Os autores gostariam de agradecer Dr. Paul Witting e Ms. Genevieve Fong por sua assistência com extração de mRNA e PCR, eo Garnett Passe e Rodney Williams Memorial Foundation para o apoio.

Declaração de Ética: Todos os procedimentos descritos a seguir foram aprovados pelo Comitê de Ética da Universidade de Sydney animal. 1. Animais NOTA: 1 e 8-9 meses de idade camundongos (C57 / BL6) foram obtidos a partir do Centro de Recursos Animal (Perth, Austrália). Os ratinhos foram alojados no mecanismo Bosch Roedor na Universidade de Sydney. <ol><li> Casa ratos em gaiolas-padrão do mouse em um ciclo claro / escuro de 12/12 horas de luz com acesso a comida e água ad libitum. </li><li> Camundongos Divida em cada faixa etária em infravermelho próximo (NIR), tratados, ou sham grupos tratados (controle) para comparação. </li></ol> 2. Infravermelho Próximo (NIR) Irradiação e Tratamento Sham <ol><li> Raspar a pele na região da cabeça e pescoço do mouse com um barbeador elétrico, tanto quanto possível para evitar a rebrota rápida de cabelo antes da realização do regime de tratamento. A fim de manter o status de livre de patógenos dos ratos, use 70% de etanolpara limpar instrumentos entre animais para maquiagem e desinfetante F10 veterinário entre animais de gaiolas separadas. Faça isto 2-3 dias antes do início do tratamento para que os animais não estão sobre-tratadas. </li><li> Conter rato com a extremidade proximal da cauda e permitir que ele relaxe na palma de uma mão ou de bancada, de modo a minimizar o stress, que pode levar a resultados falsos. </li><li> Segure o NIR (670 nm) LED (light emitting diode) dispositivo de 1-2 cm de distância da área exposta (raspada) e ligar o aparelho durante 90 segundos (Figura 1A). NOTA: A mudança de temperatura como resultado de exposição de 90 segundos foi medida como &lt;0,2 ° C em 100 ml de água. </li><li> Repita os passos de 2,2-2,3 para o grupo de tratamento simulado de animais, mas deixar o aparelho desligado (Figura 1B). </li><li> Repita os passos de 2,2-2,3 para o grupo de tratamento Nir-bloqueado de animais, mas cubra o aparelho com papel alumínio. </li><li> Repita os passos 2,2-2,5 diariamente às approximate 24 intervalos de RH durante 5 dias consecutivos. </li><li> Extraia o epitélio vestibular sensorial (3 cristas e uma mácula utricular) de ambas as orelhas no quinto dia pós-tratamento (ver secção 3 abaixo). </li></ol> 3. Extracção de tecido 20 <ol><li> Preparar 300 ml de um fluido cerebrospinal artificial (ACSF) à base de glicerol que consistem em (em mM): 26 de NaHCO3, 11 de glucose, 250 glicerol, 2,5 de KCl, 1,2 de NaH 2 PO 4, 1,2 de MgCl2, 2,5 e CaCl 2. Antes da adição de CaCl2, gás a solução com carbogénio (95% O 2 e 5% CO 2) para estabelecer um pH de 7,4 e evitar a precipitação do cálcio (turvação). Arrefeça a solução num congelador a -80 ° C durante 45 minutos de modo que uma pasta de gelo é formada. </li><li> Prepare o tampão de lise isolamento do RNA em microtubos parafuso superior marcados (pára o popping de tampas quando as alterações de pressão tubo entre congelamento e descongelamento em nitrogênio líquido), de acordocom as instruções do fabricante ou práticas de laboratório habituais. Já nitrogênio líquido pronto em um frasco de Dewar de alumínio. NOTA: Use roupas de proteção e eyeware ao manusear nitrogênio líquido. Certifique-se de que o nitrogênio líquido é usado em um quarto bem ventilado como o volume da sua forma de gás é aproximadamente 700 vezes maior do que a sua forma líquida e pode causar asfixia. </li><li> Profundamente anestesiar os ratos com cetamina (400 mg / kg) através de uma injecção intra-peritoneal. Permitir que o reflexo posterior de membros para desaparecer completamente como indicação de que os ratos são totalmente anestesiado. </li><li> Decapitar ratos com tesoura de aço inoxidável afiadas e fazer uma incisão ao longo da pele sagital do crânio usando uma lâmina de barbear (arredondado # 22). Neste momento e em toda as etapas 3,5-3,9, manter a calota craniana, cérebro e aparelho vestibular subjacente o mais fresco possível pela aplicação regular de ACSF gelada sobre o tecido. </li><li> Usando o braço pontas de tesouras padrão standard fazer uma small incisão no crânio no Lambda e cortar ao longo da sutura sagital. </li><li> Deslize suavemente um braço de um fórceps rasas debaixo do osso parietal mantendo a lâmina tão próximo quanto possível da superfície inferior do osso sem arrastar o cérebro. Seguros e afastar o osso parietal lateral e posterior do osso occipital até que o cérebro está exposto. </li><li> Use uma pequena espátula de aço inoxidável e levante o cérebro de distância do anterior e fossa craniana média para expor o nervo vestibular (CN VIII). Transecto o nervo para evitar tensão desnecessária sobre os axônios aferentes primários que inervam diretamente as células ciliadas vestibulares. </li><li> Remover o cérebro in toto após transecção do CN VIII. </li><li> Observe o labirinto ósseo contendo a cóclea e os órgãos vestibulares periféricas da fossa craniana média. Faça duas pequenas incisões ao lado de cada labirinto ósseo e extirpar toda a estrutura, mantendo o canal semicircular anterior e puxando mais tardealiado. </li><li> Mergulhar imediatamente labirintos excisadas em um prato de dissecação contendo solução ACSF gelado (como descrito no passo 3.1), enquanto que irriga continuamente com carbogéneo. </li><li> Sob um microscópio estereoscópico, mantenha pressionado o labirinto pela cóclea e fixá-lo ao fundo do prato com uma pinça. <ol><li> Use uma pinça reta fina para riscar uma pequena abertura no osso acima do canal semicircular (SSC) ampulla anterior. </li><li> Gentilmente ampliar esta abertura, alcançando imediatamente abaixo do osso e sacudindo para fora ampulífuga. Tenha cuidado aqui para não empurrar pinça na abertura e danificar o labirinto membranoso frágil abaixo. Continuar dessa maneira até que o utrículo, anterior e ampolas laterais estão todos expostos. Se possível remover a ampola posterior também. </li></ol></li><li> Usando uma pinça fina, levantar delicadamente o utrículo e ampolas de distância do labirinto ósseo até que estejam completamente isolada. Sempre que possível, mantê-lospor seus canais semicirculares associados para evitar danos ao epitélio sensorial. Em alguns casos, a parte proximal da conduta membranoso semicircular pode ter de ser cortado com tesoura de íris para libertar as ampolas a partir do osso. </li><li> Firmemente compreender os órgãos vestibulares entre a ponta de uma pinça e lugar para o tampão de lise preparado anteriormente na etapa 3.2. Agite suavemente a pinça em torno do tampão para garantir órgãos vestibulares têm destacado a pinça. Confira este for o caso, trazendo a pinça, ao microscópio estereoscópico. <ol><li> Parafuso na tampa de microtúbulos e congelar a amostra em nitrogênio líquido imediatamente. </li></ol></li></ol> 4. Extracção de ARN e RT-PCR <ol><li> Siga métodos padrão de RNA mensageiro (mRNA) extracção de acordo com as instruções do fabricante ou protocolos de laboratório preferenciais. NOTA: Um kit comercial que utilizou ARN de suporte para ajudar a isolar pequenos rendimentos de ARNm foi utilizada nesteprotocolo. Volumes de eluição inferiores podem ser utilizados para aumentar as concentrações finais de ARNm. NOTA: Negativo controles &quot;não enzima&quot; (CNE) e controles &quot;nenhum modelo de DNA&quot; (NTC) também deve ser concluído para garantir a validade dos efeitos observados. </li><li> Aplicar os métodos padrão de transcrição reversa do ARNm de ADN complementar (ADNc) e amplificação de genes alvo 21-23. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Os resultados representativos descritos aqui mostram que a entrega breve transcraniana de luz NIR (90 seg / dia durante 5 dias) é suficiente para elevar os níveis de expressão de antioxidantes em ratos mais velhos quando comparados com os ratos tratados por simulação. Enquanto calor emitido poderia representar uma fonte de mitocondrial e / ou ativação neuronal, como relatado para vestibular rato aferentes 24 - nossa medição do calor emitido pelo NIR LED dispositivo foi &lt;0,2 ° C durante 90 segundo...

O declínio da performance de equilíbrio e quedas subsequentes são comuns, e, infelizmente, muitas vezes definem características de envelhecimento natural 1. O impacto desse declínio pode ser tanto física e social, e reduz significativamente a qualidade de vida das pessoas mais velhas. Em resposta, terapias físicas e reabilitação têm sido o foco da investigação sobre as quedas, mas não têm sido associadas com redução consistente na prevalência de quedas repetidas. Ao mesmo tempo, o trabalho investiga as mudanças no sistema vestibular periférico ou central (o sistema responsável por manter o equilíbrio) é escassa, e potenciais estratégias terapêuticas dirigidas a esses sistemas e as causas subjacentes de desequilíbrio limitado. Trabalhos recentes sobre doenças neurodegenerativas associadas com a idade relacionada com a idade, incluindo degeneração macular 2-4, modelos da doença de Alzheimer, 5-8 e 9-12 doença de Parkinson têm mostrado efeitos neuroprotectores de simple aplicação não invasiva do infravermelho próximo (NIR) luz. Além disso, no sistema vestibular, NIR foi usada para aumentar a actividade dos neurónios aferentes primários vestibulares in vitro 13. Embora o mecanismo de NIR luz não é bem compreendido, a maioria dos estudos que usam NIR sugeriram que estimula NIR mitocôndrias complexo IV (citocromo c oxidase) 14-17 para facilitar o metabolismo celular. No epitélio sensorial vestibular da placa subcuticular do tipo células ciliadas I é denso em mitocôndrias 18 e, como tal, pode representar um local de ação para o tratamento terapêutico NIR. Aqui, uma breve regime de tratamento, não-invasivo de transcraniana aplicado NIR que podem ser utilizadas para medir o metabolismo celular (e implicitamente, a função mitocondrial) no rato epitélio sensorial vestibular é descrito. Também é discutida a preparação de um epitélio sensorial vestibular e é mostrado que o NIR aumenta a expressão de um ubiquitonos anti-oxidante (superóxido dismutase 1) no epitélio sensorial - anteriormente mostrado ser importante para a sobrevivência de células do cabelo cóclea 19.

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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Estratégias para atenuar o declínio na função equilíbrio com o aumento da idade são predominantemente focado em terapias físicas, incluindo tarefas de equilíbrio e exercícios. No entanto, estas abordagens não abordam as causas subjacentes do declínio do equilíbrio. Usando ratos, o impacto da luz de infravermelho próximo (NIR) sobre o metabolismo de células do epitélio sensitivo vestibular foi avaliada. Os dados coletados mostram que essa intervenção simples e segura pode proteger essas células vulneráveis ​​dos efeitos deletérios do envelhecimento natural. ARNm foi extraído a partir do epitélio sensitivo vestibular periférico isolado (ampulares crista, e mácula utricular) e posteriormente transcrita para uma biblioteca de ADNc. Esta biblioteca foi então sondadas para a expressão de antioxidante ubíqua (SOD-1). A expressão do gene antioxidante foi então usado para quantificar o metabolismo celular. Usando entrega transcraniana de Nir em jovens (4 semanas) e mais velhos (8-9 meses), ratos, e um regime de tratamento breve (90 seg / dia para 5 days), este trabalho sugere NIR sozinho pode ser suficiente para melhorar a função mitocondrial no epitélio sensitivo vestibular. Uma vez que existem atualmente não há métodos disponíveis, acessíveis e não-invasivos de terapia para melhorar a função das células ciliadas vestibular, a aplicação de radiação NIR externa fornece uma estratégia potencial para neutralizar o impacto do envelhecimento sobre o metabolismo celular inthe epitélio sensorial vestibular.

Para comparar o impacto do tratamento NIR em jovens (4 semanas) e mais velhos (8-9 meses), camundongos medimos a expressão de antioxidante superóxido dismutase 1 (SOD-1) em jovens (n ​​= 16) e mais velhos (n = 20) os ratos que foram tratados Nir, tratamento simulado, ou NIR-bloqueados. A Figura 2 mostra um aumento significativo na normalizada SOD-1 expressão β-actina de mais de 2 vezes em jovens tratados com NIR animais jovens em comparação com os animais tratados de forma simulada (p &lt;0,01...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

A disfunção mitocondrial é uma característica da senescência celular. Este trabalho usa não-invasivo do infravermelho próximo (NIR) de tratamento para melhorar a função mitocondrial no rato epitélio sensorial vestibular de envelhecimento.

Infravermelho Próximo (NIR) Luz aumenta a expressão de um marcador da função mitocondrial no mouse Vestibular Sensorial Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.9K visualizações.  University of Sydney.  A disfunção mitocondrial é uma característica da senescência celular. Este trabalho usa não-invasivo do infravermelho próximo (NIR) de tratamento para melhorar a função mitocondrial no rato epitélio sensorial vestibular de envelhecimento. 

Vídeo: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

Neuromodulação e Transportes mitocondrial: Imagens ao vivo em neurônios do hipocampo durante muito tempo

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Neurociência

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Uma preparação semi-intactas isolada do Rato Vestibular epitélio sensorial de Eletrofisiologia e de alta resolução Microscopia de dois fótons

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

Imagem in vivo da fibra óptica Nerve Integrity por ressonância magnética com contraste em Ratos

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In Vitro e In Vivo de deteção de Mitophagy em células humanas, C. Eleganse os ratos

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Medicina

A Protocol for Transcranial Photobiomodulation Therapy in Mice

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide range of neurological and psychiatric disorders, and memory enhancement in age-related cognitive decline and neurodegenerative diseases. We describe a laboratory protocol for transcranial photobiomodulation therapy (PBMT) in mice. Aged BALB/c mice (18 months old) are treated with a 660 nm laser transcranially, once daily for 2 weeks. Laser transmittance data shows that approximately 1% of the incident red light on the scalp reaches a 1 mm depth from the cortical surface, penetrating the dorsal hippocampus. Treatment outcomes are assessed by two methods: a Barnes maze test, which is a hippocampus-dependent spatial learning and memory task evaluation, and measuring hippocampal ATP levels, which is used as a bioenergetics index. The results from the Barnes task show an enhancement of the spatial memory in laser-treated aged mice when compared with age-matched controls. Biochemical analysis after laser treatment indicates increased hippocampal ATP levels. We postulate that the enhancement of memory performance is potentially due to an improvement in hippocampal energy metabolism induced by the red laser treatment. The observations in mice could be extended to other animal models since this protocol could potentially be adapted to other species frequently used in translational neuroscience, such as rabbit, cat, dog, or monkey. Transcranial photobiomodulation is a safe and cost-effective modality which may be a promising therapeutic approach in age-related cognitive impairment.

Photobiomodulation therapy is an innovative noninvasive modality for the treatment of a wide range of neurological and psychiatric disorders and can also improve healthy brain function. This protocol includes a step-by-step guide to performing brain photobiomodulation in mice by transcranial light delivery, which can be adapted for use in other laboratory rodents.

Um protocolo para a terapia de décadas transcraniana em ratos

Transcranial photobiomodulation is a potential innovative noninvasive therapeutic approach for improving brain bioenergetics, brain function in a wide ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Imagem ao vivo do Estado De Glutathione Mitocondrial Redox em Neurônios Primários usando um Indicador Ratiométrico

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

A rastreï¿½el<em&gt; In Vivo</em&gt; Ensaio para mitocondrial Modulators Usando Transgenic Bioluminescent<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Biologia

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Medição do Potencial de Membrana Mitocondrial In Vivo usando um Indicador de Voltagem Geneticamente Codificado

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

Imagem de peróxido mitocondrial em células vivas de levedura usando mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Autor em destaque: Avançando na pesquisa mitocondrial - biossensor mtHyper7 para análise subcelular 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Imagem de mtHyPer7, um biossensor ratiométrico para peróxido mitocondrial, em células vivas de levedura

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Infravermelho Próximo (NIR) Luz aumenta a expressão de um marcador da função mitocondrial no mouse Vestibular Sensorial Epithelium

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