URB597 attenuates stress-induced ventricular structural remodeling by modulating cytokines, NF-κB, and JAK2/STAT3 pathways in female and male rats.

Endocannabinoids act as a stress response system; simultaneously, the modulation of this system has emerged a novel approach for the therapy of cardiovascular disorders. We investigated the protective effects of the chronic administration of the fatty acid amide hydrolase inhibitor URB597 on morphology, pro-inflammatory and anti-inflammatory cytokine, the cytoplasm-nuclear distribution of JAK2/STAT3, and NF-κB and Nrf2/HO-1 signaling in the left ventricle of female and male rats exposed to chronic unpredictable stress. Our results show that URB597 treatment exhibits an antidepressant-like effect, decreases the heart/body weight ratio, prevents the hypertrophy of cardiomyocytes, and reduces the increased level of IL-6 in the wall of the left ventricle of stressed female and male rats. The phosphorylation levels of JAK2 and STAT3 in the ventricle of male rats treated with URB597 were declined, whereas in female rats the decrease of STAT3 was observed. In addition, URB597 reduced increased NF-κB in both females and males and increased the expression of Nrf2 and HO-1 protein in the cytosol of male rats, whereas did not affect their levels in females. Cardioprotective effects of URB597 could be linked to the ability to inhibit the JAK2 in males and the STAT3 inflammatory signaling pathways in both females and males.

Age and Sex Characteristics of the Blood Cytokine Profile in Rats Subjected to Prenatal Stress.

Parameters of blood cytokine profile in male and female rats subjected to prenatal stress on the model of swimming in cold water (10°C, 5 min, days 10-16 of gestation) were studied. Prenatal stress had no significant effects on the blood levels of IL-6 and IL-10 cytokines. The blood concentration of proinflammatory cytokine TNFα in 60-day-old rats was higher than in age-matched controls. Stress led to a lower level of anti-inflammatory IL-4 in the blood of 30-day-old males compared to controls. In female rats subjected to prenatal stress, the concentration of IL-4 decreased on day 21, but increased by day 60 of postnatal ontogeny. Specific effects of prenatal stress on the blood cytokine profile in male and female animals at different periods of ontogeny were revealed. Different and even opposite changes in blood cytokine levels could be largely mediated by sex- and age-specific features of immune functions after prenatal stress.

Panic-like responses of female Wistar rats confronted by Bothrops alternatus pit vipers, or exposure to acute hypoxia: Effect of oestrous cycle.

Anxiety-related diseases are more than twice as common in women than in men, and in women, symptoms may be exacerbated during the late luteal phase of the menstrual cycle. Despite this, most research into the underlying mechanisms, which drives drug development, have been carried out using male animals. In an effort to redress this imbalance, we compared responses of male and female Wistar rats during exposure to two unconditioned threatening stimuli that evoke panic-related defensive behaviours: confrontation with a predator (Bothrops alternatus) and acute exposure to hypoxia (7% O2 ). Threatened by venomous snake, male and female rats initially displayed defensive attention, risk assessment, and cautious interaction with the snake, progressing to defensive immobility to overt escape. Both males and females displayed higher levels of risk assessment but less interaction with the predator. They also spent more time in the burrow, displaying inhibitory avoidance, and more time engaged in defensive attention, and non-oriented escape behaviour. In females, anxiety-like behaviour was most pronounced in the oestrous and proestrus phases whereas panic-like behaviour was more pronounced during the dioestrus phase, particularly during late dioestrus. Acute hypoxia evoked panic-like behaviour (undirected jumping) in both sexes, but in females, responsiveness in late dioestrus was significantly greater than at other stages of the cycle. The results reveal that females respond in a qualitatively similar manner to males during exposure to naturally occurring threatening stimuli, but the responses of females is oestrous cycle dependent with a significant exacerbation of panic-like behaviour in the late dioestrus phase.

Neonatal pain modulates in adolescent rats the antinociceptive effects of fluoxetine and buspirone administrated to their depressive dams during gestation.

Previously, we have shown that the administration of a selective serotonin reuptake inhibitor fluoxetine or a 5-HT1A receptor agonist buspirone to stressed rats during gestation causes in the offspring alleviation of formalin-induced pain, strengthened by prenatal stress. We have also found that neonatal inflammatory pain strengthens formalin-induced pain in prenatally unstressed rats in later life. In the present study, we investigated the effect of neonatal inflammatory pain on the time-course of the biphasic pain response in the formalin test in prenatally stressed adolescent rats of both sexes to evaluate whether neonatal pain affects the antinociceptive properties of these drugs administered to their depressed mothers during gestation. Our findings demonstrate that neonatal pain modulates in prenatally stressed rats the antinociceptive effect of fluoxetine and buspirone depending on the level of organization of pain response in the central nervous system, the phase of the time-course of the formalin-induced pain, and sex of the rat.

Influence of in utero di-n-hexyl phthalate and di-cyclohexyl phthalate exposure on the endocrine glands and T3, T4, and TSH hormone levels of male and female rats: Postnatal outcomes.

The present study was designed to evaluate the effects of di-n-hexyl phthalate (DHP) and di-cyclohexyl phthalate (DCHP) on endocrine organs in rats. Oil control, 20-, 100-, and 500 mg/kg dose groups were selected and administered to pregnant rats on gestational days 6-19 by oral gavage. The neonatal stages of rats continued until postnatal day 20 and the- juvenile stages of rats continued until postnatal day of 32. The rats were allowed to mature until the neonatal and juvenile stages and there after, they were divided into four groups corresponding to the treatment levels. Body and organ weights were recorded, serum was collected, and thyroid, pancreas, pituitary gland, and adrenal gland were removed. There was a decrease in body weights in the 20- and 500mg/kg DHP and in the 20-mg/kg DCHP dose groups in neonatal male rats. In contrast, for female rats, there was an increase in body weights in the 100-mg/kg DCHP dose group and there was a decrease in body weights in the 500-mg/kg DHP dose group. Body weights were increased at 20 and 500 mg/kg in the DHP-exposed juvenile male rats. Serum thyroid-stimulating hormone (TSH) levels were increased in neonatal male rats, while they were increased in the 100-mg/kg DHP group of neonatal and juvenile female rats. Serum triiodothyronine (T3) levels were increased at the high dose of DHP for neonatal male rats and at the low and high dose levels of DCHP for female rats. Serum thyroxine (T4) levels were increased in neonatal rats for DHP. Also, some histopathological changes were observed in the thyroid, pancreas, adrenal, and pituitary gland. In conclusion, it was shown that DHP and DCHP caused negative effects on T3, T4, and TSH hormone levels.

Gender effect on the pharmacokinetics of thymoquinone: Preclinical investigation and in silico modeling in male and female rats.

Thymoquinone is the most biologically active constituent of Nigella sativa (black seed). A monoterpene compound chemically known as 2-methyl-5-isopropyl-1, 4-quinone. In this study, the gender-dependent pharmacokinetic behavior of thymoquinone in rats was investigated. Thymoquinone was administered orally (20 mg/kg) and intravenously (5 mg/kg) to male and female rats and blood samples were collected at specific time points. Plasma concentration-time curves were plotted and pharmacokinetic parameters were determined using the non-compartmental analysis. In addition, simulations of steady state concentrations of thymoquinone in male and female rats were performed using GastroPlus PK software. After oral administration, the maximum plasma concentration (Cmax) of thymoquinone was 4.52 ±â€¯0.092 µg/ml in male rats and 5.22 ±â€¯0.154 µg/ml in female rats (p = 0.002). Similarly, after intravenous administration, the Cmax was 8.36 ±â€¯0.132 µg/ml in males and 9.51 ±â€¯0.158 µg/ml in females (p = 0.550). The area under the plasma concentration-time curve (AUC)0-∞ following oral dosing was 47.38 ±â€¯0.821 µg/ml·h in females and 43.63 ±â€¯0.953 µg/ml·h in males (p = 0.014). Pharmacokinetics and plasma concentration vs. time profiles for multiple oral doses of thymoquinone in rats were predicted using a simulation model to compare the simulation results with the experimental plasma pharmacokinetic data. The differences observed in thymoquinone pharmacokinetics between male and female rats after a single dose were not evident for the simulated steady-state parameters. The findings suggest that the gender difference does not seem to play a significant role in thymoquinone disposition at steady state.

Sex differences in the sympathoexcitatory response to insulin in obese rats: role of neuropeptide Y.

KEY POINTS: Intracerebroventricular insulin increased sympathetic nerve activity (SNA) and baroreflex control of SNA and heart rate more dramatically in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced lumbar SNA (LSNA) responses were associated with reduced tonic inhibition of LSNA by neuropeptide Y (NPY) in the PVN. However, PVN NPY injection decreased LSNA similarly in obesity prone/obesity resistant/control rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the arcuate nucleus became resistant to the inhibitory effects of insulin. A high-fat diet did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced 'selective sensitization' of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. ABSTRACT: Obesity increases sympathetic nerve activity (SNA) in men but not women; however, the mechanisms are unknown. We investigated whether intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high-fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane anaesthesia. The LSNA response was magnified in OP males but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males as a result of a decrease in NPY inputs. By contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on the sympathoexcitatory actions of insulin and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.

[Toxicity of d-Arabinose in Male and Female Rats].

In order to investigate the feasibility of using the rare sugar d-arabinose as a functional food material, we examined its toxicity in rats. In an acute toxicity study, the lethal dose (LD50) was calculated to be 12.1 g/kg in males and 11.6 g/kg in females. On the other hand, in a short-term toxicity test, rats developed diarrhea when given feed containing 5% d-arabinose, which was the minimum amount added, so the maximum nontoxic amount was estimated to be less than 5%. Thus, the toxicity of d-arabinose is stronger than that of another rare sugar, d-psicose, which was reported to show no toxicity when added at 10% in the diet. Further studies are needed to establish whether d-arabinose would be safe for use as a functional ingredient.

Sex differences in the effect of acute peripheral IL-1ß administration on the brain and serum BDNF and VEGF expression in rats.

The present study was designed to evaluate, for the first time, the potential sex differences in BDNF and VEGF systems under normal conditions and in response to IL-1ß given ip. Peripheral overproduction of this cytokine mediates the pathophysiology of various acute neuroinflammatory states. Until now, the effect of IL-1ß on VEGF expression in rat brain structures and its serum level has not been examined. In male and female rats, the BDNF and VEGF mRNA expression, and BDNF level were evaluated in the amygdala, hippocampus, hypothalamus and pituitary gland. The VEGF levels were determined in the pituitary. Serum BDNF and VEGF levels were also measured. The pituitary BDNF mRNA, and BDNF and VEGF levels were higher in females than in male rats whereas in males, the BDNF levels were higher in the other brain structures. The serum BDNF concentration was similar in both groups but VEGF levels were enhanced in females. Following IL-1ß (50µg/kg ip.) administration, a higher serum IL-1ß level was detected in females than in males. In male rats, IL-1ß decreased BDNF mRNA in all the brain structures, except for the pituitary, and VEGF mRNA in the amygdala. In opposite, IL-1ß challenge in females increased the pituitary VEGF mRNA and serum BDNF and VEGF levels. These results suggest that in females BDNF and VEGF may play a more important role in the pituitary function. In males, amygdala trophic system seems to be especially sensitive to the enhanced peripheral IL-1ß production. Our findings point to the need to consider sex-related differences to be able to draw reliable conclusions about changes in BDNF and VEGF levels during inflammation.

Differences in Evolution of Epileptic Seizures and Topographical Distribution of Tissue Damage in Selected Limbic Structures Between Male and Female Rats Submitted to the Pilocarpine Model.

Epidemiological evidence shows that clinical features and comorbidities in temporal lobe epilepsy (TLE) may have different manifestations depending on the sex of patients. However, little is known about how sex-related mechanisms can interfere with the processes underlying the epileptic phenomenon. The findings of this study show that male rats with epilepsy in the pilocarpine model have longer-lasting and more severe epileptic seizures, while female rats have a higher frequency of epileptic seizures and a greater number of seizure clusters. Significant sex-linked pathological changes were also observed: epileptic brains of male and female rats showed differences in mass reduction of 41.8% in the amygdala and 18.2% in the olfactory bulb, while loss of neuronal cells was present in the hippocampus (12.3%), amygdala (18.1%), and olfactory bulb (7.5%). Another important sex-related finding was the changes in non-neuronal cells with increments for the hippocampus (36.1%), amygdala (14.7%), and olfactory bulb (37%). Taken together, our study suggests that these neuropathological changes may underlie the differences in the clinical features of epileptic seizures observed in male and female rats.

Age-related cognitive decline in rats is sex and context dependent.

Previously, we had observed age-related cognitive decline in male rats compared to adolescent and adult rats. This was shown in both a multi-branched maze test (MBM), as well as in the Morris water maze test (MWM). In the present study, we compared the behavior of similar age groups in both male and female rats using the same paradigms. The results confirmed the increase in errors and time spent in MBM in aged male rats compared to other age groups. However, no such differences were observed in female rats. In the acquisition phase of MWM, aged male rats did not differ significantly from the other two groups in terms of time spent in quadrants, whereas aged female rats spent significantly more time in quadrants compared to the other 2 age groups. Aged male rats also travelled significantly more than the other 2 age groups during the acquisition phase, whereas no such differences were observed in female rats. In both short term (30 min post acquisition) and long term (24 h after acquisition) retrieval phases of MWM, significant gender-related differences were also observed in all age groups. These findings suggest gender- and context-dependent alterations in cognitive functions during aging.

Inhibition of the fatty acid amide hydrolase changes behaviors and brain catecholamines in a sex-specific manner in rats exposed to chronic unpredictable stress.

Sex differences in the susceptibility to chronic unpredictable stress (CUS) and the effects of fatty acid amide hydrolase (FAAH) inhibitor URB597 in rats have been investigated in this study. In this context, we investigated the effects of prolonged treatment with URB597 on behavior, pro-inflammatory interleukin-6 (IL-6) and anti-inflammatory interleukin-10 (IL-10), catecholamine content and the expression of its biosynthetic and degrading enzymes in the hippocampus, hypothalamus and medial prefrontal cortex (mPFC) of rats subjected to CUS. The results show that CUS increases anxiety-like and depression-like behaviors but it was more pronounced in females. The data suggests sex differences in brain cytokines, catecholamines and their enzymes of synthesis and degradation expression in response to CUS. Our findings indicate that the FAAH inhibitor URB597 differently regulated catecholamine levels and its enzymes of synthesis and degradation in the examined brain areas of male and female rats. URB treatment failed to reduce anxiety or restore reduced norepinephrine and did not affect enzymes of catecholamine degradation in the mPFC, hippocampus and hypothalamus of CUS female rats. These studies are important because they investigate the neurochemical consequences of stress related mood disorders that might lead to the development of sex specific treatments.

Treatment with 1-benzylpiperazine (BZP) during adolescence of male and female hooded rats exposed to environmental enrichment: Subsequent behavioral outcomes.

From 30 days after birth until the completion of the study, male and female rats were caged in same-sexed twos or threes either with (enriched cages, EC) or without several objects for them to explore (standard cages, SC). From 41 to 50 days of age (late adolescence), they received a daily intraperitoneal injection of saline, or 10 or 20 mg/kg of the monoaminergic agonist drug of abuse, 1-benzylpiperazine (BZP). Ten days later (PND60+), their behavior was observed over several days in an open field, an elevated plus maze, a light-dark box and (to assess short-term memory) a Y maze in which one of the arms had been changed in brightness between two trials. These tests were repeated from 40 days after PND60+, namely PND100+. While open-arm occupancy at PND100+ in the plus maze was lower following both doses of the drug for SC rats only, other examples of BZP-related heightened anxiety were confined to EC rats. This suggested that enrichment had enhanced rather than reduced any anxiogenic effects of the drug treatment. There was no plausible evidence of BZP-associated impaired spatial memory required to recognize the changed novel Y-maze arm. Instead, changes in novelty preferences or neophobia-related anxiety were most likely. While there were also some examples of sex and age differences in the later effects of BZP, in most cases these were evident at both ages following treatment with both BZP doses. A number of overall BZP, cage, sex and age differences, independent of enrichment effects, were also observed.

Functional inactivation of the orbitofrontal cortex disrupts context-induced reinstatement of alcohol seeking in rats.

BACKGROUND: The high rate of relapse to drug use remains a central challenge to treating drug addiction. In human and rat models of addiction, environmental stimuli in contexts associated with previous drug use can provoke a relapse of drug seeking. Pre-clinical studies have used the ABA renewal procedure to study context-induced reinstatement of drug seeking. In the current study, we studied the role of the orbitofrontal cortex (OFC) in context-induced reinstatement to alcohol. METHODS: We trained male and female rats to self-administer alcohol in context A, extinguished drug-reinforced responding in a distinct context B, and assessed context-induced reinstatement in context A or B (control group). Next, we determined the effect of context-induced renewal of alcohol-seeking behavior on the expression of Fos (a neuronal activity marker) in the OFC. Finally, we determined the effect of reversible inactivation by GABAa and GABAb receptor agonists (i.e., muscimol and baclofen, respectively) in the OFC. RESULTS AND CONCLUSIONS: There were no differences between male and female rats in context-induced reinstatement of alcohol-seeking behavior. Re-exposure to Context A, but not Context B, reinstated alcohol-seeking behavior and increased expression of the neural activity marker Fos in the OFC. Reversible inactivation of the OFC with muscimol and baclofen attenuated context-induced reinstatement. Our data indicated that the OFC mediates context-induced reinstatement of alcohol-seeking behavior.

Amphetamine treatment affects the extra-hypothalamic vasopressinergic system in a sex- and nucleus-dependent manner.

The lateral septum (LS), a brain structure implicated in addictive behaviours, regulates the activation of dopaminergic neurones in the ventral tegmental area. Vasopressinergic projections from the extended amygdala to the LS, which are sexually dimorphic, could be responsible for the vulnerability to addiction in a sex-dependent manner. The present study aimed to investigate the modulatory effects of amphetamine (AMPH) on the expression of vasopressin (AVP) in the vasopressinergic extra-hypothalamic system in sensitised male and female rats. Adult male and female Sprague-Dawley rats underwent an AMPH-locomotor sensitisation protocol. Acute AMPH increased AVP mRNA expression in the medial amygdala (MeA), whereas AMPH-induced sensitisation increased AVP mRNA expression in the bed nucleus of the stria terminalis (BNST) only in females. Interestingly, the increase in AVP expression in BNST was higher in oestrus females compared to dioestrus females and acute AMPH resulted in a decrease in AVP levels in the LS, only in males. Thus, there are complex and region-specific interactions between AMPH and the extra-hypothalamic vasopressinergic system in the brain, underlying possible alterations in different behaviours caused by acute and chronic AMPH exposure.

No Evidence for Sex Differences in the Electrophysiological Properties and Excitatory Synaptic Input onto Nucleus Accumbens Shell Medium Spiny Neurons.

Sex differences exist in how the brain regulates motivated behavior and reward, both in normal and pathological contexts. Investigations into the underlying neural mechanisms have targeted the striatal brain regions, including the dorsal striatum and nucleus accumbens core and shell. These investigations yield accumulating evidence of sexually different electrophysiological properties, excitatory synaptic input, and sensitivity to neuromodulator/hormone action in select striatal regions both before and after puberty. It is unknown whether the electrical properties of neurons in the nucleus accumbens shell differ by sex, and whether sex differences in excitatory synaptic input are present before puberty. To test the hypothesis that these properties differ by sex, we performed whole-cell patch-clamp recordings on male and female medium spiny neurons (MSNs) in acute brain slices obtained from prepubertal rat nucleus accumbens shell. We analyzed passive and active electrophysiological properties, and miniature EPSCs (mEPSCs). No sex differences were detected; this includes those properties, such as intrinsic excitability, action potential afterhyperpolarization, threshold, and mEPSC frequency, that have been found to differ by sex in other striatal regions and/or developmental periods. These findings indicate that, unlike other striatal brain regions, the electrophysiological properties of nucleus accumbens shell MSNs do not differ by sex. Overall, it appears that sex differences in striatal function, including motivated behavior and reward, are likely mediated by other factors and striatal regions.