Early Dynamics of Plasma Dna in a Mouse Model of Sepsis.

Concentration of extracellular DNA (ecDNA) in plasma of septic patients is higher in comparison to healthy controls and is associated with worse prognosis in intensive care patients. Decrease of ecDNA in plasma by treatment with deoxyribonuclease (DNase) showed to have beneficial effects in animal models of sepsis. A previously published study showed that timing of DNase application is crucial for the effect of DNase. No published study monitored plasma ecDNA dynamics during sepsis in detail yet. The aim of our study was to describe the early dynamics of plasma ecDNA but also plasma DNase activity in a mouse model of sepsis. Sepsis was induced using intraperitoneal injection of E. coli and mice were euthanized every hour to obtain sufficient volume of plasma. Our results show that the concentration of plasma ecDNA is rising continuously during the first 5 h after infection and is 20-fold higher 5 h after induction of sepsis in comparison to control mice. Subcellular origin of plasma ecDNA was analyzed but fundamental differences in dynamics between nuclear and mitochondrial ecDNA were not found. DNase activity in plasma seems to rise slowly until the fourth hour, but the interindividual variability is high. In conclusion, this is the first study that describes the dynamics of plasma ecDNA and DNase activity in early sepsis in detail. Our study is the basis for further studies focused on the timing of exogenous DNase treatment in sepsis. Additional studies will be needed to monitor plasma ecDNA in later time points that are more clinically relevant.

On the Role of Testosterone in Anxiety-Like Behavior Across Life in Experimental Rodents.

Testosterone affects brain functions and might explain some of the observed behavioral sex differences. Animal models may help in elucidating the possible involvement of sex hormones in these sex differences. The effects of testosterone have been intensively investigated, especially in anxiety models. Numerous experiments have brought inconsistent results with either anxiolytic or anxiogenic effects. Besides methodological variations, contradictory findings might be explained by the divergent metabolism of testosterone and its recognition by neurons during prenatal and postnatal development. Gonadectomy and subsequent supplementation have been used to study the role of sex hormones. However, the variable duration of hypogonadism might affect the outcomes and the effect of long-term androgen deficiency is understudied. Testosterone can be metabolized to dihydrotestosterone strengthening the androgen signaling, but also to estradiol converting the androgen to estrogen activity. Moreover, some metabolites of testosterone can modulate γ-aminobutyric acid and serotonergic neurotransmission. Here we review the currently available experimental data in experimental rodents on the effects of testosterone on anxiety during development. Based on the experimental results, females are generally less anxious than males from puberty to middle-age. The anxiety-like behavior of females and males is likely influenced by early organizational effects, but might be modified by activational effects of testosterone and its metabolites. The effects of sex hormones leading to anxiogenesis or anxiolysis depend on factors affecting hormonal status including age. The biological and several technical issues make the study of effects of testosterone on anxiety very complex and should be taken into account when interpreting experimental results.

The effects of a maternal advanced glycation end product-rich diet on somatic features, reflex ontogeny and metabolic parameters of offspring mice.

Maternal exposure to a Western type diet during pregnancy might predispose the offspring to manifestation of metabolic and behavioral disturbances in later life. The Western type diet contains large amounts of advanced glycation end products (AGEs). In humans and experimental rodents, the intake of an AGE-rich diet (AGE-RD) negatively affected glucose homeostasis, and initiated the production of reactive oxygen species. Rats consuming the AGE-RD presented changes in behavior. It remains unclear whether maternal intake of the AGE-RD might affect developmental plasticity in offspring. We examined early somatic (weight, incisor eruption, ear unfolding, and eye opening) and neuromotor development, oxidative status, insulin sensitivity (HOMA index) and locomotor activity assessed in PhenoTyper cages in the offspring of mice fed during pregnancy with either the AGE-RD (25% bread crusts/75% control chow) or control chow. Until weaning, the somatic development of offspring did not differ between the two dietary groups. The AGE-RD offspring manifested physiological reflexes (auditory startle, eye lid, ear twitch and righting reflexes) earlier. As young adults, the male offspring of the AGE-RD dams were heavier and less insulin sensitive compared with their control counterparts. The AGE-RD offspring showed higher locomotor activity during the active phase. Our data indicate that the maternal AGE-RD during pregnancy might accelerate the maturation of reflexes in offspring, predispose the male progeny to weight gain and affect their glucose homeostasis. These effects manifest without the direct consumption of the AGE-RD by offspring. Further work is needed to determine the mechanisms by which the maternal AGE-RD affects neurobehavioral pathways in offspring, as well as sex differences in adverse metabolic responses.

17ß-Estradiol treatment reversed left ventricular dysfunction in castrated male rats: an echocardiographic study.

No data are available on heart function in chronic testosterone deficiency and on the effect of estrogen treatment. Eighteen 4-week-old male Lewis rats were randomly divided into 3 groups (n = 6): 1 group of sham-operated rats and 2 groups of castrated rats. Sixty-six weeks after surgery, 1 castrated group received a dose of 17ß-estradiol (10 µg/kg per day) and the remaining 2 groups received a placebo subcutaneously for 14 days. Left ventricular (LV) systolic and diastolic functions were measured by transthoracic echocardiography. Castration decreased LV ejection fraction (9%) and fractional shortening (15%) and deteriorated LV diastolic function (94%). 17ß-Estradiol treatment increased LV ejection fraction (15%) and fractional shortening (31%) and improved LV diastolic function (48%). Plasma testosterone concentrations were decreased in both castrated groups. In conclusion, chronic testosterone deficiency induced LV systolic and diastolic dysfunction; these disorders were reversed by short-term treatment with 17ß-estradiol.

Does long-term androgen deficiency lead to metabolic syndrome in middle-aged rats?

Evidence from clinical observational studies and animal experiments suggests that hypogonadism is associated with the metabolic syndrome. In most of the experiments, androgen deficiency is induced by gonadectomy in the adulthood and relatively short-term effects of hypogonadism on metabolic parameters are usually observed. The purpose of this study was to evaluate the metabolic effects of long-term androgen deficiency starting before puberty in middle-aged male rats. The components of the metabolic syndrome were examined in male, female and gonadectomized male rats at the age of 18months. Sex differences were observed in plasma testosterone, cholesterol, high-density lipoproteins and also in body weight and in glycemia dynamics during oral glucose tolerance test. Gonadectomy and long-term hypogonadism did not affect most of the analyzed metabolic parameters such as blood pressure, glycemia, plasma insulin and uric acid. The only exception was the significantly higher liver enzymes in plasma and triacylglycerol in liver found in gonadectomized males. Except low-density lipoprotein, neither treatment of middle-aged males and females with letrozole, nor supplementation of estradiol as the metabolite of testosterone in gonadectomized male rats changed any of the observed metabolic parameters. Our results suggest that long-term hypogonadism started before puberty does not induce metabolic syndrome in middle-aged male rats, but may affect the liver. Sex differences in metabolic parameters in middle-aged rats are not mediated by testosterone. Whether hypogonadism predispose to metabolic syndrome in combination with other risk factors needs further clarification.

Sex differences and sex hormones in anxiety-like behavior of aging rats.

Sex differences in the prevalence of affective disorders might be attributable to different sex hormone milieu. The effects of short-term sex hormone deficiency on behavior, especially on anxiety have been studied in numerous animal experiments, mainly on young adult rats and mice. However, sex differences in aged animals and the effects of long-term hypogonadism are understudied. The aim of our study was to analyze sex differences in anxiety-like behavior in aged rats and to prove whether they can be attributed to endogenous sex hormone production in males. A battery of tests was performed to assess anxiety-like behavior in aged female, male and gonadectomized male rats castrated before puberty. In addition, the aged gonadectomized male rats were treated with a single injection of estradiol or testosterone or supplemented with estradiol for two-weeks. Female rats displayed a less anxious behavior than male rats in most of the conducted behavioral tests except the light-dark box. Long-term androgen deficiency decreased the sex difference in anxiety either partially (open field, PhenoTyper cage) or completely (elevated plus maze). Neither single injection of sex hormones, nor two-week supplementation of estradiol in gonadectomized aged male rats significantly affected their anxiety-like behavior in the elevated plus maze. In conclusion, our results confirm sex differences in anxiety in aged rats likely mediated by endogenous testosterone production in males. Whether long-term supplementation with exogenous sex hormones could affect anxiety-like behavior in elderly individuals remains to be elucidated.

Sex-dependent effects of letrozole on anxiety in middle-aged rats.

Aromatase catalyzes the conversion of testosterone to estradiol and is involved in the physiological effects of sex hormones on brain function. Animal experiments have shown that the aromatase inhibitor, letrozole, can induce anxiety in young ovariectomized females that are used as a model of aging. Whether or not these effects would be similar in intact middle-aged animals is unknown. The aim of our study was to analyze the effects of letrozole on anxiety in middle-aged rats of both sexes. Fifteen month old male and female rats were treated daily with either letrozole or vehicle for 2 weeks. The elevated plus maze was used to test anxiety-like behaviour. Sex differences were found not only in plasma concentrations of testosterone but also in the effects of letrozole treatment on plasma testosterone (P<.05). The interaction between sex and treatment was also proven in locomotor activity (P<.05) and time spent in the open arms of the elevated plus maze (P<.05). Letrozole-treated male rats spent 95% less time in the open arms of the elevated plus maze than the control rats did (P<.05) suggesting an anxiogenic effect of aromatase inhibition. This difference was not found between letrozole-treated and vehicle-treated females. In contrast to previous experiments on young animals, letrozole seems to induce anxiety in male but not in female middle-aged rats. This sex-specific effect might be related to sex differences of oestrogen and androgen signalling in aging brains. These results should be taken into account in clinical applications of letrozole, especially in men.

Puberty marks major changes in the hippocampal and cortical c-Fos activation pattern induced by NMDA receptor antagonists.

Non-selective and subunit (GluN2B)-specific N-methyl-d-aspartate receptor (NMDAR) antagonists represent promising alternative antidepressant drugs with fast onset of the therapeutic action. The neuronal activation pattern induced by NMDAR antagonists is well characterized by c-Fos expression analysis only in the adult rodent brain. In contrast, there is little information available regarding their effects during postnatal development. Here we performed a systematic c-Fos brain mapping of the non-selective NMDAR antagonist MK-801 and the GluN2B-specific antagonist Ro 25-6981 from postnatal day 16 (P16) to P40. We found significant regional differences with gender-specificity in the activation pattern compared to the adult. Surprisingly, in the hippocampus, MK-801 triggered at pre-pubertal stages (especially at P24) very strong c-Fos expression, followed by low levels after P30, the approximate time point of puberty onset in mice. The cortical distribution of MK-801-triggered c-Fos expression before puberty differed also substantially from the adult brain, showing high levels only in deep cortical layers at pre-pubertal stages. In comparison, the cortical activation induced by Ro 25-6981 diminished from high pre-pubertal levels and was in comparison with that triggered by MK-801 low in the hippocampus. These results reveal highly dynamic changes in the c-Fos activation pattern induced by NMDAR antagonists during puberty. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

No effect of testosterone on behavior in aged Wistar rats.

In men, aging is accompanied by a gradual decline in androgen secretion. Studies suggest beneficial effects of endogenous and exogenous testosterone on affective behavior and cognitive functions. The aim of this study was to describe behavioral and cognitive sex differences and to analyze the effects of long-term androgen deficiency in aged male rats. Thirty-months old rats divided into three groups (males, females and males gonadectomized as young adults) underwent a battery of behavioral tests assessing locomotor activity, anxiety, memory, anhedonia, sociability and depression-like behavior. No major effect of gonadectomy was found in any of the analyzed behavioral measures in male rats. The only consistent sex difference was confirmed in depression-like behavior with longer immobility time observed in males. In an interventional experiment, a single dose of testosterone had no effect on gonadectomized male and female rats in the forced swim test. In contrast to previous studies this comprehensive behavioral phenotyping of aged rats revealed no major role of endogenous testosterone. Based on our results long-term hypogonadism does not alter the behavior of aged male rats, neither does acute testosterone treatment. Whether these findings have any consequences on androgen replacement therapy in aged men remains to be elucidated.

Does the non-genomic effect of testosterone on social anxiety require the presence of a classical steroid receptor?

Steroid hormones may act through a rapid mechanism that does not require an intracellular steroid receptor and its effects on gene expression. In this study we have analysed this so-called non-genomic effect of testosterone on social anxiety in rats of both sexes using androgen and oestrogen receptor blockers. Male rats were divided into four groups: SHAM-CTRL (a sham operated group treated with oil as vehicle, n=10), SHAM-TST (a sham operated group treated with testosterone at a dose of 1 mg/kg, n=10), GDX-CTRL (a castrated group treated with oil, n=10) and GDX-TST (a castrated group treated with testosterone at a dose of 1 mg/kg, n=10). Female rats were divided into two groups: OVX-CTRL (an ovariectomized group treated with oil, n=10) and OVX-TST (an ovariectomized group treated with testosterone, n=10). The intracellular androgen receptor was blocked with flutamide and both intracellular oestrogen receptors were blocked with tamoxifen (a selective oestrogen receptor modulator). Rats were tested one hour after oil or testosterone administration in the social interaction test. Although the concentration of testosterone was higher in testosterone groups, no significant difference in social interaction was observed between the groups. In summary, in this first study focusing on the non-genomic effects of testosterone on social interaction no rapid effects of testosterone in adult rats were found. Further studies should analyse potential non-genomic effects of testosterone on other forms of social behaviour.