Search

Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.

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. © 2005 Peripheral Nerve Society.

Erythropoietin (EPO) is both hematopoietic and tissue protective, putatively through interaction with different receptors. We generated receptor subtype–selective ligands allowing the separation of EPO's bioactivities at the cellular level and in animals. Carbamylated EPO (CEPO) or certain EPO mutants did not bind to the classical EPO receptor (EPOR) and did not show any hematopoietic activity in human cell signaling assays or upon chronic dosing in different animal species. Nevertheless, CEPO and various nonhematopoietic mutants were cytoprotective in vitro and conferred neuroprotection against stroke, spinal cord compression, diabetic neuropathy, and experimental autoimmune encephalomyelitis at a potency and efficacy comparable to EPO.

Erythropoietin (EPO) possesses generalized neuroprotective and neurotrophic actions. We tested the efficacy of recombinant human EPO (rhEPO) in preventing and reversing nerve dysfunction in streptozotocin (STZ)-induced diabetes in rats. Two days after STZ [60 mg/kg of body weight (b.w.), i.p.], diabetic animals were administered rhEPO (40 μg/kg of b.w.) three times weekly for 5 weeks either immediately (preventive) before or after a 5-week delay (therapeutic) after induction of hyperglycemia or at a lower dose (8 μg/kg of b.w. once per week) for 8 weeks (prolonged). Tail-nerve conduction velocities (NCV) was assessed at 5 and 11 weeks for the preventive and therapeutic schedule, respectively. Compared to nondiabetic rats, NCV was 20% lower after 5 weeks in the STZ group, and this decrease was attenuated 50% by rhEPO. Furthermore, the reduction of Na + ,K + -ATPase activity of diabetic nerves (by 55%) was limited to 24% in the rhEPO-treated group. In the therapeutic schedule, NCV was reduced by 50% after 11 weeks but by only 23% in the rhEPO-treated group. rhEPO treatment attenuated the decrease in compound muscle action potential in diabetic rats. In addition, rhEPO treatment was associated with a preservation of footpad cutaneous innervation, as assessed by protein gene product 9.5 immunostaining. Diabetic rats developed alterations in mechanical and thermal nociception, which were partially reversed by rhEPO given either in a preventative or therapeutic manner. These observations suggest that administration of rhEPO or its analogues may be useful in the treatment of diabetic neuropathy.

Experimental diabetic neuropathy has a number of features in common with the human disease, including a reduction of nerve conduction velocity (NCV) and Na+, K+-ATPase activity in nerve fibers, and alteration of the essential fatty acid metabolism leading to changes in membrane composition. N-3 polyunsaturated fatty acids (PUFA) are beneficial in different pathological conditions including diabetic complications. Since the PUFA composition of cell membranes largely depends on the diet, most studies have supplemented the diet with fish oil (FO), particularly rich in docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or DHA and EPA ethyl esters. We have prepared a new formulation of n-3 PUFA wax esters (WE) enriched in DHA and EPA that have a bioavailability comparable to oil and esters, are easily digestible and are free from most of the previous drawbacks. The present study was designed to measure Na+, K+-ATPase activity, NCV, and FA content in plasma phospholipids (PL) and to assess the preventive effect of newly formulated n-3 PUFA-WE, and individual EPA and DHA-WE (as compared with standard and FO-supplemented diets) on these abnormalities. Diabetes was induced by streptozotocin (STZ, 60 mg/kg/ip). After STZ injection, diabetic and non-diabetic control rats were fed the standard diet (St) without supplementation or supplemented with either FO or n-3 PUFA-WE or individual EPA and DHA-WE, to reach a daily dose of 0.15 g/kg, the experiment continued for five weeks. Analysis of the FA composition of plasma PL, which better reflects FA intake and content in biological membranes, showed that the sums of n-3 PUFA were double the St diet after n-3 supplement of independent of the dietary regimen, whereas those of FA of the n-6 family were not affected by n-3 supplementation. Thus, the n-6/n-3 ratios were reduced from 15:1 with the St diet to 7:1 for the FO and WE diets. Na+, K+-ATPase activity was significantly lower in diabetic rats (−30–35%), significantly restored in diabetic rats that received FO, n-3 PUFA and EPA-WE supplementation, and partially restored by DHA-WE (9% lower than with non-diabetic controls). These effects were partially associated with a significant beneficial effect on NCV which was reduced by 20% in diabetic rats. FO protected against this decrease (3%), n-3 PUFA-WE were only partially protective (11%), and EPA and DHA-WE had scant effect. This study indicates that, like FO, n-3 PUFA-EPA and DHA-WE have beneficial effects on diabetic-induced alterations in sciatic nerve ATPase+-ATPase activity and, partially, also on NCV. The newly formulated n-3 PUFA WE offer a potential advantage over products in current use, on account of their greater stability.

Erythropoietin (EPO) has neurotrophic and neuroprotective effects and its efficacy and safety has been demonstrated in patients with ischemic stroke. We investigated its efficacy in preventing and reversing established nerve disorders in streptozotocin (STZ) diabetes. After STZ injection (60 mg/kg/ip), EPO (5000 units/kg b.w. i.p. three times a week) was started in a group of rats and continued for five weeks (prevention schedule). In another group of diabetic rats, EPO was started six weeks after STZ, continued for five weeks (therapeutic schedule). Groups of non-diabetic control rats were similarly treated. Antidromic nerve conduction velocity (NCV) in the tail was assessed at five weeks for all groups and at 11 weeks for the therapeutic schedule. Compared to non-diabetic rats, NCV was 21% lower (P

Mitochondrial-mediated oxidative stress and apoptosis play a crucial role in neurodegenerative disease and aging. Both mitochondrial permeability transition (PT) and swelling of mitochondria have been involved in neurodegeneration. Indeed, knockout mice for cyclophilin-D (Cyc-D), a key regulatory component of the PT pore (PTP) that triggers mitochondrial swelling, resulted to be protected in preclinical models of multiple sclerosis (MS), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). However, how neuronal stress is transduced into mitochondrial oxidative stress and swelling is unclear. Recently, the aging determinant p66Shc that generates H2O2reacting with cytochrome c and induces oxidation of PTP and mitochondrial swelling was found to be involved in MS and ALS. To investigate the role of p66Shc/PTP pathway in neurodegeneration, we performed experimental autoimmune encephalomyelitis (EAE) experiments in p66Shc knockout mice (p66Shc−/−), knock out mice for cyclophilin-D (Cyc-D−/−), and p66Shc Cyc-D double knock out (p66Shc/Cyc-D−/−) mice. Results confirm that deletion of p66Shc protects from EAE without affecting immune response, whereas it is not epistatic to the Cyc-D mutation. These findings demonstrate that p66Shc contributes to EAE induced neuronal damage most likely through the opening of PTP suggesting that p66Shc/PTP pathway transduces neurodegenerative stresses.

Epithelial ovarian cancer (EOC) is diagnosed in more than 70% of the cases in advanced FIGO stages which renders EOC the most deadly gynecologic cancer. Up to date there are no specific symptoms and no screening tests for general population or for patients at risk. The development of early diagnostic tools that will improve clinical outcome and differentiate between benign gynecological diseases and EOC are still an unmet need. In a multicenter study financed by the Seventh Framework Program of the European Commission (TRANSCAN) called ImPECT, three European EOC centers (Medical University of Vienna, Charité in Berlin, and the European Institute of Oncology in Milan) validated a “combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer” (Pils et al. BMC Cancer, 13:178). In total, blood of 576 women were enrolled in this validation study, including healthy controls, patients with EOC, with breast cancer (to assess tumor specificity), with benign gynecologic diseases (to assess the potential for differential diagnosis), and with inflammatory diseases and diabetes (to assess non-malignant immune changes). A specific blood leukocyte fraction and plasma were isolated and RNA prepared. The expression of 23 genes (13 from the diagnostic signature, seven from an unpublished prognostic signature and three house-keeping) were measured by a multiplexed hybridization based method (Nanostring's nCounter Elements™) and the abundance of six proteins from plasma by a multiplexed bead-based technology (Luminex). Due to evidence for substantial fluctuations of gene expression values by RT-qPCR, especially of the reverse transcription (RT) reaction, we replaced the RT-qPCR method by a hybridization based method without RT. Also, one protein of the protein panel (IGF2) was replaced by another protein (HE4) for two reasons: measurement of IGF2 cannot be multiplexed with the other proteins and HE4 was shown to have a higher discriminative power compared to IGF2. In a first step the published discriminative models were validated with 96 healthy controls and 96 EOC patients. All models showed significant area under the receiver operating characteristic (ROC) curve (AUC) values (p To assess the technical reproducibility of the Nanostring method, each 84 samples were measured a second time and correlated to the first measurement: Interestingly, the median Pearson's r correlations over all 20 genes was only 0.689 (0.337-0.799) but the SVM model between controls and EOC patients was more robust: AUC of 0.811 compared to 0.848 (p=0.394). A clinical validation of these models with independent patient and control cohorts (healthy controls and patients with benign diseases) and the specificity compared to breast cancer and diseases with immune system involvement will be presented. Citation Format: Anna Bachmayr-Heyda, Stefanie Aust, Katharina Auer, Peter Borossay, Nina Pecha, Ugo Cavallaro, Ioana Braicu, Sarah Igidbashian, Costanza Savino, Franchi Dorella, Jalid Sehouli, Sonia Zaafrani, Florian Fitzal, Christian Singer, Michael Gnant, Robert Zeillinger, Dietmar Pils. Validation of a combined 13 gene six protein blood signature for earlier detection of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B28.

Although multiple sclerosis (MS) has traditionally been considered to be an inflammatory disease, recent evidence has brought neurodegeneration into the spotlight, suggesting that accumulated damage and loss of axons is critical to disease progression and the associated irreversible disability. Proposed mechanisms of axonal degeneration in MS posit cytosolic and subsequent mitochondrial Ca(2+) overload, accumulation of pathologic reactive oxygen species (ROS), and mitochondrial dysfunction leading to cell death. In this context, the role of the p66 isoform of ShcA protein (p66) may be significant. The ShcA isoform is uniquely targeted to the mitochondrial intermembrane space in response to elevated oxidative stress, and serves as a redox enzyme amplifying ROS generation in a positive feedforward loop that eventually mediates cell death by activation of the mitochondrial permeability transition pore. Consequently, we tested the hypothesis that genetic inactivation of p66 would reduce axonal injury in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). As predicted, the p66-knockout (p66-KO) mice developed typical signs of EAE, but had less severe clinical impairment and paralysis than wild-type (WT) mice. Histologic examination of spinal cords and optic nerves showed significant axonal protection in the p66-KO tissue, despite similar levels of inflammation. Furthermore, cultured p66-KO neurons treated with agents implicated in MS neurodegenerative pathways showed greater viability than WT neurons. These results confirm the critical role of ROS-mediated mitochondrial dysfunction in the axonal loss that accompanies EAE, and identify p66 as a new pharmacologic target for MS neuroprotective therapeutics.

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.

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.

Diabetes and oxidative stress concur to cardiac myocyte death in various experimental settings. We assessed whether N-acetyl-L-cysteine (NAC), an antioxidant and glutathione precursor, has a protective role in a rat model of streptozotocin (STZ)-induced diabetes and in isolated myocytes exposed to high glucose (HG). Diabetic rats were treated with NAC (0.5 g/kg per day) or vehicle for 3 months. At sacrifice left ventricle (LV) myocyte number and size, collagen deposition and reactive oxygen species (ROS) were measured by quantitative histological methods. Diabetes reduced LV myocyte number by 29% and increased myocyte volume by 20% compared to non-diabetic controls. NAC protected from myocyte loss (+25% vs. untreated diabetics, P < 0.05) and reduced reactive hypertrophy (‐16% vs. untreated diabetics, P < 0.05). Perivascular fibrosis was high in diabetic rats (+88% vs. control, P < 0.001) but prevented by NAC. ROS production and fraction of ROS-positive cardiomyocyte nuclei were drastically raised in diabetic rats (2.4- and 5.1-fold vs. control, P < 0.001) and normalized by NAC. In separate experiments, isolated adult rat ventricular myocytes were incubated in a medium containing high concentrations of glucose (HG, 25 mM) ± 0.01 mM NAC; myocyte survival (Trypan blue exclusion and apoptosis by TUNEL) and glutathione content were evaluated. The number of dead and apoptotic myocytes increased five and 6.7-fold in HG and glutathione decreased by 48% (P < 0.05). NAC normalized cell death and apoptosis and prevented glutathione loss. NAC effectively protects from hyperglycemia-induced myocyte cell death and compensatory hypertrophy through direct scavenging of ROS and replenishment of the intracellular glutathione content. © 2004 Elsevier Ltd. All rights reserved.

Animal models of human diseases affecting the peripheral nervous system are widely used to assess the pathogenesis of neurotoxicity and to compare the effect of new agents. Several behavioural, pathological and neurophysiological methods have been used, and each has advantages and disadvantages. A major goal in the study of neurotoxicity would be to assess the damage in the same way in animal models and in humans. In this study we correlated the neurophysiological results obtained in normal rats and in rats treated with cisplatin 2 mg/kg q3d × 8 with the density of intraepidermal fibers (IEF) obtained in skin biopsy specimens. The aim was to investigate the possible role of a minimally invasive procedure such as skin biopsy as an alternative method to assess the peripheral neurotoxicity of antineoplastic drugs. The nerve conduction velocity (NCV) in the tail nerve was assessed in thirty-six young adult female Wistar rats which were left untreated, or treated with erythropoietin (EPO), cisplatin (CDDP) or EPO + CDDP. CDDP and CDDP + EPO-treated rats had a significantly reduced NCV vs. age-matched untreated rats. At sacrifice, skin specimens were obtained. The density of IEF was calculated by 2 independent blinded examiners and the correlation existing between NCV and IEF was highly significant (r = 0.670, p