The cytokine profile of women with severe anxiety and depression during pregnancy.

BACKGROUND: Controversial findings regarding the association between pro-inflammatory cytokines and depression have been reported in pregnant subjects. Scarce data about anxiety and its relationships with cytokines are available in pregnant women. To understand the association between anxiety and cytokines during pregnancy, we conducted the present study in women with or without depression. METHODS: Women exhibiting severe depression (SD) and severe anxiety (SA) during the 3rd trimester of pregnancy (n = 139) and control subjects exhibiting neither depression nor anxiety (n = 40) were assessed through the Hamilton Depression Rating Scale (HDRS) and the Hamilton Anxiety Rating Scale (HARS). Serum cytokines were measured by a multiplex bead-based assay. Correlation tests were used to analyze the data and comparisons between groups were performed. A general linear model of analysis of variance was constructed using the group as a dependent variable, interleukin concentrations as independent variables, and HDRS/HARS scores and gestational weeks as covariables. RESULTS: The highest levels of Th1- (IL-6, TNF-α, IL-2, IFN-γ), Th17- (IL-17A, IL-22), and Th2- (IL-9, IL-10, and IL-13) related cytokines were observed in women with SD + SA. The SA group showed higher concentrations of Th1- (IL-6, TNF-α, IL-2, IFN-γ) and Th2- (IL-4, and IL-10) related cytokines than the controls. Positive correlations were found between HDRS and IL-2, IL-6, and TNF-α in the SA group (p < 0.03), and between HDRS and Th1- (IL-2, IL-6, TNF-α), Th2- (IL-9, IL-10, IL-13) and Th17- (IL-17A) cytokines (p < 0.05) in the SD + SA group. After controlling the correlation analysis by gestational weeks, the correlations that remained significant were: HDRS and IL-2, IL-6, IL-9, and IL-17A in the SD + SA group (p < 0.03). HARS scores correlated with IL-17A in the SA group and with IL-17A, IL-17F, and IL-2 in the SD + SA group (p < 0.02). The linear model of analysis of variance showed that HDRS and HARS scores influenced cytokine concentrations; only IL-6 and TNF-α could be explained by the group. CONCLUSIONS: We found that the cytokine profiles differ when comparing pregnant subjects exhibiting SA with comorbid SD against those showing only SA without depression.

Metabolism Regulation by Estrogens and Their Receptors in the Central Nervous System Before and After Menopause.

Estrogens through their intracellular receptors regulate various aspects of glucose and lipid metabolism. The effects of estrogens in metabolism can be mediated by their receptors located in different areas of the brain such as the hypothalamus, which is involved in the control of food intake, energy expenditure, and body weight homeostasis. Alterations in the metabolic regulation by estrogens participate in the pathogenesis of the metabolic syndrome and cardiovascular diseases in women. The metabolic syndrome is an important disease around the world, consisting in a combination of characteristics including abdominal obesity, dyslipidemia, hypertension, and insulin resistance. It increases the risk of cardiovascular disease and type 2 diabetes. It has been suggested that there is an increase in the incidence of metabolic syndrome during menopause due to estrogens deficiency. Estrogens replacement improves insulin sensitivity and reduces the risk of diabetes in rats. In the brain, estrogens through the interaction with their receptors regulate the activity of neurons involved in energy homeostasis, including appetite and satiety. Thus, estradiol and their receptors in the hypothalamus play a key role in metabolic syndrome development during menopause.

The role of cytokines in the regulation of neurotransmission.

Cytokines are highly inducible, secretory proteins that mediate intercellular communication in the immune system. They are grouped in several protein families, namely tumor necrosis factors, interleukins, interferons and colony-stimulating factors. In recent years, evidence has elucidated that some of these proteins as well as their receptors are also produced in the central nervous system (CNS) by specific neural cell lineages under physiological and pathological conditions. Cytokines regulate a variety of processes in the CNS, including neurotransmission. The current data let us to suggest that cytokines play an important role in the regulation of both excitatory and inhibitory neurotransmission in the CNS. This knowledge could be fundamental for the proposal of new therapeutic approaches to neurological and psychiatric disorders.

Changes in progesterone receptor mRNA content in the rabbit lung during early pregnancy and after sex steroid hormone treatment.

In this work we determined progesterone receptor (PR) mRNA content in female rabbit lung during the first 5 days of pregnancy and in ovariectomized animals after subcutaneous injection of oestradiol benzoate (25 micrograms/kg) for 2 days or oestradiol benzoate (25 micrograms/kg) for 2 days plus a single dose of progesterone (5 mg/kg) on day three. On each day (0-5) of pregnancy and 24 h after the last dose in the case of the treated animals, animals were killed and lung was excised; total RNA was extracted and processed for Northern blot analysis. The results showed three main PR mRNA transcripts (6.1, 4.4 and 1.8 kb) in rabbit lung. The 4.4 kb species was the most abundant. PR mRNA content was markedly increased by oestradiol benzoate and downregulated by progesterone. It significantly increased on the first day of pregnancy and then diminished progressively, reaching its lowest value on day 5. These findings suggest that PR mRNA content in the rabbit lung is regulated by sex steroid hormones and changes according to the physiological concentrations of oestradiol and progesterone.

Immunohistochemical localization of intracellular progesterone and glucocorticoid receptors in the rabbit lung.

We have determined the presence and distribution of intracellular progesterone receptors (PRs) and glucocorticoid receptors (GRs) in the lung of adult female rabbits using immunohistochemistry. The effects of ovariectomy and administration of oestradiol benzoate (10 micrograms for 3 consecutive days) upon PR and GR immunoreactivity were also studied. The results demonstrated the presence of both steroid hormone receptors in the female rabbit lung. PR and GR immunoreactivity was predominantly nuclear and located in alveolar epithelial cells and various interstitial cells such as polymorphonuclear leucocytes. Tissue distribution of both receptors was similar in all cases. Oestradiol treatment induced a marked increase in the number of PR immunoreactive cells compared with intact and ovariectomized female animals. Neither ovariectomy nor oestradiol treatment modified the number of GR immunoreactive cells. The presence and localization of intracellular PRs and GRs in several lung cell types suggest that they may play an important role in mediating the effects of progesterone and glucocorticoids in various physiological processes in the rabbit lung. The data also indicated an oestrogen regulation of PRs in the rabbit lung.

The effect of total sleep deprivation on plasma melatonin and cortisol in healthy human volunteers.

Twelve healthy volunteers were included in this study. Baseline curves for melatonin and cortisol were obtained after one night of adaptation to laboratory conditions. From 10:00 p.m. to 6:00 a.m., blood samples were drawn every hour. On the third night, the subjects were kept awake at the sleep unit. Curves for the two hormones were then obtained after 36 h of total sleep deprivation (SD). The levels of these hormones were evaluated by calculating the area under the curve at each hour in both situations (basal and after sleep deprivation). It was found that the melatonin levels were increased after sleep deprivation, whereas the cortisol levels remained the same. These results suggest a mechanism by which a reset of abnormal rhythms can occur in depression.

Changes in progesterone receptor isoforms content in the rat brain during the oestrous cycle and after oestradiol and progesterone treatments.

We studied the effects of oestradiol and progesterone on progesterone receptor (PR) isoform content in the brain of ovariectomized rats and in intact rats during the oestrous cycle by Western blot analysis. In the hypothalamus and the preoptic area of ovariectomized rats, PR-A and PR-B content was increased by oestradiol, whereas progesterone significantly diminished the content of both PR isoforms after 3 h of treatment in the hypothalamus, but not in the preoptic area. In the hippocampus, only PR-A content was significantly increased by oestradiol while progesterone significantly diminished it after 12 h of treatment. In the frontal cortex, no treatment significantly modified PR isoform content. During the oestrous cycle, the lowest content of PR isoforms in the hypothalamus was observed on diestrus day and, by contrast, in the preoptic area, the highest content of both PR isoforms was observed on diestrus day. We observed no changes in PR isoform content in the hippocampus during the oestrous cycle. These results indicate that the expression of PR isoforms is differentially regulated by sex steroid hormones in a regionally specific manner.

Intracellular progesterone receptors are differentially regulated by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit.

The aim of this study was to examine the role of sex steroid hormones in the regulation of intracellular progesterone receptors (PR) in the rabbit central nervous system. We determined PR concentration in cytosol preparations from the hypothalamus, the frontal, tempo-parietal and occipital cortex, by using the specific binding of the synthetic progestin [3H]ORG 2058. PR concentration was higher in the hypothalamus of intact adult females than in that of adult males and prepubertal females, whereas no significant differences were observed in the cerebral cortex of these animals. PR concentration was similar in the three cortical regions analyzed, indicating a homogeneous distribution of PR in the cerebral cortex. The administration of estradiol to ovariectomized animals increased PR concentration in the hypothalamus but not in the cortex. The administration of progesterone to ovariectomized rabbits did not modify PR concentration in any region, however when progesterone was administered after estradiol, it induced a significant diminution in hypothalamic PR concentration without effects in the cortex. These findings suggest that in the rabbit, PR are estrogen regulated in the hypothalamus but not in the cerebral cortex. In the latter, PR are not regulated by progesterone, whereas in the former the estrogen-induced PR are down-regulated by progesterone. Interestingly, hypothalamic PR constitutively expressed in ovariectomized animals are progesterone-insensitive.

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

Progesterone receptor isoforms are differentially regulated by sex steroids in the rat forebrain.

We studied the effects of estradiol (E2) and progesterone (P4) on expression of genes coding for PR isoforms in the forebrain of ovariectomized rats by RT-PCR analysis. In the hypothalamus the expression of both PR isoforms was induced by E2 and down-regulated by P4. In the preoptic area these changes were only observed in the PR-B isoform. In contrast, in the hippocampus PR induction by E2 was only observed for PR-A. In this region P4 did not modify the expression of any PR isoform. These results indicate that PR isoforms expression is differentially regulated by sex steroid hormones in distinct forebrain regions and suggest that the tissue-specific regulation of either PR-A or PR-B may be involved in the physiological actions of P4 upon the rat brain.

Infusion of 3alpha,5alpha-THP to the pontine reticular formation attenuates PTZ-induced seizures.

Whether progesterone (P(4)) and its metabolite, 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) have anti-seizure effects through actions in the pontine reticular formation (PRF) was investigated. Concentrations of P(4) and 3alpha, 5alpha-THP in the PRF were greater in proestrous and hormone-primed rats, that are typically more resistant to seizure-induction, than diestrous and males rats. Ovx, Long-Evans rats with unilateral microinjections into the PRF of 3alpha,5alpha-THP (5 microg/0.2 microl), but not P(4) (11 microg/0.2 microl) or vehicle (beta-cyclodextrin), had a greater latency and lower incidence of tonic-clonic seizures induced by pentylenetetrazol (PTZ; 70 mg/kg, IP) administration. Infusions that missed the PRF were not effective. These data suggest 3alpha,5alpha-THP has anti-seizure effects in part through actions in the PRF.

c-Fos gene expression pattern in the hypothalamus and the preoptic area of defeminized rats.

The object of the present study was to determine the c-fos gene expression pattern in the hypothalamus (HYP) and the preoptic area (POA) after estradiol and testosterone priming during the critical period of sexual differentiation of the rat brain. Three-day-old female rats were injected s.c. with a single dose of 17beta-estradiol (200 microg), testosterone enantate (200 microg) or vehicle (corn oil). HYP and POA were dissected 2 h, 24 h and 14 days after treatments and on the day of vaginal opening (VO). Other animals, previously treated as above, were acutely injected with 17beta-estradiol (5 microg) on the day of VO; HYP and POA were obtained 3 h later. Total RNA was extracted and processed for semiquantitative RT-PCR. We observed that c-fos gene expression was markedly increased in POA of the animals treated with estradiol or testosterone 2 h after treatments, while a non-significant increase in c-fos gene expression was observed in the HYP of these animals. We found a significant increase in c-fos expression in HYP and POA on the day of VO in both estradiol and testosterone defeminized rats. Interestingly, the acute estradiol administration on the day of VO did not induce c-fos gene expression in either HYP or POA of defeminized animals, instead a diminution in its expression was observed in animals treated with testosterone in POA. The overall results suggest that estradiol and testosterone imprinting during critical postnatal period of sexual differentiation of the brain permanently modifies the regulation of c-fos gene expression.

Microinjections of muscimol and bicuculline into the pontine reticular formation modify the sleep-waking cycle in the rat.

The role of gamma-aminobutyric acid (GABA) in the sleep-waking cycle was evaluated by means of microinjections of muscimol and bicuculline into the rat pontine reticular formation (PRF). Muscimol (20 ng) produced a marked increase in wakefulness (70%), a decrease in slow-wave sleep (SWS) (35%) and a remarkable delay in the onset of both SWS and paradoxical sleep, without modifying the percentage of the latter. Bicuculline (4 ng) shortened SWS latency by about 70%. These results suggest that GABAergic transmission in the PRF is involved in the regulation of sleep-waking cycle in the rat.

Regulation of progesterone receptor gene expression by sex steroid hormones in the hypothalamus and the cerebral cortex of the rabbit.

The effects of estradiol benzoate (EB) and progesterone (P4) upon progesterone receptor (PR) gene expression in the cerebral cortex and the hypothalamus of the rabbit were studied. Ovariectomized adult rabbits were subcutaneously treated with EB (25 micrograms/kg) for 2 days, and with EB (25 micrograms/kg) + a single dose of P4 (5 mg/kg) on day 3. Twenty-four hours after the last dose, the frontal cortex, the hypothalamus and the uterus were excised, total RNA was extracted and processed for reverse transcription-polymerase chain reaction. PR gene expression was induced by EB and down-regulated by P4 both in the frontal cortex and the hypothalamus in a manner similar to that observed in the uterus. The finding that PR gene transcription is regulated by steroid hormones in the cerebral cortex suggests that post-transcriptional processes are involved in the insensitivity of cortical PR protein to steroids regulation previously reported with binding techniques.

Progesterone microinjections into the pontine reticular formation modify sleep in male and female rats.

It has been reported that progesterone (P4) induces changes in sleep, but the brain regions involved in these actions are unknown. We studied the effects of P4 microinjections into the pontine reticular formation (PRF) upon rat sleep. Intact adult male and ovariectomized female rats were unilaterally injected with P4 into the PRF and the sleep-waking cycle was recorded for 6 h. P4 (1.0 and 5.0 microg/0.2 microl) did not modify sleep, but at a higher dose (7.5 microg/0.2 microl) it produced a marked decrease in rapid eye movement sleep (REM) latency in both male (55%) and female (63%) rats. A non-significant increase in the number of REM episodes was observed after P4 administration. These findings suggest that P4 should participate in the mechanisms related to REM initiation in the rat through its effects in the PRF.

Mating modifies c-fos expression in the brain of male and female rabbits.

Copulation in rabbits provokes behavioral and neuroendocrine changes in both sexes. To investigate if the activity of particular brain regions is modified accordingly we quantified, by the reverse transcription-polymerase chain reaction method, c-fos expression in the preoptic area, hypothalamus, hippocampus, and frontal cortex of male and female rabbits before mating, immediately afterwards, and 1 h later. Mating immediately increased c-fos expression in the hypothalamus of both sexes, the frontal cortex of females, and the preoptic area of males. c-fos expression did not change in the hippocampus after mating in either sex but decreased in the preoptic area of females following mating. Results show that mating provokes changes in brain activity, in a gender- and region-specific manner, which may underlie the behavioral and endocrine consequences of copulation in rabbits.

Differential methylation in steroid 5 alpha-reductase isozyme genes in epididymis, testis, and liver of the adult rat.

DNA methylation has been largely involved in the regulation of tissue-specific gene expression. The aim of the study was to determine the methylation pattern of steroid 5 alpha-reductase genes 1 and 2 in two reproductive tissues (testis and epididymis) and a nonreproductive tissue (liver) that exhibit different contents of steroid 5 alpha-reductase isozymes. These isozymes induce the bioconversion of testosterone to dihydrotestosterone that in mammals is a key molecule for external genitalia development. Genomic DNA from the testis, the epididymis, and the liver from normal adult rats was used to determine cytosine and adenine methylation pattern of steroid 5 alpha-reductase genes 1 and 2 by restriction fragment length polymorphism (RFLP) analysis using restriction enzymes sensitive to adenine (Mbo I and Sau3A I) and cytosine (Hpa II and MSP I) methylation. We also evaluated the expression of both steroid 5 alpha-reductase genes by northern blot. When genomic DNA was digested with Hpa II or Msp I, we found that steroid 5 alpha-reductase gene 2 was less cytosine methylated in the epididymis and in the testis than in the liver. In contrast, when genomic DNA was digested with Mbo I or Sau3A I, we observed that gene 2 was more adenine methylated in the epididymis and in the testis than in the liver. 5 alpha-Reductase gene 1 presented the same adenine- and cytosine-methylation pattern in the studied tissues. We also found a differential expression of steroid 5 alpha-reductase genes. Gene 2 was expressed both in the testis and the epididymis but not in the liver; whereas gene 1 was only expressed in the latter. Our results suggest that the differential methylation pattern in 5 alpha-reductase gene 2 in reproductive and nonreproductive tissues should be involved in the regulation of its expression.

Progesterone receptor isoforms expression pattern in human astrocytomas.

Progesterone receptors (PR) have been detected in human astrocytomas; however, the expression pattern of PR isoforms in these brain tumors is unknown. Progesterone receptor isoforms expression was studied in 13 biopsies of astrocytomas (6 grade III, and 7 grade IV) from adult Mexican patients by using reverse transcription-polymerase chain reaction and immunohistochemistry. Progesterone receptor expression was observed at mRNA and at protein levels in 66% and 83% of astrocytomas grade III, respectively, whereas 100% of astrocytomas grade IV expressed PR. Almost all PR mRNA content in astrocytomas grades III and IV corresponded to PR-B. The number of immunoreactive cells expressing PR-B was higher than that expressing PR-A in 73% of the cases. Estrogen receptor-alpha protein was only observed in 33% of astrocytomas grade III, whereas no astrocytomas grade IV expressed it. These data suggest that PR-B is the predominant isoform expressed in human astrocytomas grades III and IV, and that estrogen receptor-alpha is not expressed in astrocytomas grade IV.

Progesterone receptor isoforms expression in the prepuberal and adult male rat brain.

Progesterone receptor (PR) isoforms expression was determined in several regions of the prepuberal and adult male rat brain by using reverse transcription coupled to polymerase chain reaction. Rats under a 14:10-h light-dark cycle, with lights on at 0600 h were used. We found that in the hypothalamus of prepuberal animals the expression of both PR isoforms was similar, whereas PR-A expression was higher than that of PR-B in adults. In the cerebellum PR-B expression was predominant in both prepuberal and adult rats. In both ages PR-A and PR-B exhibited a non-significant tendency to be predominant in the hippocampus and the preoptic area respectively. In the frontal cortex and the olfactory bulb PR isoforms were expressed at a similar level. These results indicate a differential expression pattern of PR isoforms in the male rat brain and suggest that the tissue-specific expression of PR-A and PR-B is important for the appropriate response of each cerebral region to progesterone.

Differential effects of NMDA antagonists microinjections into the nucleus reticularis pontis caudalis on seizures induced by pentylenetetrazol in the rat.

It has been shown that NMDA antagonists block the tonic but not the clonic component of seizures when they are injected in the oral region of the rat pontine reticular formation (PRF). The participation of the caudal PRF in the effects of NMDA antagonists upon the tonic and the clonic components of generalized seizures induced by pentylenetetrazol (PTZ) is unknown. The aim of the present study was to evaluate the effects of unilateral microinjections of competitive and non-competitive NMDA antagonists, 2-amino-7-phosphonoheptanoic acid (AP-7) and dizocilpine (MK-801), respectively, into the nucleus reticularis pontis caudalis of the rat PRF upon seizures induced by PTZ (70 mg/kg i.p.). MK-801 induced a dose-related decrease both in the incidence of generalized tonic-clonic seizures (GTCS) and in the presence of spikes in the EEG. MK-801 also increased GTCS latency. On the contrary, AP-7 did not have effects on GTCS. Interestingly, it induced ipsilateral circling behavior. These results suggest that in the caudal region of the rat PRF only non-competitive NMDA antagonists should block the generation of tonic and clonic components of generalized seizures.