L�ATTIVAZIONE DI TSPO E DI LXR STIMOLA LA NEUROSTEROIDOGENESI ED E� PROTETTIVA IN UN MODELLO DI NEUROPATIA PERIFERICA DI RATTO DIABETICO
Creators: name: GIATTI, SILVIA
Publisher: name: Universit� degli Studi di Milano
Year: 2011

description: Diabetes mellitus (DM) is one of the first leading cause of death in most high-income countries and there is evidence that it is epidemic in many economically developing and newly industrialized nations. Beside these considerations, there are epidemiologic evaluations that show a dramatic increase of the number of diabetic and with impaired glucose tollerance patients in the next years. It is also important to underline that complications from diabetes, such as coronary artery and peripheral vascular disease, stroke, diabetic neuropathy, renal failure and blindness result in increasing disability, reduced life expectancy and enormous health costs for virtually every society. In particular, diabetic peripheral neuropathy can account for the 50-75% of non traumatic amputations; diabetes is therefore undoubtedly one of the most challenging health problems of our century. Recently, in an animal model of diabetic neuropathy (i.e. the rat raised diabetic with the injection of streptozotocin - STZ), it has been demonstrated that, in plasma and in sciatic nerve, the levels of neuroactive steroids are dramatically decreased. Promising results have been obtained in the same experimental model after treatments with progesterone, testosterone and their derivatives. The use of molecules able to increase the neuroactive steroid levels directly in the nervous system could be an interesting therapeutic approach because it may avoid the endocrine side effects usually observed after systemic treatment with steroids. In order to evaluate the possible neuroprotective effects mediated by an increase of neuroactive steroid levels in the peripheral nervous system, using the model of the STZ rat we here analyzed the effects of two different pharmacological approaches. In the first part of this thesis, we analyzed the effect of Ro5-4864, a ligand of the translocator protein of 18 kDa (TSPO). TSPO is a key regulator of neuroactive steroid synthesis and it is involved in the rate limitig step of this process (i.e. the entrance of cholesterol into the mitochondria). Indeed, it has been recently demonstrated that injection of TSPO ligands, like for instance Ro5-4864, was able to increase the capability of steroidogenic machinery in adrenal cortex, placenta, testis, ovary and glial cells. In the second part of this thesis, the effect of GW3965, a ligand of the Liver X Receptor (LXR), was analyzed. The rationale of this approach was based on the finding that cholesterol homeostasis is partially controlled by the regulation of different genes activated by LXR. Moreover, LXR activation is able to increase the steroidogenesis in the adrenal glands. At 2 months after STZ injection, two different protocols of TSPO ligand administration were assessed; in particular, animals were injected every 2 days or once a week whit the ligand (i.e., they received 16 or 4 injections). In the case of GW3965 treatment, at 2 months, diabetic animals were treated once a week (i.e., they received 4 injections). Functional and behavioural parameters, like for instance hot plate test and the recording of nerve conduction velocity, as well as biochemical parameters, like Na+,K+-ATPase activity and myelin protein gene expression, were analyzed in order to assess the neuroprotective effects of treatments. Moreover, in the case of Ro5-4864 treatment, the intra-epidermal nerve fiber density was quantified in the skin of the hindpaw footpad. Furthermore, in the experiment with GW3965 treatment, the levels of gene expression of LXR targets and of steroidogenic enzimes were determined in the sciatic nerve of diabetic animals. Data obtained showed that activation of TSPO and of LXR using their ligands once a week resulted in an increased levels of neuroactive steroids and protection of the sciatic nerve from neuropathy induced by diabetes. Conversely, 16 treatments with Ro5-4864 did not produced any protective effects. The increase of neuroactive steroid levels observed in the sciatic nerve after the treatment with the two molecules was in agreement with the improve of functional and biochimical parameters observed in diabetic rats, like for istance the thermal nociceptive activity, nerve conduction velocity, and Na+,K+-ATPase activity. Moreover, a recover of the intra-epidermal nerve fiber density after Ro5-4864 injection and a significant increase of the gene expression of some enzymes of steroidogenesis after GW3965 administration were detected. In contrast to what observed after TSPO activation, treatment with GW3965 did not consistently countered the diabetes-induced decrease in expression of the myelin protein zero observed in the sciatic nerve of STZ-treated animals. This observation suggests somewhat different mechanisms of action for LXR and TSPO ligands, an issue that it should be explored in the future. In conclusion the data here reported show that Ro5-4864 and GW3965 are neuroprotective in an animal model of diabetic peripheral neuropathy. Since a pharmacological treatment for this pathology is not yet available, data here provide suggest that these two molecules may represent promising candidates for the treatment of diabetic neuropathy.