Cosimo Massafra

Nucleated red blood cell count at birth as an index of perinatal brain damage

OBJECTIVE The prognostic value of the nucleated red blood cell count at birth with respect to perinatal brain damage and neonatal outcome was assessed in infants at high risk of having neurologic damage. STUDY DESIGN The nucleated red blood cell count at birth, pulsed Doppler ultrasonography in the cerebral arteries, cranial fontanelle sonograms, and neurodevelopmental status were evaluated in 337 newborn infants. RESULTS The nucleated red blood cell count at birth was significantly higher (1) in neonates with abnormal Doppler ultrasonographic parameters for the cerebral arteries at 48 to 72 hours after birth than in healthy neonates, (2) in 6-month-old infants with sequelae of hypoxic-ischemic encephalopathy than in healthy infants, and (3) in 3-year-old children with abnormal developmental status than in those with no abnormalities at follow-up. Significant correlations were observed between the nucleated red blood cell count and gestational age, Apgar score at 1 and 5 minutes, pH, base deficit, fraction of inspired oxygen, blood oxygen content, and birth weight. CONCLUSIONS The nucleated red blood cell count at birth not only reflects a response of the infant to perinatal hypoxia but is also a reliable index of perinatal brain damage.

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 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.

The behavioral and neuronal effects induced by repetitive nociceptive stimulation are affected by gonadal hormones in male rats.

&NA; The role of gonadal hormones in inducing long‐term modifications in response to transient nociceptive stimuli was investigated in adult male rats. Three weeks after gonadectomy or sham surgery, animals were randomly divided into groups to be exposed to sham (only a prick in the dorsal hind paw) or formalin treatment (50 &mgr;l, 5% s.c. in the dorsal hind paw) once a week for the following 3 weeks. In gonadectomized animals the formalin‐induced responses (licking, flexing and jerking of the injected paw) did not differ from those of intact animals after the first formalin injection. However, their levels were higher after the second or third injections. Indeed, in intact animals the formalin‐induced responses progressively decreased, being significantly lower after the third injection than after the first; in gonadectomized animals, the formalin‐induced responses did not change with repetition of the formalin treatment. In intact rats, c‐Fos expression in the paraventricular nucleus of the thalamus and arcuate nucleus of the hypothalamus remained at control levels or decreased in animals injected two or three times with formalin; in gonadectomized rats, c‐Fos expression increased with repetition of the noxious stimulation, reaching the highest levels in animals injected three times with formalin. These results show that male gonadal hormones have an inhibitory, adaptive effect on the behavioral and neuronal responses to repeated nociceptive stimulation.

Long-term ovariectomy changes formalin-induced licking in female rats: the role of estrogens

Gonadal hormones have been shown to exert modulatory effects on nociception and analgesia. To investigate the role of gonadal hormones in the response by female rats to both phasic and persistent nociceptive stimulation, we evaluated the effects of long-term ovariectomy (OVX, 6 months) on the thermal pain threshold and on formalin-induced responses. The thermal pain threshold was evaluated with the plantar test apparatus, while persistent pain was induced by a subcutaneous injection of dilute formalin (50 microliter, 10%) in the dorsal hind paw. The formalin test was carried out in an open field apparatus where the animals spontaneous behavior and formalin-induced responses (licking duration, flinching frequency and flexing duration of the injected paw) were recorded for 60 min. Estradiol and corticosterone plasma levels were determined in blood collected from the anesthetized animals at the end of the test. In OVX females, the duration of formalin-induced licking was longer than in Intact females during both the first and the second phase; flinching and flexing did not differ from Intact. The thermal pain threshold was only slightly affected by OVX. Estradiol and corticosterone were lower in OVX females than Intact ones. These data indicate that long-term depletion of gonadal hormones in female rats modulates the pain-induced behavioral responses related to supraspinal neural circuits (licking of the injected paw) rather than more spinally mediated responses such as formalin-induced flinching and withdrawal latency in the plantar test.

Gender‐related differences in erythrocyte glutathione peroxidase activity in healthy subjects

objective The antioxidant property of oestrogens may partly explain the gender differences in atherosclerotic heart disease and a reduction in the incidence of coronary heart disease, as well as mortality from cardiovascular disease in women undergoing postmenopausal oestrogen therapy. In the present study, we tested the hypothesis that the antioxidant glutathione peroxidase (GSH‐Px) erythrocyte activity is gender related and is correlated with oestradiol serum levels.

Variations in erythrocyte antioxidant glutathione peroxidase activity during the menstrual cycle

Antioxidant enzymes are an important part of the defence mechanisms against free radical damage. Little is known, however, about the relationship between sex steroid hormones and cellular antioxidant systems. We have investigated the effect of physiological sex steroid changes on the erythrocyte antioxidant glutathione peroxidase (GSH‐Px) activity during the menstrual cycle in a population of healthy normomenorrhoic women.

ANTIOXIDANT ERYTHROCYTE ENZYME ACTIVITIES DURING ORAL CONTRACEPTION

Nineteen healthy young women, non-smokers and of normal weight, used a combined oral contraceptive, consisting of 20 micrograms ethinylestradiol and 150 micrograms desogestrel, for 9 cycles and were investigated before starting oral contraceptive use, and during the 3rd, 6th and 9th cycle. In all cases, the antioxidant erythrocyte enzyme activities, superoxide-dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px), were determined. Pyruvate-kinase (PK) activity was also considered as a predictable index of the age and constancy of the erythrocyte population. A steady, significant increase in GSH-Px and CAT enzyme activities was found. No significant variations were found in SOD and PK enzyme levels. These findings clearly suggest that the use of combined oral contraceptives leads to an increase in antioxidant defenses. The specific physiological and biochemical mechanisms of this response merit further investigation.

Estrogen and μ-opioid receptor antagonists counteract the 17β-estradiol-induced licking increase and interferon-γ reduction occurring during the formalin test in male rats

&NA; Women have a higher incidence of chronic pain syndromes than men and are generally more sensitive to experimental pain. Numerous studies have shown that the female gonadal hormones, estrogens, can profoundly affect the nervous and immune systems, including mechanisms involved in pain and nociception. In the present study, we used antagonists of estrogen receptors (ER) or &mgr;‐opioid receptors (&mgr;OR) to evaluate the effects of estrogens on formalin‐induced behavioural and immune responses in male rats. After two days of priming with 17&bgr;‐estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 &mgr;l, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a &mgr;OR antagonist (&bgr;‐funaltrexamine, FNA) or saline. The spontaneous behaviours, pain‐related behaviours and interferon‐&ggr; (IFN‐&ggr;) production by peripheral blood mononuclear cells were studied in all groups. We found that central administration of estradiol increased the amount of licking of the formalin‐injected paw in the second phase of the formalin test. Whereas ICI and FNA had no effect on pain behaviour in saline‐pre‐treated animals, both antagonists reversed the estradiol‐induced increase in licking. The immune system was differently affected by formalin and estradiol treatment. Indeed, formalin injection per se decreased IFN‐&ggr; production; estradiol had no effect on sham‐injected animals but strongly reduce the decrease of IFN‐&ggr; production in formalin‐injected animals. The results demonstrate that centrally acting estrogens affect ER‐ and &mgr;OR‐mediated pain processing and influence immune function.