Ilaria Ceccarelli

Exposure to the estrogenic pollutant bisphenol A affects pain behavior induced by subcutaneous formalin injection in male and female rats.

We investigated the effects of perinatally administered bisphenol A (BPA), an environmental contaminant with estrogenic activity, on formalin-induced nociceptive responses. Male and female offspring of mother rats treated with BPA or oil were cross-fostered after birth to obtain three homogeneous groups: BPA-prenatal, receiving BPA via the placenta; BPA-postnatal, receiving BPA through suckling; OIL, control, from mothers receiving only peanut oil (vehicle). All groups underwent a pain test with s.c. formalin injection (50 microl, 10%) or were sham injected (pricking with a syringe needle) in the dorsal hind paw. They were immediately placed in an open field apparatus where pain responses (licking, flexing and paw-jerk) were recorded for 60 min. Corticosterone, testosterone and estradiol serum levels were determined in blood obtained at the end of the experiment. BPA-prenatal treatment induced an increase in licking duration in females and in flexing duration in both sexes in the first half of the test (0-30 min after formalin injection). BPA-postnatal treatment induced a decrease in paw-jerk frequency in males and females during the second part of the test (30-60 min after formalin injection). Plasma concentrations of corticosterone, estradiol and testosterone did not differ significantly between groups. These results indicate that exposure to BPA modified the activity of neural pathways and/or centers involved in nociception and pain in a sex-related and exposure-related manner.

Endocrine consequences of opioid therapy

Gonadal hormones are known to be affected by morphine and other opioids. In this paper, we summarize data collected in recent years which clearly indicate that the opioid-induced effects on steroid hormones depend on the opioid used and in some cases on the sex of the subject. Indeed morphine is able to reduce hormones like testosterone and cortisol in both male and female subjects in just a few hours, probably acting directly on peripheral glands. These depressant effects of morphine on hormones are also present in the treatment of surgical pain and are quickly reversible once opioid administration is suspended. Similar actions were also found to occur in experimental animals and in vitro in glial cells, further confirming the morphine-induced reduction of testosterone cell content. Testosterone and its metabolites are well known substances involved in the development and maintenance of the brain and all body structures. Thus when treating pain with opioids, their effects on hypothalamo-pituitary-gonadal and hypothalamo-pituitary-adrenal-related hormones must be considered and, where possible, hormone replacement therapy should be started.

Cross-sex hormone administration changes pain in transsexual women and men.

Abstract Chronic pain is gender‐related, since there is a clear predominance of one sex with respect to the other in most pain syndromes. Gonadal hormones are known to affect the occurrence and incidence of pain. Transsexuals receive cross‐sex hormones to develop and maintain somatic characteristics of the opposite sex: male to female transsexuals (MtF) are administered estrogens and anti‐androgens, while female to male transsexuals (FtM) are administered androgens. Hence, these subjects represent a model to study the relationship between sex hormones and pain. Questionnaires dealing with sociodemographic data and pain (occurrence, frequency, duration, intensity, location and associated symptoms) were administered to both MtF and FtM transsexuals under hormone treatment for sex reassignment for at least 1 year. Forty‐seven MtF and 26 FtM completed the questionnaires. Fourteen of the 47 MtF (29.8%) reported painful conditions, which in 11 subjects were not present before the beginning of hormone treatment. Pain consisted mainly of headaches and breast and musculoskeletal pain. Five subjects suffered from more than one pain condition. Sixteen of the 26 FtM (61.5%) reported pain. In 11 subjects, the pain was present before the beginning of hormone intake, and in 6 of them it improved after testosterone administration. These data suggest that marked changes in sex hormones affect the occurrence of pain in a high percentage of humans but not in all of them. Whether these effects are due to peripheral or central actions of sex steroids is unknown.

Gender-related effects of chronic non-malignant pain and opioid therapy on plasma levels of macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a cytokine produced by neuroendocrine and immune tissues that possesses several characteristics of a neuroendocrine mediator. Chronic pain is known to affect and to be affected by neuroendocrine and immune mechanisms. In the present study, the plasma levels of MIF and several hormones (cortisol, estradiol, testosterone) were determined to evaluate their mutual behaviour in controls and in chronic pain patients. Blood samples were collected from males and females divided into groups depending on their age (younger or older than 55) and health condition: (1) pain‐free control subjects; (2) chronic non‐malignant pain subjects. Moreover, two additional groups were added to evaluate the effects of short‐ and long‐term opioid administration: (3) short‐term opioid‐treated chronic pain patients and (4) long‐term opioid‐treated chronic pain patients (longer than 6 months). MIF in control/younger men was higher than in all the other control and chronic pain groups. MIF was lower in pain patients than in controls of both sexes. MIF was not changed by morphine administration; its levels remained lower in opioid‐treated subjects than in controls after both short‐ and long‐lasting administration. Chronic pain changed hormone plasma levels differently in male and female patients. MIF was positively correlated with testosterone and negatively with estradiol. These results demonstrate sex differences in the younger men and women and a strong pain‐induced decrease of MIF availability. These findings suggest the involvement of this cytokine in the sex differences observed in chronic pain conditions.

Bisphenol-A differently affects estrogen receptors-α in estrous-cycling and lactating female rats

The effect of long-term exposure to bisphenol-A (BPA) on estrogen receptor-alpha (ER) immunoreactivity was studied in the medial preoptic area, arcuate nucleus and the ventromedial nucleus of the hypothalamus of estrous cycling and lactating female rats. Pregnant/lactating or estrous cycling rats were exposed to BPA (40 mg/Kg/day) or peanut oil. Lactating females showed fewer ER-immunoreactive cells than non-lactating females in the medial preoptic area and ventromedial nucleus of the hypothalamus. BPA induced an increase in ER-immunoreactive cells in the medial preoptic nucleus irrespective of lactation. BPA only induced a decrease in ER-immunoreactive cells in the arcuate nucleus of the lactating group; oil induced an increase in ER-immunoreactive cells in the lactating with respect to non-lactating group. The results demonstrate that exposure of adult females to BPA modifies the number of ERs.

Behavioural and hormonal effects of restraint stress and formalin test in male and female rats.

The formalin test was used to measure the analgesia induced by restraint in male and female rats. Animals were restrained for 30 min or left undisturbed in their cage and then (1) killed immediately to collect blood for hormonal determinations; or (2) subcutaneously injected with formalin in the hind paw (or sham-injected), introduced to an open field for recording of behaviour, and killed at the end of this procedure. In both experiments, corticosterone was found to be higher in females. In Experiment 1, the ability of restraint to be stressful was confirmed by the increase in corticosterone in both sexes and by the decrease of testosterone in males. In Experiment 2, restraint-treatment induced a reduction in licking and flexing that was limited to the second phase. The reduction occurred in different periods and to a different degree in the two sexes; it was greater in females. Spontaneous behaviours showed sex differences in restraint-treated but not in formalin-treated animals. The results show that the hormonal effects observed after restraint are not present after the formalin test and that the marked analgesia observed with phasic painful stimuli does not occur with a longer-lasting one such as that induced by formalin, after which only partial and short-lasting effects were observed.

Testosterone affects formalin-induced responses differently in male and female rats ☆

To evaluate the role of testosterone in pain modulation, we subcutaneously injected male and female rats with testosterone propionate (TP, 5 mg/kg in oil) or oil for 6 days; on the seventh day, all rats were subjected to the formalin test (10%, 50 microl). Behaviours were recorded in an open field (60 min). At the end of the formalin test, the rats were anaesthetized to collect blood from the abdominal aorta. Among the formalin-induced responses, licking was higher in females than males and was decreased by TP in females; jerking and flexing were only slightly affected by treatment. TP increased testosterone plasma levels in both sexes. These results indicate a role of testosterone in modulating formalin-induced responses. The effects appear to be different in males and females.

Single opioid administration modifies gonadal steroids in both the CNS and plasma of male rats

While morphine remains one of the most widely used opioids for the treatment of painful conditions, other opioids are also commonly employed. Because of the interactions between opioids and gonadal hormones, in particular opioid-induced hypogonadism, this study investigated the effects of widely used opioids on plasma testosterone and estradiol levels and brain testosterone levels in male rats. Animals were s.c. injected with two concentrations of morphine (5 or 10 mg/kg), fentanyl (0.05 or 0.1 mg/kg), tramadol (10 or 40 mg/kg), buprenorphine (0.05 or 0.1 mg/kg) or saline (0.7 ml/kg). Four or 24 h after treatment, the rats were deeply anesthetized to collect blood samples from the abdominal aorta and to perfuse the brains with saline. Plasma and brain hormone levels were measured by radioimmunoassay. In rats studied 4 h after treatment, all the opioids except tramadol 10 mg/kg decreased plasma testosterone in comparison with saline administration. At the same time, plasma estradiol levels were lower than control in the groups treated with the low doses of morphine, tramadol and buprenorphine, while estradiol remained at control levels in the other groups. Twenty-four hours after treatment, plasma testosterone levels were different (higher) than control in the animals treated with the low doses of morphine, fentanyl and buprenorphine. Estradiol was lower than control in the low dose groups, while the high doses did not produce any changes with respect to control. Four hours after treatment, brain testosterone was drastically decreased in all groups except buprenorphine, in which it remained at control levels. All groups returned to control levels at 24 h after treatment. In conclusion, opioids exert important effects on plasma and CNS sex hormone levels. The different magnitude and time-course of the effects of the different opiates on testosterone and estradiol levels are likely due to their different mechanism of action.

Effects of long-term exposure of lemon essential oil odor on behavioral, hormonal and neuronal parameters in male and female rats

Behavioral, hormonal and neuronal responses to prolonged exposure to the volatile components of essential oil (EO) extracted from citrus lemon were investigated in male and female rats. Animals were exposed to the lemon essence for 2 weeks while in their cage. Anxiety was then determined with the elevated plus-maze apparatus while nociception was evaluated with a phasic thermal pain stimulus (plantar test) and with a chemical pain stimulus (formalin test). At the end of the experimental sessions, brain areas were dissected to measure beta-endorphin (beta-EP) concentrations in the hypothalamus and periaqueductal gray matter (PAG). Blood samples were collected to determine corticosterone plasma levels. In both sexes, prolonged EO exposure decreased the time spent in the open arms of the plus-maze apparatus. EO-exposed males and females showed higher thermal nociceptive thresholds than controls when tested with the plantar test apparatus. EO exposure induced female-specific decreases in formalin-induced pain behaviors during the formalin test. beta-EP concentrations in the hypothalamus and PAG were affected by EO. Corticosterone was lower in EO-exposed animals of both sexes than in their controls. These results suggest that long-term exposure to lemon EO can induce significant, at times sex-specific, changes in neuronal circuits involved in anxiety and pain.

Effects of novelty and pain on behavior and hippocampal extracellular ACh levels in male and female rats

In vivo microdialysis was used to assess the effects of novelty and pain on hippocampal ACh release in male and female rats. Experiments were carried out during the dark phase and consisted of 2 days of tests: on Day 1, after Baseline 1, animals were exposed to a new cage (Novelty) to which, 30 min later, a plastic cylinder (Object) was introduced. On Day 2, after Baseline 2, the Formalin test (50 microl of formalin 10%, s.c. injected in the dorsal hindpaw) was carried out in the animals home cage. All behaviors were recorded. The extracellular levels of ACh in the dorsal hippocampus were estimated, in 10-min samples, by assay of ACh in the dialysates by HPLC. On Day 1 the raw values of ACh were higher in females than in males, but no sex difference was present when the percentage of change was considered. In both sexes the Novelty and Object tests induced an increase in ACh levels with respect to Baseline. Higher levels of exploration were present in females than males during the first 10 min of Novelty. On Day 2, ACh release increased in both sexes during the Formalin test. No sex difference in either ACh raw values or the percentages of change were found. Females showed higher levels of licking and lower levels of activity than males. The present study shows that novelty and pain induce similar hippocampal cholinergic activation in male and female rats but different behaviors. The results are discussed in light of the several anatomical and functional sex differences present in the hippocampus.