15.5 (E15.5). Several PRRX1-positive cells then invaded the anterior lobe, together with a few PRRX2-positive cells, on E16.5. Some PRRX1-positive cells were also positive for mesenchymal stem cell marker NESTIN. Moreover, some PRRX1/NESTIN double-positive cells showed characteristics of vascular endothelial cells with an Isolectin-B4-binding capacity. PRRX1 co-localized with vascular smooth muscle cell/pericyte marker α-smooth muscle actin in the deep area of Atwell's recess. We confirmed the presence of PRRX2/NESTIN double-positive cells at an entry area in Atwell's recess and at the periphery of the pituitary, but PRRX2 did not co-localize with Isolectin B4 or α-smooth muscle actin. These data suggest that PRRX1- and PRRX2-positive mesenchymal stem/progenitor cells are present at the periphery of the embryonic pituitary and at the entry from Atwell's recess and participate in pituitary vasculogenesis by differentiation into vascular endothelial cells and pericytes, whereas the presence of PRRX2 indicates much higher stemness than PRRX1.

Quantitative analysis of somatosensory cortex development in metatherians and monotremes, with comparison to the laboratory rat.

Metatherians and monotremes are born in an immature state, followed by prolonged nurturing by maternal lactation. Quantitative analysis of isocortical sections held in the collections at the Museum für Naturkunde, Berlin was used to compare the pace of somatosensory cortex development relative to body size and pallial thickness between metatherian groups, monotremes, and the laboratory rat. Analysis indicated that the pace of pallial growth in the monotremes is much lower than that in the metatherians or laboratory rat, with an estimated 8.6-fold increase in parietal cortex thickness between 10 and 100 mm body length, compared to a 10- to 20-fold increase among the metatherians and the rat. It was found that aggregation of cortical plate neurons occurs at similar embryo size in the mammals studied (around 8-14 mm body length) and a similar pallial thickness (around 200 µm), but that proliferative zone involution occurs at a much higher body size and pallial thickness in the monotremes compared to the metatherians and the laboratory rat. The observations suggest that cortical development in the monotremes is slower and subject to different regulatory signals to the therians studied. The slow pace may be related to either generally slower metabolism in monotremes or less efficient nutrient supply to the offspring due to the lack of teats.

Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes.

In this study, we investigated the expression of the pathway, SRF-microRNA-1/microRNA-133a-Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF-microRNA-1/microRNA-133a-Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.

Efficient collection and cryopreservation of embryos in F344 strain inbred rats.

In rats, it is now possible to produce genetically engineered strains, not only as transgenic animals but also using gene knockout techniques. Reproductive technologies have been used as indispensable tools to produce and maintain these novel valuable strains. Although studies for collecting and cryopreserving embryos have been reported using outbred rats, efficient methods have not been established in inbred strains. The F344 inbred strain is important in rat breeding and has been used for the production of transgenic/knockout strains and for genome sequencing. Here we studied the optimal conditions for oocyte collection by induction of superovulation, and the development of embryos after cryopreservation in F344 rats. The response to pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) was examined by injection of 150 IU/kg PMSG + 75 IU/kg hCG or 300 IU/kg PMSG + 300 IU/kg hCG. Superovulation was achieved at high efficiency by an injection of 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, superovulation in this strain showed similar high response as Wistar rats. Of 2-cell embryos cryopreserved by vitrification in a solution containing 10% propylene glycol, 30% ethylene glycol, 20% Percoll and 0.3 M sucrose, more than 90% survived after warming and 32% developed to offspring. However, the freezability of pronuclear stage embryos was extremely low. This study demonstrated that sufficient unfertilized oocytes and embryos can be collected from F344 rats by the induction of superovulation with 150 IU/kg PMSG + 75 IU/kg hCG. Furthermore, cryopreservation of 2-cell embryos using this vitrification protocol can now be applied to maintaining valuable rat strains derived from the F344 inbred strain as genetic resources.

Whole-rat conditional gene knockout via genome editing.

Animal models with genetic modifications under temporal and/or spatial control are invaluable to functional genomics and medical research. Here we report the generation of tissue-specific knockout rats via microinjection of zinc-finger nucleases (ZFNs) into fertilized eggs. We generated rats with loxP-flanked (floxed) alleles and a tyrosine hydroxylase promoter-driven cre allele and demonstrated Cre-dependent gene disruption in vivo. Pronuclear microinjection of ZFNs, shown by our data to be an efficient and rapid method for creating conditional knockout rats, should also be applicable in other species.

Engineering an in-vitro model of rodent cartilage.

OBJECTIVES: The purpose of this study was to identify a cell source, scaffold substrate and culture environment suitable for use in engineering an in-vitro model of rodent cartilage. METHODS: The chondrogenic activity and stability of cells isolated at Day 18 of gestation was assessed under normoxia and hypoxia using a cytokine stimulation assay and gene expression analysis. The ability of the selected cells seeded in fibrous electrospun scaffolds to form cartilaginous tissue during longterm static and dynamic culture was assessed using immunocytochemistry and biochemical analysis. KEY FINDINGS: Rodent fetal chondrocytes appear to have enhanced phenotypic stability compared with other cell sources. Following 16 weeks under static culture, the engineered constructs were found to have greater cellularity and collagen content that native rodent cartilage. CONCLUSIONS: A cell source, scaffold and culture environment have been identified that support the generation of in-vitro rodent cartilage. In future work, cytokine treatment of the engineered tissues will take place to generate in-vitro osteoarthritis models.

Toxicokinetics of domoic acid in the fetal rat.

Domoic acid (DA) is a potent neurotoxin that has both marine wildlife and human health impacts, including developmental effects during prenatal exposure in rodent models. However, little is known regarding DA toxicokinetics in the fetal unit during maternal-fetal transfer. Tissue distribution and toxicokinetics of DA were investigated in pregnant rats and their pups just prior to birth at gestational day 20. Pregnant Sprague Dawley rats were given an intravenous dose of 1.0 mg DA/kg and samples of maternal plasma, fetal plasma, placenta, amniotic fluid and fetal brain were taken at intervals over 24 h. Toxicokinetic parameters were determined using WinNonLin software analysis. Maternal plasma DA log concentration-time curves fit a two compartment pharmacokinetic profile, with alpha and beta half-lives of elimination of 26.9 and 297 min, respectively. Placenta had a C(max) of 752 ng/mL and a terminal half-life of 577 min. Maternal-fetal transfer between the plasma compartments was 31% with a fetal plasma C(max) of 86 ng/mL at 60 min and terminal half-life of 553 min. Amniotic fluid and fetal brain had overall averages of 27±12 ng/mL and 8.12 ng/g, respectively, and did not show evidence of elimination over 24 h. The longer fetal retention of DA, particularly in amniotic fluid, indicates that the fetus may be continually re-exposed during gestation, which could potentially lead to a disease state even at small exposure dose. This has implications for the California sea lions (Zalophus californianus), which exhibit an epilepsy-like disease that arises months after DA producing blooms.

Chorioallantoic and yolk sac placentation in Thrichomys laurentinus (Echimyidae) and the evolution of hystricognath rodents.

The evolutionary history of Hystricognathi is associated with major transformations in their placental system. Data so far indicate that key characters are independent from size dimensions in medium to very large species. To better understand the situation in smaller species, we analyzed placental development in a spiny rat, Thrichomys laurentinus. Fourteen individuals ranging from early implantation to near term were investigated by histology, immunohistochemistry, proliferation activity and electron microscopy. Placentation in Thrichomys revealed major parallels to the guinea pig and other hystricognath rodents with respect to the early and invasive implantation, the process of trophoblast invasion, the internal organization of the labyrinth and the trophospongium as well as the establishment of the complete inverted yolk sac placenta. In contrast to systematically related small-sized species, the placental regionalization in Thrichomys was characterized by a remarkable lobulated structure and associated growing processes. Reverse to former perspectives, these conditions represented ancient character states of hystricognaths. The subplacenta was temporarily supplied by both the maternal and fetal blood systems, a rare condition among hystricognaths. The extraplacental trophoblast originating from the subplacenta was partly proliferative in mid gestation. In conclusion, the presented results indicated that only minor variations occurred in small-sized hystricognath species, independent of their systematic interrelationships. Previous views were supported that placentation in hystricognaths followed an extraordinary stable pattern, although the group had distinct habitats in South America and Africa that were separated 30-40 million years ago.

Efectos del éstres crónico sobre la apoptosis y la proliferación celular en la corteza adrenal de ratas gestantes / Effects of chronic stress on apoptosis and cell proliferation in the adrenal cortex of pregnant rats

La homeostasis de los tejidos que constituyen a los seres vivos es regulada por los procesos de proliferación y apoptosis celular. El modelo de estrés crónico intermitente por inmovilización durante la gestación puede alterar diversos mecanismos que mantienen la homeostasis del organismo, los cuales son motivo de estudios actuales. En estudios previos se comprobó variaciones significativas en el peso de las glándulas adrenales y en los niveles plasmáticos de PRL, estrógenos y CORT. Bajo la hipótesis que el estrés crónico produce modificaciones en los procesos de proliferación y apoptosis en las glándulas adrenales de ratas gestantes, los objetivos fueron cuantificar las células en proliferación y apoptosis mediante la utilización de técnicas inmunocitoquímicas en la corteza adrenal de ratas en la segunda mitad de la gestación, y comprobar las características ultraestructurales del proceso apoptótico con microscopía electrónica de transmisión. Material y Métodos: En condiciones de bioterio controladas, se estudió la glándula adrenal de ratas de 12, 17 y 21 días de gestación, controles (RC) y experimentales (RE) sometidas a un estrés crónico, intermitente, homotípico e intenso. Se usaron técnicas combinadas de inmunomarcación, análisis estereológico y cuantificación de imágenes de cortes alternados para determinar las variaciones del fenómeno apoptótico y proliferativo que presentan las diferentes poblaciones celulares de la corteza adrenal. El tratamiento estadístico se realizó con los Softwares InfoStat y el SAS 9.1. Se aplicó ANOVA de una y tres vías y la distribución de Poisson de un modelo logarítmico lineal con efecto de grupo, tiempo e interacciones. Las diferencias fueron consideradas significativas si p<0.05. Resultados Apoptosis: a. En Ratas Gestantes Controles y Estresadas: 1. El índice apoptótico disminuyó a medida que progresó la gestación

SUMMARY: Homeostasis of tissues is regulated by cellular proliferation and apoptosis processes. Intermittent chronic stress by immobilization during gestation may alter several mechanisms that maintain the organism homeostasis. Those mechanisms are object of several studies. Previous studies using the immobilization model have shown significant variations in adrenal glands weight and plasmatic levels of PRL, estrogens and CORT. Under the hypothesis that chronic stress causes changes in proliferation and apoptosis processes in adrenal glands of gestant rats, the main objectives of this study were: 1) to identify and quantify cell nuclei in proliferation and apoptosis using immunocytochemical techniques in adrenal cortex samples of rats in the second half of gestation and, 2) to check the ultraestructural haracteristics of apoptotic process by means of transmission electronic microscopy. Materials and Methods: In controlled lab conditions, the adrenal glands of control (RC) and experimental (RE) rats at 12, 17 and 21 days of gestation were studied. Experimental rats were subject to an intermittent, chronic, homotypic and intense stress in order to avoid habituation. A combination of labeling techniques, stereological analysis and image quantification of alternate sections was used to determine the changes in proliferation and apoptosis in the different cell populations of the adrenal cortex. Statistical analyses were performed with InfoStat and SAS 9.1 softwares. Data was analyzed with one and three-way ANOVAs and, a linear logarithmic model with Poisson distribution including group, time and interactions effects

Efectos del éstres crónico sobre la apoptosis y la proliferación celular en la corteza adrenal de ratas gestantes / Effects of chronic stress on apoptosis and cell proliferation in the adrenal cortex of pregnant rats

La homeostasis de los tejidos que constituyen a los seres vivos es regulada por los procesos de proliferación y apoptosis celular. El modelo de estrés crónico intermitente por inmovilización durante la gestación puede alterar diversos mecanismos que mantienen la homeostasis del organismo, los cuales son motivo de estudios actuales. En estudios previos se comprobó variaciones significativas en el peso de las glándulas adrenales y en los niveles plasmáticos de PRL, estrógenos y CORT. Bajo la hipótesis que el estrés crónico produce modificaciones en los procesos de proliferación y apoptosis en las glándulas adrenales de ratas gestantes, los objetivos fueron cuantificar las células en proliferación y apoptosis mediante la utilización de técnicas inmunocitoquímicas en la corteza adrenal de ratas en la segunda mitad de la gestación, y comprobar las características ultraestructurales del proceso apoptótico con microscopía electrónica de transmisión. Material y Métodos: En condiciones de bioterio controladas, se estudió la glándula adrenal de ratas de 12, 17 y 21 días de gestación, controles (RC) y experimentales (RE) sometidas a un estrés crónico, intermitente, homotípico e intenso. Se usaron técnicas combinadas de inmunomarcación, análisis estereológico y cuantificación de imágenes de cortes alternados para determinar las variaciones del fenómeno apoptótico y proliferativo que presentan las diferentes poblaciones celulares de la corteza adrenal. El tratamiento estadístico se realizó con los Softwares InfoStat y el SAS 9.1. Se aplicó ANOVA de una y tres vías y la distribución de Poisson de un modelo logarítmico lineal con efecto de grupo, tiempo e interacciones. Las diferencias fueron consideradas significativas si p<0.05. Resultados Apoptosis: a. En Ratas Gestantes Controles y Estresadas: 1. El índice apoptótico disminuyó a medida que progresó la gestación(AU)

SUMMARY: Homeostasis of tissues is regulated by cellular proliferation and apoptosis processes. Intermittent chronic stress by immobilization during gestation may alter several mechanisms that maintain the organism homeostasis. Those mechanisms are object of several studies. Previous studies using the immobilization model have shown significant variations in adrenal glands weight and plasmatic levels of PRL, estrogens and CORT. Under the hypothesis that chronic stress causes changes in proliferation and apoptosis processes in adrenal glands of gestant rats, the main objectives of this study were: 1) to identify and quantify cell nuclei in proliferation and apoptosis using immunocytochemical techniques in adrenal cortex samples of rats in the second half of gestation and, 2) to check the ultraestructural haracteristics of apoptotic process by means of transmission electronic microscopy. Materials and Methods: In controlled lab conditions, the adrenal glands of control (RC) and experimental (RE) rats at 12, 17 and 21 days of gestation were studied. Experimental rats were subject to an intermittent, chronic, homotypic and intense stress in order to avoid habituation. A combination of labeling techniques, stereological analysis and image quantification of alternate sections was used to determine the changes in proliferation and apoptosis in the different cell populations of the adrenal cortex. Statistical analyses were performed with InfoStat and SAS 9.1 softwares. Data was analyzed with one and three-way ANOVAs and, a linear logarithmic model with Poisson distribution including group, time and interactions effects(AU)