RESUMO
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.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas Adaptadoras da Sinalização Shc/deficiência , Animais , Axônios/patologia , Axônios/ultraestrutura , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Adjuvante de Freund/efeitos adversos , Glicoproteínas/efeitos adversos , Peróxido de Hidrogênio/farmacologia , Infiltração Leucêmica/tratamento farmacológico , Infiltração Leucêmica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Glicoproteína Mielina-Oligodendrócito , Fibras Nervosas Mielinizadas/patologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervo Óptico/imunologia , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Fragmentos de Peptídeos/efeitos adversos , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
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 microg/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.
Assuntos
Cisplatino/efeitos adversos , Eritropoetina/análogos & derivados , Eritropoetina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Doenças do Sistema Nervoso Periférico/prevenção & controle , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Cisplatino/farmacocinética , Cisplatino/uso terapêutico , Eritropoetina/administração & dosagem , Feminino , Gânglios Espinais/metabolismo , Rim/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/fisiopatologia , Síndromes Neurotóxicas/prevenção & controle , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/fisiopatologia , Ratos , Ratos Wistar , Nervo Isquiático/metabolismo , Cauda/efeitos dos fármacos , Cauda/inervação , Cauda/fisiopatologiaRESUMO
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.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/prevenção & controle , Eritropoetina/análogos & derivados , Eritropoetina/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Análise de Variância , Animais , Peso Corporal/efeitos dos fármacos , Doença Crônica , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Glicoproteínas , Hematócrito/métodos , Humanos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Índice de Gravidade de Doença , Medula Espinal/efeitos dos fármacos , Baço/metabolismo , Estatísticas não Paramétricas , Fatores de Tempo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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 H2O2 reacting 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.
Assuntos
Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Degeneração Neural/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Animais , Apoptose , Linhagem Celular , Peptidil-Prolil Isomerase F , Ciclofilinas/deficiência , Ciclofilinas/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Ensaio de Imunoadsorção Enzimática , Deleção de Genes , Humanos , Camundongos , Camundongos Knockout , Poro de Transição de Permeabilidade Mitocondrial , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Proteínas Adaptadoras da Sinalização Shc/deficiência , Medula Espinal/metabolismo , Medula Espinal/patologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
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.
Assuntos
Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Animais , Contagem de Células/métodos , Cisplatino , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Pé , Imuno-Histoquímica , Células de Langerhans/metabolismo , Células de Langerhans/patologia , Masculino , Fibras Nervosas/efeitos dos fármacos , Condução Nervosa/fisiologia , Condução Nervosa/efeitos da radiação , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Análise de Regressão , Ubiquitina Tiolesterase/metabolismoRESUMO
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.
Assuntos
Acetilcisteína/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Glucose/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Cardiomegalia/prevenção & controle , Núcleo Celular/química , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Glutationa/análise , Glutationa/metabolismo , Ventrículos do Coração/citologia , Miocárdio/química , Miocárdio/metabolismo , Miócitos Cardíacos/química , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismoRESUMO
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 microg/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 microg/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.
Assuntos
Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/prevenção & controle , Eritropoetina/uso terapêutico , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/fisiopatologia , Eletrofisiologia , Humanos , Masculino , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/patologia , Nociceptores/efeitos dos fármacos , Nociceptores/fisiopatologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Recombinantes , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
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.