Roberto Bianchi

Progesterone and its derivatives are neuroprotective agents in experimental diabetic neuropathy: A multimodal analysis

One important complication of diabetes is damage to the peripheral nervous system. However, in spite of the number of studies on human and experimental diabetic neuropathy, the current therapeutic arsenal is meagre. Consequently, the search for substances to protect the nervous system from the degenerative effects of diabetes has high priority in biomedical research. Neuroactive steroids might be interesting since they have been recently identified as promising neuroprotective agents in several models of neurodegeneration. We have assessed whether chronic treatment with progesterone (P), dihydroprogesterone (DHP) or tetrahydroprogesterone (THP) had neuroprotective effects against streptozotocin (STZ)-induced diabetic neuropathy at the neurophysiological, functional, biochemical and neuropathological levels. Using gas chromatography coupled to mass-spectrometry, we found that three months of diabetes markedly lowered P plasma levels in male rats, and chronic treatment with P restored them, with protective effects on peripheral nerves. In the model of STZ-induced of diabetic neuropathy, chronic treatment for 1 month with P, or with its derivatives, DHP and THP, counteracted the impairment of nerve conduction velocity (NCV) and thermal threshold, restored skin innervation density, and improved Na(+),K(+)-ATPase activity and mRNA levels of myelin proteins, such as glycoprotein zero and peripheral myelin protein 22, suggesting that these neuroactive steroids, might be useful protective agents in diabetic neuropathy. Interestingly, different receptors seem to be involved in these effects. Thus, while the expression of myelin proteins and Na(+),K(+)-ATPase activity are only stimulated by P and DHP (i.e. two neuroactive steroids interacting with P receptor, PR), NCV, thermal nociceptive threshold and intra-epidermal nerve fiber (IENF) density are also affected by THP, which interacts with GABA-A receptor. Because, a therapeutic approach with specific synthetic receptor ligands could avoid the typical side effects of steroids, future experiments will be devoted to evaluating the role of PR and GABA-A receptor in these protective effects.

Intraepidermal nerve fiber density in rat foot pad: neuropathologic–neurophysiologic correlation

Abstract  Quantification of cutaneous innervation in rat footpad is a useful tool to investigate sensory small‐diameter nerve fibers, which are affected early in peripheral neuropathies. The aim of this work was to provide normative reference data on the density of intraepidermal nerve fibers (IENFs) and Langerhans cells in the hindpaw footpad of Sprague–Dawley and Wistar rats. We also evaluated the sensibility of IENF density by comparing neuropathologic findings with neurophysiologic examination and the presence of peripheral neuropathy in two well‐characterized animal models of neuropathy. IENF density was quantified in 22 Sprague–Dawley rats and 13 Wistar rats and compared with 19 age‐matched Sprague–Dawley rats with streptozotocin‐induced diabetic neuropathy and 30 age‐matched Wistar rats with cisplatin‐ or paclitaxel‐induced neuropathy. Antidromic tail sensory nerve conduction velocity (SNCV) was assessed in all animals. IENF and Langerhans cell densities were constant in healthy Sprague–Dawley rats at any age, and they were similar to those observed in healthy Wistar rats. In neuropathic rats, both SNCV and IENF density were significantly reduced with respect to controls. Quantification of IENF density was significantly correlated with changes in conduction velocity. Diabetic neuropathy rats alone showed a significantly higher density of Langerhans cells compared with controls. Our study demonstrated that IENF density quantification correlates with SNCV changes and suggests that this might represent a useful outcome measurement in experimental neuropathies.

Delayed administration of erythropoietin and its non-erythropoietic derivatives ameliorates chronic murine autoimmune encephalomyelitis

Erythropoietin (EPO) mediates a wide range of neuroprotective activities, including amelioration of disease and neuroinflammation in rat models of EAE. However, optimum dosing parameters are currently unknown. In the present study, we used a chronic EAE model induced in mice by immunization with the myelin oligodendrocyte glycoprotein peptide (MOG35-55) to compare the effect of EPO given with different treatment schedules. EPO was administered intraperitoneally at 0.5, 5.0 or 50 microg/kg three times weekly starting from day 3 after immunization (preventive schedule), at the onset of clinical disease (therapeutic schedule) or 15 days after the onset of symptoms (late therapeutic schedule). The results show that EPO is effective even when given after the appearance of clinical signs of EAE, but with a reduced efficacy compared to the preventative schedule. To determine whether this effect requires the homodimeric EPO receptor (EPOR2)-mediated hematopoietic effect of EPO, we studied the effect of carbamylated EPO (CEPO) that does not bind EPOR2. CEPO, ameliorated EAE without changing the hemoglobin concentration. Another non-erythropoietic derivative, asialoEPO was also effective. Both EPO and CEPO equivalently decreased the EAE-associated production of TNF-alpha, IL-1beta and IL-1Ra in the spinal cord, and IFN-gamma by peripheral lymphocytes, indicating that their action involves targeting neuroinflammation. The lowest dosage tested appeared fully effective. The possibility to dissociate the anti-neuroinflammatory action of EPO from its hematopoietic action, which may cause undesired side effects in non-anemic patients, present new avenues to the therapy of multiple sclerosis.

Protective Effect of Erythropoietin and Its Carbamylated Derivative in Experimental Cisplatin Peripheral Neurotoxicity

Purpose: Antineoplastic drugs, such as cisplatin (CDDP), are severely neurotoxic, causing disabling peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. Cotreatment with neuroprotective agents and CDDP has been proposed for preventing or reversing the neuropathy. Erythropoietin given systemically has a wide range of neuroprotective actions in animal models of central and peripheral nervous system damage. However, the erythropoietic action is a potential cause of side effects if erythropoietin is used for neuroprotection. We have successfully identified derivatives of erythropoietin, including carbamylated erythropoietin, which do not raise the hematocrit but retain the neuroprotective action exerted by erythropoietin. Experimental Design: We have developed previously an experimental chemotherapy-induced peripheral neurotoxicity that closely resembles CDDP neurotoxicity in humans. The present study compared the effects of erythropoietin and carbamylated erythropoietin (50 μg/kg/d thrice weekly) on CDDP (2 mg/kg/d i.p. twice weekly for 4 weeks) neurotoxicity in vivo. Results: CDDP given to Wistar rats significantly lowered their growth rate (P < 0.05), with slower sensory nerve conduction velocity (P < 0.001) and reduced intraepidermal nerve fibers density (P < 0.001 versus controls). Coadministration of CDDP and erythropoietin or carbamylated erythropoietin partially but significantly prevented the sensory nerve conduction velocity reduction. Both molecules preserved intraepidermal nerve fiber density, thus confirming their neuroprotective effect at the pathologic level. The protective effects were not associated with any difference in platinum concentration in dorsal root ganglia, sciatic nerve, or kidney specimens. Conclusions: These results widen the spectrum of possible use of erythropoietin and carbamylated erythropoietin as neuroprotectant drugs, strongly supporting their effectiveness.

Neuroprotective effects of a ligand of translocator protein-18kDa (Ro5-4864) in experimental diabetic neuropathy

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.

Activation of the Liver X Receptor Increases Neuroactive Steroid Levels and Protects from Diabetes-Induced Peripheral Neuropathy

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 LRXα and β 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 5α-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 3α-diol) in the sciatic nerve, and with neuroprotective effects on thermal nociceptive activity, nerve conduction velocity, and Na+, K+-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.

Decrease of nerve Na+,K+-ATPase activity in the pathogenesis of human diabetic neuropathy ☆

A decrease in Na+,K(+)-ATPase activity is claimed to play a central role in the pathogenesis of electrophysiological and morphological abnormalities that characterize the neuropathic complications in different animal models of diabetes mellitus. The peripheral nerves from 17 patients with either type I or type II diabetes mellitus were studied to assess the importance of changes in Na+,K(+)-ATPase activity in chronic human diabetic neuropathy. Sixteen nerves from age- and sex-matched normal individuals, and 12 nerves from non-diabetic neuropathic subjects undergoing vascular or orthopedic surgery served as negative and positive controls, respectively. All specimens were processed blind. Ouabain-sensitive ATPase activity was measured by a modified spectrophotometric coupled-enzyme assay. Standard histology, fiber teasing and electron microscopy were used to establish the normal or neuropathological patterns of surgical material. Morphometric analysis permitted calculation of fiber density in each nerve specimen and correlation of this figure with the relevant enzymatic activity. Na+,K(+)-ATPase activity was approximately 59% lower in nerves from diabetic patients than in normal controls (P < 0.01) and approximately 38% lower in nerves from non-diabetic patients with neuropathy (P < 0.01). Although nerves from both neuropathic conditions had significantly fewer fibers than those from normal individuals (diabetic -33%, and non-diabetic -22%), the decreases in Na+,K(+)-ATPase activity and fiber density were not correlated only in specimens from diabetic patients (r2 = 0.096; P = 0.22). Taken together with data from experimental animal models, these results suggest that the reduction in Na+,K(+)-ATPase activity in diabetic nerves is not an epiphenomenon secondary to fiber loss; rather, it may be an important factor in the pathogenesis and self-maintenance of human diabetic neuropathy.

ATPase Activity Defects in Alloxan-Induced Diabetic Sciatic Nerve Recovered by Ganglioside Treatment

ATPase activities were measured in sciatic nerves from rats with alloxan-induced diabetes (ALX-D) of various duration (2 wk, 5 wk, 9 wk, and 6 mo). Our data confirm that sciatic nerve Na+-K+-ATPase abnormalities are present very early in ALX-D rats, similar to results previously described in streptozocin-induced diabetic rats, spontaneously diabetic BB Wistar rats, and ALX-D rabbits. Na+-K+-ATPase activity decreased by 26–47% in ALX-D rats compared with age-matched controls. Ganglioside treatment (10 mg/kg i.p. for 10 or 30 days starting 1 wk after ALX injection) completely impeded the enzyme reduction. The effect observed at the end of either 10 or 30 days of treatment lasted ≥1 mo. Chronic diabetic groups treated for 30 days before killing also presented normal ATPase activity at the end of treatment. Therefore, gangliosides are effective on Na+-K+-ATPase even in animals with a longer duration of diabetes. The maintenance of fairly normal ATPase activity by ganglioside treatment could mirror a more general recovery from early metabolic dysfunction and/or late structural abnormalities in diabetic nerve fibers.

Testosterone derivatives are neuroprotective agents in experimental diabetic neuropathy.

Abstract.In this study we have assessed the effect of testosterone (T), dihydrotestosterone (DHT) and 5αandrostan-3α, 17β-diol (3α-diol) therapies on diabetic neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin and resulted in decreased T and increased 3α-diol levels in plasma and in decreased levels of pregnenolone and DHT in the sciatic nerve. Moreover, a reduced expression of the enzyme converting Tinto DHT (i.e., the 5α-reductase) also occurs at the level of sciatic nerve, suggesting that the decrease of DHT levels could be due to an impairment of this enzyme. Chronic treatment for 1 month with DHT or 3α-diol increased tail nerve conduction velocity and partially counteracted the increase of thermal threshold induced by diabetes. Treatment with DHT increased tibial Na+,K+-ATPase activity and the expression of myelin protein P0 in the sciatic nerve.DHT, 3α-diol and T reversed the reduction of intra-epidermal nerve fiber density induced by diabetes. These observations indicate that T metabolites can reverse behavioral, neurophysiological, morphological and biochemical alterations induced by peripheral diabetic neuropathy.

Docetaxel-induced peripheral neuropathy: protective effects of dihydroprogesterone and progesterone in an experimental model

Abstract  Peripheral neurotoxicity is a frequent complication limiting docetaxel chemotherapy in patients with cancer. We developed an experimental model that closely mimics the course of neuropathy in patients, aiming to investigate both the mechanisms of neurotoxicity at biochemical, functional and morphological levels and the potential neuroprotective role of neuroactive steroids. We demonstrated that treatment with dihydroprogesterone (DHP) or progesterone (P) counteracts docetaxel‐induced neuropathy, preventing nerve conduction and thermal threshold changes, and degeneration of skin nerves in the foodpad. Neuroactive steroids also counteract the changes in gene expression of several myelin proteins and calcitonin gene‐related peptide induced by docetaxel in sciatic nerve and lumbar spinal cord, respectively. Most nerve abnormalities observed during the treatment with docetaxel spontaneously recovered after drug withdrawal, similarly to what occurs in patients. However, results of midterm follow‐up experiments indicated that animals cotreated with DHP or P have a faster recovery of the neuropathy compared with docetaxel‐treated rats. Our study confirmed that neuroactive steroids exert a protective effect on peripheral nerves at different levels, suggesting that they might represent a new therapeutic frontier for patients with chemotherapy‐induced neuropathy.