Genomic effects of a nanostructured alumina insecticide in human peripheral blood lymphocytes in vitro.

Nanotechnology is providing new tools for precision agriculture, such as agrochemical agents and innovative delivery mechanisms to improve cropping efficiency. Powder nanoinsecticides, such as experimental nanostructured alumina (NSA), show great potential for sustainable agriculture as an alternative to conventional synthetic pesticides because their mechanism of insecticide action is based on physical rather than on biochemical phenomena. However, even in highly non-reactive and hardly soluble substances such as alumina, reduced particle size may lead to an increased toxicity of the material. In order to determine whether NSA induces DNA and chromosomal damage, its toxicity was assessed in human peripheral blood lymphocytes (PBL) and contrasted with commercial nanostructured alumina, natural insecticide powders and a conventional pesticide. PBL from healthy donors were exposed for 24 h to increasing concentrations (50, 100 and 200 µg/mL) of NSA particle agglomerates (<350 nm); positive and negative NSA-particles, respectively; bulk Al2O3 (4.5 µm) or Diatomaceous Earth (SiO2, <4.5 µm). Alkaline comet assay and micronuclei (MNi) test were used to assess DNA damage and chromosomal breakage, respectively. Cell viability was tested with resazurin assay. Comet assay results revealed no significant increase in DNA damage by NSA compared to other natural substances. As expected, DNA breaks were significantly higher in cells exposed to an organophosphate [OPP] control (P < 0.05). No statistically significant differences were found in terms of cellular viability at 50 and 100 µg/mL of NSA but cell survival decreased at 200 µg/mL as well as in OPP group. Positively charged NSA particles significantly reduced cell viability and increased DNA migration and oxidative DNA damage (8-oxoG). NSA as well as the electrically charged NSA particles had no significant effect on MNi induction. Our results indicate that NSA particles are non-cytotoxic and non-genotoxic at the tested doses and do not cause obvious DNA damage in human PBL in vitro.

Differential effects of hypo- and hyperthyroidism on remodeling of contacts between neurons expressing the neuropeptide EI and tyrosine hydroxylase in hypothalamic areas of the male rat.

The Neuropeptide EI (NEI, glutamic acid- isoleucine amide) participates in neuroendocrine function. Previously we demonstrated that NEI concentration is regulated by thyroid hormones in discrete hypothalamic areas in rats. We observed that the thyroid status affects the dopaminergic regulation of the pituitary hormones. In this study we explored possible interactions between NEI and tyrosine hydroxylase (TH) containing elements in selected hypothalamic areas of male rats. Neuronal somas, terminals and boutons were assessed by confocal microscopy, in hypo- and hyperthyroid animals. We observed a remodeling of the contacts between the TH and NEI immunoreactive elements in the incerto-hypothalamic area (IHy, also known as rostromedial zona incerta) according to thyroid function. However, in the dorsolateral zone of the peduncular part of the lateral hypothalamus (DL-PLH) the thyroid hormones affect the dendritic trees of the neurons without perturbing the overall NEI/TH contacts. Also, we demonstrated that TRH Receptor 1 (TRH-R1) is colocalized in NEI immunoreactive neurons in the peduncular part of the lateral hypothalamus (PLH) and NEI precursor mRNA expression increased by hypothyroidism indicating that NEI neurons are responsive to the feedback mechanisms of the Hypothalamic Pituitary-Thyroid Axis (HPT). In conclusion, the hypothyroid status seems to increase the interactions between the NEI neurons and the dopaminergic pathways while hyperthyroidism either decreases or displays no effects. Altogether these observations support the participation of the IHy and PLH NEI as a modulating component of the HPT suggesting that altered neuroendocrine, behavioral and cognitive dysfunctions induced by dysthyroidism could be in part mediated by NEI.

Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth.

Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.

Reversion by vitamin E treatment of the oxidative damage but not of the advancement in reproductive senescence produced by neonatal hypoxia or hypoxia-ischemia in female rats.

BACKGROUND/AIMS: Few studies address the long-term consequences of perinatal hypoxia (H), a frequent birth complication. Previously we described advanced reproductive senescence (premature loss of regular cyclicity) in female rats subjected to perinatal H or H plus unilateral ischemia (HI) associated with changes in the hypothalamic expression of estrogen and opioid receptors. Our aim is to explore whether hypothalamic inflammation and oxidative damage mediate these reproductive alterations. METHODS: Female rats were subjected on postnatal day (PND) 7 to H (6.5% O2 for 50 min) or HI (H + right carotid artery ligature) and inflammation/oxidative damage markers, such as iNOS, nNOS, insulin-like growth factor (IGF) system expression, glial reaction and macrophage invasion in the medial basal hypothalamus-preoptic area (GFAP Western blot and immunohistochemistry, ED1 immunohistochemistry), were determined. The effect of antioxidant treatment with vitamin E (VE; 1.5 mg/rat on PND 4, 6 and 8) was also explored. RESULTS: No significant cellular inflammatory reactions were observed although GFAP protein was significantly increased at early times after injury. Forty-eight hours after injury iNOS, nNOS and IGF-I mRNA decreased in the HI group, and nNOS in the H group. IGFBP-3 mRNA increased in HI rats at 48 h and 30 days, while it fell at 7 days postinjury in both groups. VE treatment prevented the effects of HI on oxidation/inflammation markers, but did not prevent the premature onset of reproductive senescence or the altered hormone receptors expression. CONCLUSION: These results suggest that the oxidative and inflammatory damage caused by perinatal H or HI may not be responsible for the late-onset reproductive abnormalities.

Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids.

BACKGROUND: The opioid system modulates prolactin release during late pregnancy. Its role and the participation of ovarian hormones in this modulation are explored in ether stress-induced prolactin release. METHODS/RESULTS: Estrous, 3-day and 19-day pregnant rats were used. We administered the antagonist mifepristone (Mp) and tamoxifen to evaluate progesterone and estradiol action in naloxone (NAL, opioid antagonist) or saline treated rats. Ether stress had no effect on serum prolactin levels in controls but increased prolactin release in NAL-treated rats. Prolactin response to stress in NAL-treated rats was blocked by l-DOPA administration. Mp treatment on day 18 of pregnancy increased prolactin levels after stress without alterations by NAL. Tamoxifen on days 14 and 15 of pregnancy completely blocked Mp and NAL effects on prolactin release at late pregnancy. In contrast, stress significantly increased prolactin levels in estrous rats and pretreatment with NAL prevented this. On day 3 of pregnancy, at 6.00 p.m., stress and NAL treatment inhibited prolactin levels in saline-treated rat. No effect of stress or NAL administration was detected on day 3 of pregnancy at 9.00 a.m. icv administration of specific opioids antagonist, B-Funaltrexamine but not Nor-Binaltorphimine or Naltrindole, caused a significant increase in stress-induced prolactin release. CONCLUSIONS: Opioid system suppression of prolactin stress response during late pregnancy was observed only after progesterone withdrawal, involving a different opioid mechanism from its well-established stimulatory role. This mechanism acts through a mu opioid receptor and requires estrogen participation. The opioid system and progesterone may modulate stress-induced prolactin release, probably involving a putative prolactin-releasing factor.

Luteal expression of thyroid hormone receptors during gestation and postpartum in the rat.

BACKGROUND: Progesterone (P4) is the main steroid secreted by the corpora lutea (CL) and is required for successful implantation and maintenance of pregnancy. Although adequate circulating levels of thyroid hormone (TH) are needed to support formation and maintenance of CL during pregnancy, TH signaling had not been described in this gland. We determined luteal thyroid hormone receptor isoforms (TR) expression and regulation throughout pregnancy and under the influence of thyroid status, and in vitro effects of triiodothyronine (T3) exposure on luteal P4 synthesis. METHODS: Euthyroid female Wistar rats were sacrificed by decapitation on gestational day (G) 5, G10, G15, G19, or G21 of pregnancy or on day 2 postpartum (L2). Hyperthyroidism and hypothyroidism were induced in female Wistar rats by daily administration of thyroxine (T4; 0.25 mg/kg subcutaneously) or 6-propyl-2-thiouracil (PTU; 0.1 g/L in drinking water), respectively. Luteal TR expression of mRNA was determined using real-time reverse-transcription quantitative polymerase chain reaction, and of protein using Western blot and immunohistochemistry. Primary cultures of luteal cells and of luteinized granulosa cells were used to study in vitro effects of T3 on P4 synthesis. In addition, the effect of T3 on P4 synthesis under basal conditions and under stimulation with luteinizing hormone (LH), prolactin (PRL), and prostaglandin E2 (PGE2) was evaluated. RESULTS: TRα1, TRα2, and TRß1 mRNA were present in CL, increasing during the first half and decreasing during the second half of pregnancy. At the protein level, TRß1 was abundantly expressed during gestation reaching a peak at G19 and decreasing afterwards. TRα1 was barely expressed during early gestation, peaked at G19, and diminished thereafter. Expression of TRß1 and TRα1 at the protein and mRNA level were not influenced by thyroid status. T3 neither modified P4 secretion from CL of pregnancy nor its synthesis in luteinized granulosa cells in culture. CONCLUSIONS: This study confirms for the first time the presence of TR isoforms in the CL during pregnancy and postpartum, identifying this gland as a TH target during gestation. TR expression is modulated in this tissue in accordance with the regulation of P4 metabolism, and the abrupt peripartum changes suggest a role of TH during luteolysis. However, TH actions on the CL do not seem to be related to a direct regulation of P4 synthesis.

Cytoprotective mechanisms in rats lung parenchyma with zinc deprivation.

Suboptimal intake of Zinc (Zn) is one of the most common worldwide nutritional problems. The aim of this study is to provide new evidence on the relation between moderate Zn restriction, and cytoprotective functions in airway epithelium. We analyzed the effect of moderate Zn deficiency (ZD) on the expression of several pro and anti-apoptotic proteins and cytoprotective factors (Hsp27 and Hsp 70i), as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control group, Zn-deficient group and Zn-refed group. Our previous findings showed an important oxidative and nitrosative stress during ZD, this situation is accompanied by inflammation and alterations in the expression of matrix extracellular proteins. We observed a strong immunopositive area of anti and pro-apoptotics proteins in ZD groups. The mRNA levels of Nrf-2, Bax and Bad were increased in ZD, while in ZD refed group its levels were similar to the control values. The increased expression of Nrf-2 is likely to be critical for protection of lung under inflammatory process triggered during ZD. Hsp27 and Hsp 70i showed an increase of immunostaining area but they were not significant. During the supplementation period, heat-shock proteins increased significantly. In conclusion, our results provide further evidence of the pathways involved in cytoprotection and apoptosis caused by ZD. Additional studies are required in order to investigate whether Hsp27 and Hsp70 are consistently associated with cellular stress and inflammation in lung. There may be a beneficial role for improved Zn nutrition or Zn supplements early in lung pathology.

TNFRp55 controls regulatory T cell responses in Yersinia-induced reactive arthritis.

In addition to its well-known pro-inflammatory effects, tumor necrosis factor (TNF) displays anti-inflammatory activities through mechanisms poorly understood. Previously, we reported the development of severe chronic Yersinia enterocolitica-induced reactive arthritis (ReA) in mice lacking the TNF receptor (TNFR)p55. As regulatory T (T(reg)) cells limit chronic inflammation, here we aim to investigate the expansion and function of CD4(+)CD25(+)FoxP3(+) T(reg) cells in the ReA animal model. The number of T(reg) cells as well as the FoxP3 mRNA expression and interleukin (IL)-10 levels were significantly decreased in joint regional lymph nodes (RLNs) of TNFRp55(-/-) mice vs wild-type (WT) mice at the arthritis onset. However, at chronic phase of arthritis, the number of T(reg) cell in TNFRp55(-/-) was similar to WT mice. To explore the in vivo function of T(reg) cells at this chronic phase in WT and TNFRp55-deficient mice, we adoptively transferred CD4(+) T cells from TNFRp55-deficient mice of day 21, into naïve WT or TNFRp55(-/-) mice. When knockout mice were used as recipients we observed higher delayed-type hypersensitivity (DTH) responses and joint inflammation after heat-killed Yersinia (HKY) stimulation. Accordingly, we found higher levels of IL-17, interferon (IFN)-γ, IL-6, transforming growth factor (TGF)-ß1 and IL-12/23p40 and lower IL-10 levels in RLN of paws challenged with HKY in TNFRp55(-/-) recipient mice. In addition, we found that CD4(+) T cells from TNFRp55(-/-) mice controlled antigen-specific IL-12/23(p40) production in recipient WT mice. Our results show that TNFRp55 controls the induction and function of T(reg) cells through differential regulation of cytokine production, suggesting a novel molecular target for immune intervention in ReA.

Alterations of the extracellular matrix of lung during zinc deficiency.

Suboptimal intake of Zn is one of the most common nutritional problems worldwide. Previously, we have shown that Zn deficiency (ZD) produces oxidative and nitrosative stress in the lung of rats. We analyse the effect of moderate ZD on the expression of several intermediate filaments of the cytoskeleton, as well as the effect of restoring Zn during the refeeding period. Adult male rats were divided into three groups: Zn-adequate control (CO) group; ZD group; Zn-refeeding group. CerbB-2 and proliferating cell nuclear antigen (PCNA) expression was increased in the ZD group while the other parameters did not change. During the refeeding time, CerbB-2, cytokeratins, vimentin and PCNA immunostaining was higher than that in the CO group. The present findings indicate that the overexpression of some markers could lead to the fibrotic process in the lung. Perhaps ZD implications must be taken into account in health interventions because an inflammation environment is associated with ZD in the lung.

Lactation deficit in OFA hr/hr rats may be caused by differential sensitivity to stress compared with Wistar and Sprague Dawley rats.

OFA hr/hr (OFA) rats present a major lactation deficit that impairs offspring survival. To explore whether abnormal stress responsiveness causes this deficit, we compared their hormonal (prolactin, progesterone, and corticosterone) responses to stress (room change and 2-min ether exposure) with those of Wistar and Sprague Dawley (SD) rats. We tested responses during the estrous cycle, pregnancy, lactation, after ovariectomy, and ovarian steroid hormone priming, and responses to suckling. We evaluated hypothalamic expression of receptors for prolactin (PRLRlong) and the isoforms of receptors for progesterone (PRA and B) and estrogen (ERα and ß) in late pregnancy. We tested whether administration of an anxiolytic (diazepam) improved lactation. Ether exposure increased circulating levels of the three hormones in the three strains of rats, cycling and ovariectomized, but was less effective in pregnancy and lactation. Elevated estrogen level (estrus and estradiol-treated ovariectomized rats) potentiated the prolactin response more in SD and OFA rats than in Wistar rats. Elevated progesterone level (late pregnancy, lactation, progesterone-treated ovariectomized rats) inhibited the prolactin response less in OFA than in SD or Wistar rats. Ether exposure inhibited the prolactin and oxytocin responses to suckling only in OFA rats. Diazepam treatment increased pup survival rate and the prolactin response to suckling. Hypothalamic total PR mRNA content, assayed by RT-PCR, was higher in pregnant OFA rats compared with SD and Wistar rats, but the PRB/PRA protein ratio determined by Western blot was lowest in Wistar rats, intermediate in OFA rats, and highest in SD rats. The heightened sensitivity to stress of lactating OFA rats may contribute to their lactational deficit and be caused by a combination of hypoprolactinemia and reduced inhibitory capacity of progesterone.

Effect of progesterone withdrawal on hypothalamic mechanisms related to prolactin release in late pregnant rats.

BACKGROUND/AIMS: Progesterone (P(4)) fall provoked by spontaneous or prostaglandin F2α (PGF2α)-induced luteolysis in late pregnant rats triggers a prolactin (PRL) surge 12-24 h later. METHODS: To investigate the hypothalamic mechanism mediating this response, we determined expression of tyrosine hydroxylase (TH), PRL receptors (long form, PRLR(long)), estrogen-α (ERα) and ERß, P(4) (PR) A and B receptors, and STAT5a, STAT5b, suppressors of cytokine signaling 1 (SOCS1), SOCS3 and CIS at mRNA (by semiquantitative and real-time RT-PCR) and protein (by Western blot only for TH, ERα and PRs) levels, and dopamine and DOPAC (by high-performance liquid chromatography) contents in the mediobasal hypothalamus (MBH) 24 h after luteolysis induced by a PGF2α analogue (cloprostenol, 25 µg/rat s.c. at 8 and 12 h on day 19 of pregnancy). RESULTS: PGF2α treatment decreased circulating P(4) and estradiol and increased PRL and the estradiol/P(4) ratio. MBH DOPAC and DOPAC/dopamine ratio fell, indicating decreased dopaminergic transmission. PRLR(long), PRB and ERα mRNA increased. ERα and PR proteins were not modified. However, TH protein and mRNA did not change. PRA, the small PR isoform, was much more abundant than PRB, the isoform considered to mediate P(4) genomic actions. STAT5a, SOCS1 and SOCS3 mRNA were also increased. CONCLUSION: The P(4) fall induced by PGF2α treatment induces PRL release through diminution in MBH dopaminergic transmission without change in TH expression. The increased PRLR along with elevated circulating PRL may be responsible for maintaining high TH expression through activation of short-loop feedback mechanisms, counteracting the effect of the fall in circulating P(4). In parallel, SOCS expression contributes to limit PRL signaling.

Estrogens regulate the expression of NHERF1 in normal colon during the reproductive cycle of Wistar rats.

In breast cancer cell lines, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) gene is regulated at the transcriptional level by estrogens, the protein expression levels correlate with the presence of estrogen receptors and the effect is blocked by anti-estrogens. However, there is limited information regarding the regulation of NHERF1 by estrogens in normal colon tissue. The NHERF1 protein has an important role in the maintenance of the intestine ultrastructure. NHERF1-deficient mice showed defects in the intestinal microvilli as well as molecular alterations in brush border membrane proteins. Here, we have studied the expression of NHERF1 in normal rat colon and uterus during the reproductive cycle of Wistar rats. We found that NHERF1 expression in rat colon during the estral cycle is modified by estrogen levels: higher expression of NHERF1 was observed during the proestrous and estrous stages and lower expression in diestrous 1 when estrogen levels decreased. In uterus, NHERF1 was expressed in the apical region of the luminal epithelium and glands in all stages of the estral cycle, and in both colon and uterus, the expression was independent of the proliferation status. Our results show that NHERF1 expression is regulated by estrogens in colon during the rat estral cycle.

Lack of TNFR p55 results in heightened expression of IFN-γ and IL-17 during the development of reactive arthritis.

Reactive arthritis (ReA) is a type of arthritis originating from certain gastrointestinal or genitourinary infections. In previous studies, we reported the development of progressive Yersinia enterocolitica-induced ReA in mice lacking TNFR p55; however, the mechanisms underlying this effect are still uncertain. In this study, we investigated the impact of TNFR p55 deficiency in modulating Ag-specific Th1 and Th17 responses during this arthritogenic process. We found more severe ReA in TNFRp55(-/-) mice compared with their wild-type (WT) counterparts. This effect was accompanied by increased levels of Yersinia LPS in the joints of knockout mice. Analysis of the local cytokine profile revealed greater amounts of IFN-γ and IL-17 in arthritic joints of TNFRp55(-/-) mice compared with WT mice at day 21 postinfection. Moreover, altered IL-17 and IFN-γ production was observed in mesenteric and inguinal lymph nodes of Yersinia-infected TNFRp55(-/-) mice, as well as in spleen cells obtained from infected mice and restimulated ex vivo with bacterial Ags. Increased levels of cytokine secretion were associated with a greater frequency of CD4(+)IL-17(+), CD4(+)IFN-γ(+), and IL-17(+)IFN-γ(+) cells in TNFRp55(-/-) mice compared with WT mice. Remarkably, Ab-mediated blockade of IL-17 and/or IFN-γ resulted in reduced joint histological scores in TNFRp55(-/-) mice. A mechanistic analysis revealed the involvement of p40, a common subunit of heterodimeric IL-12 and IL-23, in the generation of augmented IFN-γ and IL-17 production under TNFR p55 deficiency. Taken together, these data indicate that, in the absence of TNFR p55 signaling, Th1 and Th17 effector cells may act in concert to sustain the inflammatory response in bacterial-induced arthritogenic processes.

Alteration in the expression of inflammatory parameters as a result of oxidative stress produced by moderate zinc deficiency in rat lung.

Suboptimal intake of dietary zinc (Zn) is one of the most common nutritional problems worldwide. Previously, the authors have shown that zinc deficiency (ZD) produces oxidative and nitrosative stress in lung of male rats. The goal of this study is to test the effect of moderate ZD on insulin-like growth factor (IGF)-1, IGF-binding protein (IGFBP)-5, NADH oxidase (NOX)-2, tumor necrosis factor alpha (TNFalpha), as well as the effect of restoring zinc during the refeeding period. Adult male rats were divided into 3 groups: Zn-adequate control group, Zn-deficient group, and Zn-refeeding group. eNOS, metallothionein (MT) II, and NOX-2 was increased in ZD group. The authors observed an increased gene transcription of superoxide dismutase (SOD)-2 and gluthathione peroxidase (GPx)-1 in ZD group, as well as in ZD-refeeding group, but catalase (CAT) transcription did not change in the treated groups. Proinflammatory factors, such as TNFalpha and vascular cell adhesion molecular (VCAM)-1 increased in ZD, whereas it decreased in ZD refeeding. However, peroxisome proliferator-activated receptor gamma (PPARgamma) and IGF-1 gene transcription decreased in ZD, whereas IGFBP-5 decreased in the ZD group. These parameters are associated to alterations in the lung histoarchitecture. The zinc supplementation period is brief (only 10 days), but it is enough to inhibit some proinflammatory factors. Perhaps, zinc deficiency implications must be taken into account in health interventions because inflammation and prooxidant environment are associated with ZD in lung.

Advancement of reproductive senescence and changes in the early expression of estrogen, progesterone and micro-opioid receptors induced by neonatal hypoxia in the female rat.

Perinatal hypoxia is a frequent birth complication, and although its early consequences on brain development have been well studied, few studies address any long-term effects. Postnatal insults producing small disturbances in endocrine function can have marked and long-lasting effects. In the present work we studied the effects of two types of perinatal brain injury: global hypoxia (H, 6.5% O2 for 50 min) and hypoxia plus ischemia (HI, ligature of the right carotid artery) on female rat reproductive performance and expression of mediobasal hypothalamus-preoptic area (MBH-PO) estrogen, progesterone and micro-opioid receptors at different times after injury, measuring the mRNA (by semiquantitative RT-PCR) and protein (by Western blot). H or HI advanced approximately 3 months after the appearance of blunted preovulatory LH surges and cyclic irregularities (prolonged estrus) characteristic of the early stages of reproductive senescence. 48 h after H or HI we observed decreases in ERbeta, microOR and PR (only in the H group) mRNAs and in total ER and microOR proteins, followed by increased PR levels (mRNA and protein) 7 days post-injury and by increased microOR protein and ERbeta mRNA in the H group and ERalpha, ERbeta and microOR mRNAs and ER protein in the HI group 30 days post-injury. Thus, an episode of hypoxia suffered during early postnatal life induces premature reproductive senescence on the female rats, accompanied by early changes in some MBH-PO hormone receptors (microOR, ER and PR), whose expression is intimately involved in the regulation of gonadotropin secretion and female sexual cyclicity.

Hormonal profile and reproductive performance in lactation deficient (OFA hr/hr) and normal (Sprague-Dawley) female rats.

Lactation deficiency may have important consequences on infant health, particularly in populations of low socioeconomic status. The OFA hr/hr (OFA) strain of rats, derived from Sprague-Dawley (SD) rats, has deficient lactation and is a good model of lactation failure. We examined the reproductive performance and hormonal profiles in OFA and SD strains to determine the cause(s) of the lactation failure of the OFA strain. We measured hormonal (PRL, GH, gonadotropins, oxytocin, and progesterone) levels by RIA in cycling, pregnant, and lactating rats and in response to suckling. Dopaminergic metabolism was assessed by determination of mediobasal hypothalamic dopamine and dihydroxyphenylacetic acid (DOPAC) concentrations by HPLC and tyrosine hydroxylase expression by immunocytochemistry and western blot. OFA rats have normal fertility but 50% of the litters die of malnutrition on early lactation; only 6% of the mothers show normal lactation. The OFA rats showed lower circulating PRL during lactation, increased hypothalamic dopamine and DOPAC, and impaired milk ejection with decreased PRL and oxytocin response to suckling. Before parturition, PRL release and lactogenesis were normal, but dopaminergic metabolism was altered, suggesting activation of the dopaminergic system in OFA but not in SD rats. The number of arcuate and periventricular neurons expressing tyrosine hydroxylase was higher in SD rats, but hypothalamic expression of TH was higher in OFA rats at the end of pregnancy and early lactation. These results suggest that the OFA rats have impaired PRL release linked with an augmented dopaminergic tone which could be partially responsible for the lactational failure.

Acute sublethal global hypoxia induces transient increase of GAP-43 immunoreactivity in the striatum of neonatal rats.

We assessed immunoreactivity (IR) in the cerebral cortex (CC), hippocampus (Hipp), and striatum (ST) of a growth-associated protein, GAP-43, and of proteins of the synaptic vesicle fusion complex: VAMP-2, Syntaxin-1, and SNAP-25 (SNARE proteins) throughout postnatal development of rats after submitting the animals to acute global postnatal hypoxia (6.5% O(2), 70 min) at postnatal day 4 (PND4). In the CC only the IR of the SNARE protein SNAP-25 increased significantly with age. The hypoxic animals showed the same pattern of IR for SNAP-25, although with lower levels at PND11, and also a significant increase of VAMP-2. SNAP-25 (control): PND11 P < 0.001 vs. PND18, 25, and 40, SNAP-25 (hypoxic): P < 0.001 vs. PND18, 25, and 40; VAMP-2 (hypoxic): P < 0.05 PND11 vs. PND18, and P < 0.01 vs. PND25 and PND40; one-way ANOVA and Bonferroni post-test. In the Hipp, SNAP-25 and syntaxin-1 increased significantly with age, reaching a plateau at PND25 through PND40 in control animals (one-way ANOVA: syntaxin-1: P = 0.043; Bonferroni: NS; SNAP-25: P = 0.013; Bonferroni: P < 0.01 PND11 vs. PND40). Hypoxic rats showed higher levels of significance in the one-way ANOVA than controls (syntaxin-1: P = 0.009; Bonferroni: P < 0.05 PND11 vs. PND25 and P < 0.001 PND11 vs. PND40). In the ST, GAP-43 differed significantly among hypoxic and control animals and the two-way ANOVA revealed significant differences with age (F = 3.23; P = 0.037) and treatment (F = 4.84; P = 0.036). VAMP-2 expression also reached statistical significance when comparing control and treated animals (F = 6.25, P = 0.018) without changes regarding to age. Elevated plus maze test performed at PND40 indicated a lower level of anxiety in the hypoxic animals. At adulthood (12 weeks) learning, memory and locomotor abilities were identical in both groups of animals. With these results, we demonstrate that proteins of the presynaptic structures of the ST are sensitive to acute disruption of homeostatic conditions, such as a temporary decrease of the O(2) concentration. Modifications in the activity of these proteins could contribute to the long term altered responses to stress due to acute hypoxic insult in the neonatal period.

Inflammatory responses of the substantia nigra after acute hypoxia in neonatal rats.

The neocortex and the striatum are the brain regions most known to be particularly vulnerable to acute insults like hypoxia or ischemia. In this work, we assess the possibility of cellular damage to the substantia nigra (SN) after hypoxia-reoxygenation in the new born rat. The aim of the present paper was to evaluate the expression of growth factor IGF-I, and growth factor binding proteins IGFBP-3 and IGFBP-5 genes and induction of NOS family members (nNOS, eNOS and iNOS) and TNF-alpha genes together with glia activation, in the SN at 5 and 48 h after severe hypoxia in the 7 day-old rat, a model for the term human fetus. At early time, while IGFs remain unchanged, we found a transient increase in eNOS and nNOS. Two days after the injury, nNOS expression remained high, iNOS and TNF-alpha increased and also GFAP protein expression was observed together with a profusion of reactive astrocytes distributed throughout the SN. This study on the acute effects of hypoxia on the developing brain provides additional insights into the vulnerability of the SN, a brain region involved in neurodegenerative pathologies.