Significant levels of neuroactive steroids are still detected in the nervous system of rodents after the removal of peripheral steroidogenic glands. However, the influence of the plasma levels of gonadal steroids on the levels of neuroactive steroids in the nervous system has not so far been clarified in detail. Accordingly, by liquid chromatography tandem mass spectrometry, we have analysed the levels of neuroactive steroids in the sciatic nerve, in three central nervous system (CNS) regions (i.e. cerebellum, cerebral cortex and spinal cord) and in the plasma of male and female animals. The levels present in gonadally intact animals were compared with those present in short- and long-term gonadectomised animals. We observed that: (i) changes in neuroactive steroid levels in the nervous system after gonadectomy do not necessarily reflect the changes in plasma levels; (ii) long-term gonadectomy induces changes in the levels of neuroactive steroids in the peripheral nervous system (PNS) and the CNS that, in some cases, are different to those induced by short-term gonadectomy; (iii) the effect of gonadectomy on neuroactive steroid levels is different between the PNS and the CNS and within different CNS regions; and (iv) the effects of gonadectomy on neuroactive steroid levels in the nervous system show sex differences. Altogether, these observations indicate that the nervous system adapts its local levels of neuroactive steroids in response to changes in gonadal hormones with sex and regional specificity and depending on the duration of the peripheral modifications.

Neuroactive steroids act in the peripheral nervous system as physiological regulators and as protective agents for acquired or inherited peripheral neuropathy. In recent years, modulation of neuroactive steroids levels has been studied as a potential therapeutic approach to protect peripheral nerves from damage induced by diabetes. Nuclear receptors of the liver X receptor (LXR) family regulate adrenal steroidogenesis via their ability to control cholesterol homeostasis. Here we show that rat sciatic nerve expresses both LRX␣ and ␤ isoforms and that these receptors are functional. Activation of liver X receptors using a synthetic ligand results in increased levels of neurosteroids and protection of the sciatic nerve from neuropathy induced by diabetes. LXR ligand treatment of streptozotocin-treated rats increases expression in the sciatic nerve of steroidogenic acute regulatory protein (a molecule involved in the transfer of cholesterol into mitochondria), of the enzyme P450scc (responsible for conversion of cholesterol into pregnenolone), of 5␣-reductase (an enzyme involved in the generation of neuroactive steroids) and of classical LXR targets involved in cholesterol efflux, such as ABCA1 and ABCG1. These effects were associated with increased levels of neuroactive steroids (e.g., pregnenolone, progesterone, dihydroprogesterone and 3␣-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na ϩ , K ϩ -ATPase activity. These results suggest that LXR activation may represent a new pharmacological avenue to increase local neuroactive steroid levels that exert neuroprotective effects in diabetic neuropathy.

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The aim of the present study was to confirm that olive oil phenols reduce human platelet aggregability and to verify the hypothesis that cAMP-and cGMP-phosphodiesterases (PDE) could be one of the targets of the biological effect. Four extracts from oils characterized by a high phenol content (HPE), and low phenol levels (LPE) were prepared and analyzed quali-and quantitatively by HPLC-UV and electrospray ionization -MS/MS. Human washed platelets stimulated with thrombin were used for the aggregation assay. Human platelet cAMP-PDE and recombinant PDE5A1 were used as enzyme source. Platelet aggregation and enzyme activity were assayed in the presence of HPE, LPE and individual phenols. The phenol content of HPE ranged between 250 and 500 mg/kg, whereas the LPE content was 46 mg/kg. The compounds identified were hydroxytyrosol (HT), tyrosol (TY), oleuropein aglycone (OleA) and the flavonoids quercetin (QU), luteolin (LU) and apigenin (AP). OleA was the most abundant phenol (range 23·3 to 37·7 %) and LU was the most abundant flavonoid in the extracts. Oil extracts inhibited platelet aggregation with an 50% inhibitory concentration interval of 1·23 -11·2 mg/ml. The inhibitory effect of individual compounds (10 mM) including homovanillyl alcohol (HVA) followed this order: OleA . LU . HT ¼ TY ¼ QU ¼ HVA, while AP was inactive. All the extracts inhibited cAMP-PDE, while no significant inhibition of PDE5A1 (50mg/ml) was observed. All the flavonoids and OleA inhibited cAMP-PDE, whereas HT, TY, HVA (100 mM) were inactive. Olive oil extracts and part of its phenolic constituents inhibit platelet aggregation; cAMP-PDE inhibition is one mechanism through which olive oil phenols inhibit platelet aggregation.

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