Peripheral Neuroimmunoendocrine Interactions: Contribution of TNFRp55 to the Circadian Synchronization of Progesterone and Cytokine Production in Joints of Mice in Late Pregnancy.

OBJECTIVE: Circadian rhythms are generated by the suprachiasmatic nucleus of the hypothalamus and involve rhythmic expression of clock genes and proteins. This rhythmicity is transferred to peripheral tissues by neural and hormonal signals. Late pregnancy is considered a state of inflammation which impacts on peripheral tissues such as joints. Tumor necrosis factor (TNF) mediates inflammatory and circadian responses through its p55 receptor (TNFRp55). Neuroimmunoendocrine interactions in joints have not been studied completely. The purpose of this study was to analyze these interactions, investigating the circadian rhythms of progesterone (Pg) and pro- and anti-inflammatory cytokines in the joints at the end of pregnancy (gestational day 18). Moreover, the impact of TNFRp55 deficiency on these temporal oscillations was explored. METHODS: Wild-type and TNFRp55-deficient (KO) C57BL/6 mice were kept under constant darkness in order to study their endogenous circadian rhythms. The expression of the clock genes Bmal1 and Per1 at circadian time 7 was studied by reverse transcription polymerase chain reaction in the ankle joints of nonpregnant and pregnant (gestational day 18) mice. In late pregnancy, Pg and the cytokines interleukin 17 (IL-17), IL-6, and IL-10 were measured in the joints throughout a 24-h period by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: A significant increase in Bmal1 and Per1 mRNA expression was detected in the joints of pregnant KO mice. Furthermore, KO mice displayed a desynchronization of articular Pg and cytokine production. CONCLUSIONS: Our results show that TNF, via TNFRp55 signaling, modulates articular Pg and cytokine circadian rhythms in late pregnancy. These findings suggest a temporal neuroimmunoendocrine association in peripheral tissues in late pregnancy.

Circadian rhythms of factors involved in luteal regression are modified in p55 tumour necrosis factor receptor (TNFRp55)-deficient mice.

The rhythm of factors involved in luteal regression is crucial in determining the physiological duration of the oestrous cycle. Given the role of tumour necrosis factor (TNF)-α in luteal function and circadian regulation and that most of the effects of TNF-α are mediated by p55 TNF receptor (TNFRp55), the aims of the present study were to analyse the following during the luteal regression phase in the ovary of mice: (1) whether the pattern of expression of progesterone (P4) and the enzymes involved in the synthesis and degradation of P4 is circadian and endogenous (the rhythm persists in constant conditions, (i.e., constant darkness) with a period of about 24 hours); (2) circadian oscillations in clock gene expression; (3) whether there are daily variations in the expression of key genes involved in apoptosis and antioxidant mechanisms; and (4) the consequences of TNFRp55 deficiency. P4 was found to oscillate circadianally following endogenous rhythms of clock factors. Of note, TNFRp55 deficiency modified the circadian oscillation in P4 concentrations and its enzymes involved in the synthesis and degradation of P4, probably as a consequence of changes in the circadian oscillations of brain and muscle ARNT-Like protein 1 (Bmal1) and Cryptochrome 1 (Cry1). Furthermore, TNFRp55 deficiency modified the circadian rhythms of apoptosis genes, as well as antioxidant enzymes and peroxidation levels in the ovary in dioestrus. The findings of the present study strengthen the hypothesis that dysregulation of TNF-α signalling may be a potential cause for altered circadian and menstrual cycling in some gynaecological diseases.

Rhythmic Bdnf and TrkB expression patterns in the prefrontal cortex are lost in aged rats.

Aging brain undergoes several changes leading to a decline in cognitive functions. Memory and learning-related genes such as Creb, Bdnf and its receptor TrkB, are expressed in different brain regions including prefrontal cortex. Those genes' proteins regulate a wide range of functions such as synaptic plasticity and long-term potentiation. In this work, our objectives were: 1) to investigate whether Creb1, Bdnf and TrkB genes display endogenous circadian expression rhythms, in the prefrontal cortex of rats maintained under constant darkness conditions; 2) to study the synchronization of those temporal patterns to the local cellular clock and 3) to evaluate the aging consequences on both cognition-related genes and activating clock transcription factor, BMAL1, rhythms. A bioinformatics analysis revealed clock-responsive (E-box) sites in regulatory regions of Creb1, Bdnf and TrkB genes. Additionally, cAMP response elements (CRE) were found in Bdnf and TrkB promoters. We observed those key cognition-related factors expression oscillates in the rat prefrontal cortex. Creb1 and TrkB mRNAs display a circadian rhythm with their highest levels occurring at the second half of the 24h period. Interestingly, the cosinor analysis revealed a 12-h rhythm of Bdnf transcript levels, with peaks occurring at the second half of the subjective day and night, respectively. As expected, the BMAL1 rhythm's acrophase precedes Creb1 and first Bdnf expression peaks. Noteworthy, Creb1, Bdnf and TrkB expression rhythms are lost in the prefrontal cortex of aged rats, probably, as consequence of the loss of BMAL1 protein circadian rhythm and altered function of the local cellular clock.

Participation of the extrinsic cholinergic innervation in the action of nitric oxide on the ovarian steroidogenesis in the first proestrous in rats.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system and an ovary without peripheral neural influence from virgin rats in the first proestrous were used to test whether ovarian extrinsic innervation and nitric oxide (NO) affects steroidogenesis in the ovary. The CG and the ovary were placed in separate buffered-compartments, connected by the SON. Stimulation of the CG was achieved by 10(-6)M acetylcholine (Ach). The ovary without peripheral neural influence was placed alone in a buffered-compartment. To test a possible role of NO in the ovarian response to peripheral neural influence, 100µM sodium nitroprusside (SNP, an NO donor) and 100µM N(G)-nitro-l-arginine methyl ester (l-NAME, an inhibitor of NO synthase) were added to the ovarian compartment separately. In the CG-SON-O system, SNP into the ovarian compartment increased the concentration of NO, reduced the release of progesterone and increased the release of estradiol (E2), increasing the mRNAs related to their synthesis enzyme. The addition of l-NAME to the ovarian compartment caused an opposite effect. In the ovary alone, NO manifested an antisteroidogenic effect on both hormones. These results show that the ovarian extrinsic innervation maintains a direct relationship between NO and E2, both needed at high levels during the follicular phase, allowing the continuity of the estrous cycle.

Oxidative stress and altered steroidogenesis in the ovary by cholinergic stimulation of coeliac ganglion in the first proestrous in rats. Implication of nitric oxide.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system from virgin rats in the first proestrous was used to test whether cholinergic stimulation of CG affects oxidative status and steroidogenesis in the ovary. The CG and the O were placed in separate buffered-compartments, connected by the SON, and the CG was stimulated by acetylcholine (Ach). To test a possible role of nitric oxide (NO) in the ovarian response to cholinergic stimulation of CG, aminoguanidine (AG) - an inhibitor of inducible-NO synthase was added to the O compartment. After 180 min incubation, the oxidative status was assessed in O whereas nitrite and steroidogenesis were assessed at 30, 120 and 180 min. Ach in CG decreased the total antioxidant capacity, but increased NO production and protein carbonization in O. Ach stimulation of CG increased estradiol, but decreased progesterone release in O by reducing the mRNAs related to their synthesis and degradation. The addition of AG to the O compartment caused an opposite effect, which was more pronounced in the presence of Ach in the CG compartment than in its absence. These results show that the stimulation of the extrinsic-cholinergic innervation of the O increases the concentration of NO, causes oxidative stress and modulates steroidogenesis in the first rat proestrous.

Circadian rhythms of locomotor activity and hippocampal clock genes expression are dampened in vitamin A-deficient rats.

The main external time giver is the day-night cycle; however, signals from feeding and the activity/rest cycles can entrain peripheral clocks, such as the hippocampus, in the absence of light. Knowing that vitamin A and its derivatives, the retinoids, may act as regulators of the endogenous clock activity, we hypothesized that the nutritional deficiency of vitamin A may influence the locomotor activity rhythm as well as the endogenous circadian patterns of clock genes in the rat hippocampus. Locomotor activity was recorded during the last week of the treatment period. Circadian rhythms of clock genes expression were analyzed by reverse transcription-polymerase chain reaction in hippocampus samples that were isolated every 4 hours during a 24-hour period. Reduced glutathione (GSH) levels were also determined by a kinetic assay. Regulatory regions of clock PER2, CRY1, and CRY2 genes were scanned for RXRE, RARE, and RORE sites. As expected, the locomotor activity pattern of rats shifted rightward under constant dark conditions. Clock genes expression and GSH levels displayed robust circadian oscillations in the rat hippocampus. We found RXRE and RORE sites on regulatory regions of clock genes. Vitamin A deficiency dampened rhythms of locomotor activity as well as modified endogenous rhythms of clock genes expression and GSH levels. Thus, vitamin A may have a role in endogenous clock functioning and participate in the circadian regulation of the cellular redox state in the hippocampus, a peripheral clock with relevant function in memory and learning.

Daily rhythms of catalase and glutathione peroxidase expression and activity are endogenously driven in the hippocampus and are modified by a vitamin A-free diet.

OBJECTIVES: Alterations in enzymatic antioxidant defense systems lead to a deficit of cognitive functions and altered hippocampal synaptic plasticity. The objectives of this study were to investigate endogenous rhythms of catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as CREB1 mRNA, in the rat hippocampus, and to evaluate to which extent the vitamin A deficiency could affect those temporal patterns. METHODS: Rats from control and vitamin A-deficient (VAD) groups received a diet containing 4000 IU of vitamin A/kg diet, or the same diet devoid of vitamin A, respectively, during 3 months. Rats were maintained under 12-hour-dark conditions, during 10 days before the sacrifice. Circadian rhythms of CAT, GPx, RXRγ, and CREB1 mRNA levels were determined by reverse transcriptrase polymerase chain reaction in hippocampus samples isolated every 4 hours during a 24-hour period. CAT and GPx enzymatic activities were also determined by kinetic assays. Regulatory regions of clock and antioxidant enzymes genes were scanned for E-box, RXRE, and CRE sites. RESULTS: E-box, RXRE, and CRE sites were found on regulatory regions of GPx and CAT genes, which display a circadian expression in the rat hippocampus. VAD phase shifted CAT, GPx, and RXRγ endogenous rhythms without affecting circadian expression of CREB1. DISCUSSION: CAT and GPx expression and enzymatic activity are circadian in the rat hippocampus. The VAD affected the temporal patterns antioxidant genes expression, probably by altering circadian rhythms of its RXR receptors and clock factors; thus, it would impair the temporal orchestration of hippocampal daily cognitive performance.

Retinoic acid receptors move in time with the clock in the hippocampus. Effect of a vitamin-A-deficient diet.

An endogenous time-keeping mechanism controls circadian biological rhythms in mammals. Previously, we showed that vitamin A deficiency modifies clock BMAL1 and PER1 as well as BDNF and neurogranin daily rhythmicity in the rat hippocampus when animals are maintained under 12-h-light:12-h-dark conditions. Retinoic acid nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), have been detected in the same brain area. Our objectives were (a) to analyze whether RARα, RARß and RXRß exhibit a circadian variation in the rat hippocampus and (b) to investigate the effect of a vitamin-A-deficient diet on the circadian expression of BMAL1, PER1 and retinoic acid receptors (RARs and RXRß) genes. Holtzman male rats from control and vitamin-A-deficient groups were maintained under 12-h-light:12-h-dark or 12-h-dark:12-h-dark conditions during the last week of treatment. RARα, RARß, RXRß, BMAL1 and PER1 transcript and protein levels were determined in hippocampus samples isolated every 4 h in a 24-h period. Regulatory regions of RARs and RXRß genes were scanned for clock-responsive sites, while BMAL1 and PER1 promoters were analyzed for retinoic acid responsive elements and retinoid X responsive elements. E-box and retinoid-related orphan receptor responsive element sites were found on regulatory regions of retinoid receptors genes, which display an endogenously controlled circadian expression in the rat hippocampus. Those temporal profiles were modified when animals were fed with a vitamin-A-deficient diet. Similarly, the nutritional vitamin A deficiency phase shifted BMAL1 and abolished PER1 circadian expression at both mRNA and protein levels. Our data suggest that vitamin A deficiency may affect the circadian expression in the hippocampus by modifying the rhythmic profiles of retinoic acid receptors.

Cadmium-induced oxidative stress and histological damage in the myocardium. Effects of a soy-based diet.

Cd exposure has been associated to an augmented risk for cardiovascular disease. We investigated the effects of 15 and 100 ppm of Cd on redox status as well as histological changes in the rat heart and the putative protective effect of a soy-based diet. Male Wistar rats were separated into 6 groups and treated during 60 days as follows: groups (1), (2) and (3) were fed a casein-based diet; groups (4), (5) and (6), a soy-based diet; (1) and (4) were given tap water; (2) and (5) tap water containing 15 ppm of Cd²âº; and (3) and (6) tap water containing 100 ppm of Cd²âº. Serum lipid peroxides increased and PON-1 activity decreased in group (3). Lipoperoxidation also increased in the heart of all intoxicated groups; however protein oxidation only augmented in (3) and reduced glutathione levels diminished in (2) and (3). Catalase activity increased in groups (3) and (6) while superoxide dismutase activity increased only in (6). Glutathione peroxidase activity decreased in groups (3) and (6). Nrf2 expression was higher in groups (3) and (6), and MTI expression augmented in (3). Histological examination of the heart tissue showed the development of hypertrophic and fusion of cardiomyocytes along with foci of myocardial fiber necrosis. The transmission electron microscopy analysis showed profound ultra-structural damages. No protection against tissue degeneration was observed in animals fed the soy-based diet. Our findings indicate that even though the intake of a soy-based diet is capable of ameliorating Cd induced oxidative stress, it failed in preventing cardiac damage.

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

Daily patterns of clock and cognition-related factors are modified in the hippocampus of vitamin A-deficient rats.

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRß expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα, and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRß mRNA level as well as the amplitude of PER1, REVERB gene, and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein, and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus, and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area.

Androstenedione acts on the coeliac ganglion and modulates luteal function via the superior ovarian nerve in the postpartum rat.

Androstenedione can affect luteal function via a neural pathway in the late pregnant rat. Here, we investigate whether androstenedione is capable of opposing to regression of pregnancy corpus luteum that occurs after parturition, indirectly, from the coeliac ganglion. Thus, androstenedione was added into the ganglionar compartment of an ex vivo coeliac ganglion-superior ovarian nerve-ovary system isolated from non-lactating rats on day 4 postpartum. At the end of incubation, we measured the abundance of progesterone, androstenedione and oestradiol released into the ovarian compartment. Luteal mRNA expression and activity of progesterone synthesis and degradation enzymes, 3ß-hydroxysteroid-dehydrogenase (3ß-HSD) and 20α-hydroxysteroid-dehydrogenase (20α-HSD), respectively, as well as the aromatase, Bcl-2, Bax, Fas and FasL transcript levels, were also determined. Additionally, we measured the ovarian release of norepinephrine, nitric oxide and luteal inducible nitric oxide synthase (iNOS) mRNA expression. The presence of androstenedione in the ganglion compartment significantly increased the release of ovarian progesterone, androstenedione and oestradiol without modifying 3ß-HSD and 20α-HSD activities or mRNA expression. The ovarian release of oestradiol in response to the presence of androstenedione in the ganglion compartment declined with time of incubation in accord with a reduction in the aromatase mRNA expression. Androstenedione added to the ganglion compartment decreased FasL mRNA expression, without affecting luteal Bcl-2, Bax and Fas transcript levels; also increased the release of norepinephrine, decreased the release of nitric oxide and increased iNOS mRNA. In summary, on day 4 after parturition, androstenedione can mediate a luteotropic effect acting at the coeliac ganglion and transmitting to the ovary a signaling via a neural pathway in association with increased release of norepinephrine, decreased nitric oxide release, and decreased expression of FasL.

Temporal patterns of lipoperoxidation and antioxidant enzymes are modified in the hippocampus of vitamin A-deficient rats.

Animals can adapt their behavior to predictable temporal fluctuations in the environment through both, memory-and-learning processes and an endogenous time-keeping mechanism. Hippocampus plays a key role in memory and learning and is especially susceptible to oxidative stress. In compensation, antioxidant enzymes activity, such as Catalase (CAT) and Glutathione peroxidase (GPx), has been detected in this brain region. Daily rhythms of antioxidant enzymes activity, as well as of glutathione and lipid peroxides levels, have been described in brain. Here, we investigate day/night variations in lipoperoxidation, CAT, and GPx expression and activity, as well as the temporal fluctuations of two key components of the endogenous clock, BMAL1 and PER1, in the rat hippocampus and evaluate to which extent vitamin A deficiency may affect their amplitude or phase. Holtzman male rats from control, vitamin A-deficient, and vitamin A-refed groups were sacrificed throughout a 24-h period. Daily levels of clock proteins, lipoperoxidation, CAT and GPx mRNA, protein, and activity, were determined in the rat hippocampus obtained every 4 or 5 h. Gene expression of RARalpha and RXRbeta was also quantified in the hippocampus of the three groups of rats. Our results show significant daily variations of BMAL1 and PER1 protein expression. Rhythmic lipoperoxidation, CAT, and GPx, expression and activity, were also observed in the rat hippocampus. Vitamin A deficiency reduced RXRbeta mRNA level, as well as the amplitude of BMAL1 and PER1 daily oscillation, phase-shifted the daily peak of lipoperoxidation, and had a differential effect on the oscillating CAT and GPx mRNA, protein, and activity. Learning how vitamin A deficiency affects the circadian gene expression in the hippocampus may have an impact on the neurobiology, nutritional and chronobiology fields, emphasizing for the first time the importance of nutritional factors, such as dietary micronutrients, in the regulation of circadian parameters in this brain memory-and-learning-related region.