RESUMO
Non-exudative age-related macular degeneration (NE-AMD) is the leading blindness cause in the elderly. Clinical and experimental evidence supports that early alterations in macular retinal pigment epithelium (RPE) mitochondria play a key role in NE-AMD-induced damage. Mitochondrial dynamics (biogenesis, fusion, fission, and mitophagy), which is under the central control of AMP-activated kinase (AMPK), in turn, determines mitochondrial quality. We have developed a NE-AMD model in C57BL/6J mice induced by unilateral superior cervical ganglionectomy (SCGx), which progressively reproduces the disease hallmarks circumscribed to the temporal region of the RPE/outer retina that exhibits several characteristics of the human macula. In this work we have studied RPE mitochondrial structure, dynamics, function, and AMPK role on these parameters' regulation at the nasal and temporal RPE from control eyes and at an early stage of experimental NE-AMD (i.e., 4 weeks post-SCGx). Although RPE mitochondrial mass was preserved, their function, which was higher at the temporal than at the nasal RPE in control eyes, was significantly decreased at 4 weeks post-SCGx at the same region. Mitochondria were bigger, more elongated, and with denser cristae at the temporal RPE from control eyes. Exclusively at the temporal RPE, SCGx severely affected mitochondrial morphology and dynamics, together with the levels of phosphorylated AMPK (p-AMPK). AMPK activation with metformin restored RPE p-AMPK levels, and mitochondrial dynamics, structure, and function at 4 weeks post-SCGx, as well as visual function and RPE/outer retina structure at 10 weeks post-SCGx. These results demonstrate a key role of the temporal RPE mitochondrial homeostasis as an early target for NE-AMD-induced damage, and that pharmacological AMPK activation could preserve mitochondrial morphology, dynamics, and function, and, consequently, avoid the functional and structural damage induced by NE-AMD.
Assuntos
Proteínas Quinases Ativadas por AMP , Modelos Animais de Doenças , Degeneração Macular , Camundongos Endogâmicos C57BL , Mitocôndrias , Dinâmica Mitocondrial , Epitélio Pigmentado da Retina , Animais , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Camundongos , Degeneração Macular/patologia , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Humanos , Metformina/farmacologiaRESUMO
Non-exudative age-related macular degeneration (NE-AMD), the main cause of blindness in people above 50 years old, lacks effective treatments at the moment. We have developed a new NE-AMD model through unilateral superior cervical ganglionectomy (SCGx), which elicits the disease main features in C57Bl/6J mice. The involvement of oxidative stress in the damage induced by NE-AMD to the retinal pigment epithelium (RPE) and outer retina has been strongly supported by evidence. We analysed the effect of enriched environment (EE) and visual stimulation (VS) in the RPE/outer retina damage within experimental NE-AMD. Exposure to EE starting 48 h post-SCGx, which had no effect on the choriocapillaris ubiquitous thickness increase, protected visual functions, prevented the thickness increase of the Bruch's membrane, and the loss of the melanin of the RPE, number of melanosomes, and retinoid isomerohydrolase (RPE65) immunoreactivity, as well as the ultrastructural damage of the RPE and photoreceptors, exclusively circumscribed to the central temporal (but not nasal) region, induced by experimental NE-AMD. EE also prevented the increase in outer retina/RPE oxidative stress markers and decrease in mitochondrial mass at 6 weeks post-SCGx. Moreover, EE increased RPE and retinal brain-derived neurotrophic factor (BDNF) levels, particularly in Müller cells. When EE exposure was delayed (dEE), starting at 4 weeks post-SCGx, it restored visual functions, reversed the RPE melanin content and RPE65-immunoreactivity decrease. Exposing animals to VS protected visual functions and prevented the decrease in RPE melanin content and RPE65 immunoreactivity. These findings suggest that EE housing and VS could become an NE-AMD promising therapeutic strategy.
Assuntos
Degeneração Macular/fisiopatologia , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , CamundongosRESUMO
Nonexudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. Currently, there are no available treatments for NE-AMD. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks. Several lines of evidence strongly support the involvement of oxidative stress in NE-AMD-induced retinal pigment epithelium (RPE) and outer retina damage. Melatonin is a proven and safe antioxidant. Our aim was analysing the effect of melatonin in the RPE/outer retina damage within experimental NE-AMD. The treatment with melatonin starting 48 h after SCGx, which had no effect on the ubiquitous choriocapillaris widening, protected visual functions and avoided Bruch´s membrane thickening, RPE melanin content, melanosome number loss, retinoid isomerohydrolase (RPE65)-immunoreactivity decrease, and RPE and hotoreceptor ultrastructural damage induced within experimental NE-AMD exclusively located at the central temporal (but not nasal) region. Melatonin also prevented the increase in outer retina/RPE oxidative stress markers and a decrease in mitochondrial mass at 6 weeks post-SCGx. Moreover, when the treatment with melatonin started at 4 weeks post-SCGx, it restored visual functions and reversed the decrease in RPE melanin content and RPE65-immunoreactivity. These findings suggest that melatonin could become a promising safe therapeutic strategy for NE-AMD.
Assuntos
Degeneração Macular/patologia , Melatonina/farmacologia , Retina/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacosRESUMO
Non-exudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. The macular retinal pigment epithelium (RPE) lies in a high oxidative environment because its high metabolic demand, mitochondria concentration, reactive oxygen species levels, and macular blood flow. It has been suggested that oxidative stress-induced damage to the RPE plays a key role in NE-AMD pathogenesis. The fact that the disease limits to the macular region raises the question as to why this area is particularly susceptible. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks exclusively circumscribed to the temporal region of the RPE/outer retina. The aim of this work was analyzing RPE regional differences that could explain AMD localized susceptibility. Lower melanin content, thicker basal infoldings, higher mitochondrial mass, and higher levels of antioxidant enzymes, were found in the temporal RPE compared with the nasal region. Moreover, SCGx induced a decrease in the antioxidant system, and in mitochondria mass, as well as an increase in mitochondria superoxide, lipid peroxidation products, nuclear Nrf2 and heme oxygenase-1 levels, and in the occurrence of damaged mitochondria exclusively at the temporal RPE. These findings suggest that despite the well-known differences between the human and mouse retina, it might not be NE-AMD pathophysiology which conditions the localization of the disease, but the macular RPE histologic and metabolic specific attributes that make it more susceptible to choroid alterations leading initially to a localized RPE dysfunction/damage, and secondarily to macular degeneration.
Assuntos
Degeneração Macular/fisiopatologia , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/fisiopatologia , Animais , Modelos Animais de Doenças , Ganglionectomia/métodos , Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Peroxidação de Lipídeos , Degeneração Macular/etiologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/inervação , Epitélio Pigmentado da Retina/metabolismo , Gânglio Cervical Superior/lesões , Gânglio Cervical Superior/cirurgia , Fatores de TempoRESUMO
Non-exudative age-related macular degeneration, a prevalent cause of blindness, is a progressive and degenerative disease characterized by alterations in Bruch's membrane, retinal pigment epithelium, and photoreceptors exclusively localized in the macula. Although experimental murine models exist, the vast majority take a long time to develop retinal alterations and, in general, these alterations are ubiquitous, with many resulting from non-eye-specific genetic manipulations; additionally, most do not always reproduce the hallmarks of human age-related macular degeneration. Choroid vessels receive sympathetic innervation from the superior cervical ganglion, which, together with the parasympathetic system, regulates blood flow into the choroid. Choroid blood flow changes have been involved in age-related macular degeneration development and progression. At present, no experimental models take this factor into account. The aim of this work was to analyze the effect of superior cervical gangliectomy (also known as ganglionectomy) on the choroid, Bruch's membrane, retinal pigment epithelium and retina. Adult male C57BL/6J mice underwent unilateral superior cervical gangliectomy and a contralateral sham procedure. Although superior cervical gangliectomy induced ubiquitous choroid and choriocapillaris changes, it induced Bruch's membrane thickening, loss of retinal pigment epithelium melanin content and retinoid isomerohydrolase, the appearance of drusen-like deposits, and retinal pigment epithelium and photoreceptor atrophy, exclusively localized in the temporal side. Moreover, superior cervical gangliectomy provoked a localized increase in retinal pigment epithelium and photoreceptor apoptosis, and a decline in photoreceptor electroretinographic function. Therefore, superior cervical gangliectomy recapitulated the main features of human non-exudative age-related macular degeneration, and could become a new experimental model of dry age-related macular degeneration, and a useful platform for developing new therapies.
Assuntos
Degeneração Macular/etiologia , Gânglio Cervical Superior/cirurgia , Animais , Lâmina Basilar da Corioide/patologia , Lâmina Basilar da Corioide/ultraestrutura , Corioide/patologia , Degeneração Macular/patologia , Masculino , Melaninas/metabolismo , Camundongos Endogâmicos C57BL , Células Fotorreceptoras de Vertebrados/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Gânglio Cervical Superior/patologia , cis-trans-Isomerases/metabolismoRESUMO
The participation of sympathetic nerve fibers in the innervation of the nasal-associated lymphoid tissues (NALT) was investigated in hamsters. Vesicular monoamine transporter 2 (VMAT2), an established sympathetic marker, is expressed in all neurons of superior cervical ganglia (SCG). In addition, VMAT2 -immunoreactive nerve fibers were localized in the NALT as well as in adjacent anatomical structures of the upper respiratory tract. Unilateral surgical ablation of the SCG abolished VMAT2 innervation patterns ipsilaterally while the contra lateral side is unaffected. These results provide the anatomical substrate for a neuroimmune connection in the NALT.
Assuntos
Tecido Linfoide/citologia , Nariz/inervação , Gânglio Cervical Superior/citologia , Sistema Nervoso Simpático/fisiologia , Animais , Cricetinae , Ganglionectomia , Regulação da Expressão Gênica/fisiologia , Masculino , Mesocricetus , Nariz/anatomia & histologia , Sistema Respiratório/metabolismo , Gânglio Cervical Superior/metabolismo , Gânglio Cervical Superior/cirurgia , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Th1 lymphocytes are crucial in the immune response against Mycobacterium tuberculosis. Nevertheless, IFN-γ alone is not sufficient in the complete eradication of the bacteria, suggesting that other cytokines might be required for pathogen removal. Th17 cells have been associated with M. tuberculosis infection, but the role of IL-17-producing cells in human TB remains to be understood. Therefore, we investigated the induction and regulation of IFN-γ and IL-17 during the active disease. TB patients were classified as High and Low Responder individuals according to their T cell responses against the antigen, and cytokine expression upon M. tuberculosis stimulation was investigated in peripheral blood and pleural fluid. Afterwards, the potential correlation among the proportions of cytokine-producing cells and clinical parameters was analyzed. In TB patients, M. tuberculosis induced IFN-γ and IL-17, but in comparison with BCG-vaccinated healthy donors, IFN-γ results were reduced significantly, and IL-17 was markedly augmented. Moreover, the main source of IL-17 was represented by CD4(+)IFN-γ(+)IL-17(+) lymphocytes, a Th1/Th17 subset regulated by IFN-γ. Interestingly, the ratio of antigen-expanded CD4(+)IFN-γ(+)IL-17(+) lymphocytes, in peripheral blood and pleural fluid from TB patients, was correlated directly with clinical parameters associated with disease severity. Indeed, the highest proportion of CD4(+)IFN-γ(+)IL-17(+) cells was detected in Low Responder TB patients, individuals displaying severe pulmonary lesions, and longest length of disease evolution. Taken together, the present findings suggest that analysis of the expansion of CD4(+)IFN-γ(+)IL-17(+) T lymphocytes in peripheral blood of TB patients might be used as an indicator of the clinical outcome in active TB.
Assuntos
Regulação da Expressão Gênica , Interferon gama/biossíntese , Interleucina-17/biossíntese , Mycobacterium tuberculosis , Células Th1/metabolismo , Células Th17/metabolismo , Tuberculose/sangue , Adulto , Feminino , Humanos , Interferon gama/imunologia , Interleucina-17/imunologia , Masculino , Índice de Gravidade de Doença , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
En ratones sometidos a gangliectomia simpática cervical superior (SCGx) unilateral se estudió el efecto de la desnervación local sobre la respuesta imune de ganglios linfáticos submaxilares (SmLN). El contenido de norepinefrina (NE) de SmLN disminuyó en un 90% a los 7-20 dias luego de la SCGx uni o bilateral. Los SmLN ipsilaterales de ratones SCGx unilateralmente 10-20 días antes e inyectados i.d. o i.p. con eritrocitos de carnero mostraron una respuesta de células formadoras de placa (PFC) significantivamente mayor que el control invervado contralateral. En ratones inyectados con eritrocitos de carnero en al fase temprana de parálisis neural simpática (2 h luego de la SCGx), se detectó una respuesta PFC aumentada mientras que durante la fase de degeneración anterógrada de los terminales (6-24 h luego de SCGx), se observó una disminucióm crecimente de PFC. En ratones crónicamente SCGx se detectó también un aumento significativo en la hipersensibilidad por contacto y reacción alogeneica retardada en al oreja ipsilateral a la operación. Al inyectar células de SmLN desnervados a F1 de (BALB/c x C57Bl/6) o de (BALB/c x AKR) se obtuvbieron índices esplénicos significativamente mayores que los observados luego de inyectar células controles. La estimulación mitogénica en diferentes protocolos experimentales no resultó en diferencias significativas entre SmLN desnervados e inervados. Luego de la descentralización local por sección unilateral del tronco lingual-cuerda del tímpano, los SmLN ipsilaterales exhibieron menor respuesta PFC 8-28 días luego de la cirugía. Estos resultados indican función modulatoria del sistema nervioso autónomo en la respuesta inmune
Assuntos
Camundongos , Animais , Doenças da Glândula Submandibular/imunologia , Ganglionectomia , Linfonodos/imunologia , Sistema Nervoso Autônomo/fisiologia , Simpatectomia , Doenças da Glândula Submandibular/metabolismo , Linfonodos/metabolismo , Soros Imunes , Imunização , Camundongos Endogâmicos AKR , Camundongos Endogâmicos BALB C , Nervo da Corda do Tímpano/cirurgia , Norepinefrina/metabolismoRESUMO
En ratones sometidos a gangliectomia simpática cervical superior (SCGx) unilateral se estudió el efecto de la desnervación local sobre la respuesta imune de ganglios linfáticos submaxilares (SmLN). El contenido de norepinefrina (NE) de SmLN disminuyó en un 90% a los 7-20 dias luego de la SCGx uni o bilateral. Los SmLN ipsilaterales de ratones SCGx unilateralmente 10-20 días antes e inyectados i.d. o i.p. con eritrocitos de carnero mostraron una respuesta de células formadoras de placa (PFC) significantivamente mayor que el control invervado contralateral. En ratones inyectados con eritrocitos de carnero en al fase temprana de parálisis neural simpática (2 h luego de la SCGx), se detectó una respuesta PFC aumentada mientras que durante la fase de degeneración anterógrada de los terminales (6-24 h luego de SCGx), se observó una disminucióm crecimente de PFC. En ratones crónicamente SCGx se detectó también un aumento significativo en la hipersensibilidad por contacto y reacción alogeneica retardada en al oreja ipsilateral a la operación. Al inyectar células de SmLN desnervados a F1 de (BALB/c x C57Bl/6) o de (BALB/c x AKR) se obtuvbieron índices esplénicos significativamente mayores que los observados luego de inyectar células controles. La estimulación mitogénica en diferentes protocolos experimentales no resultó en diferencias significativas entre SmLN desnervados e inervados. Luego de la descentralización local por sección unilateral del tronco lingual-cuerda del tímpano, los SmLN ipsilaterales exhibieron menor respuesta PFC 8-28 días luego de la cirugía. Estos resultados indican función modulatoria del sistema nervioso autónomo en la respuesta inmune (AU)