Name: near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium
Uploaded: 2015-03-14T14:00:00.000+00:00
Duration: 6 min 56 s
Description: 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

Gli autori desiderano ringraziare il Dr. Paul Witting e la signora Genevieve Fong per la loro assistenza con estrazione mRNA e PCR, e Garnett Passe e Rodney Williams Memorial Foundation per il supporto.

Etica Dichiarazione: Tutte le procedure descritte qui di seguito sono stati approvati dalla University of Sydney Comitato etico degli animali. 1. Gli animali NOTE: 1 e 8-9 mesi topi (C57 / BL6) sono stati ottenuti dalla Animal Resources Centre (Perth, Australia). I topi sono stati alloggiati in Facility Bosch Roditore presso l&#39;Università di Sydney. <ol><li> Casa topi in gabbie standard del mouse su un 12/12 luce hr / ciclo scuro con accesso a cibo e acqua ad libitum. </li><li> Topi Divide in ogni fascia d&#39;età in vicino infrarosso (NIR) trattati, o sham trattati (controllo) gruppi per il confronto. </li></ol> 2. vicino infrarosso (NIR) irradiazione e Sham trattamento <ol><li> Accorciare il pelo sulla regione della testa e del collo del mouse con un rasoio elettrico più vicino possibile impedire rapida ricrescita dei capelli prima del completamento del regime di trattamento. Per mantenere lo stato privo di patogeni dei topi, utilizzare etanolo al 70%per pulire strumenti tra gli animali da trucco e F10 veterinaria disinfettante tra animali di gabbie separate. Fare questo 2 - 3 giorni prima l&#39;inizio del trattamento in modo gli animali non sono più manico. </li><li> Trattenere il mouse tenendo l&#39;estremità prossimale della coda e permettono di rilassarsi nel palmo di una mano o superiore del banco di modo da ridurre al minimo lo stress che potrebbe portare a risultati falsi. </li><li> Tenere il NIR (670 nm) LED (light emitting diode) Dispositivo 1 - 2 cm di distanza dal (rasato) area esposta e accendere il dispositivo per 90 secondi (Figura 1A). NOTA: La variazione di temperatura a seguito di esposizione 90 sec stata misurata come &lt;0,2 ° C in 100 ml di acqua. </li><li> Ripetere i passaggi 2,2-2,3 per il gruppo di trattamento sham degli animali, ma lasciare l&#39;apparecchio spento (Figura 1B). </li><li> Ripetere i passaggi 2,2-2,3 per il gruppo di trattamento Nir-bloccato di animali, ma coprire il dispositivo con un foglio di alluminio. </li><li> Ripetere i passaggi 2,2-2,5 giorni alle approximate 24 intervalli hr per 5 giorni consecutivi. </li><li> Estrarre l&#39;epitelio vestibolare sensoriale (3 creste e 1 macula utricular) da entrambe le orecchie per il quinto giorno dopo il trattamento (vedere paragrafo 3). </li></ol> 3. Tissue estrazione 20 <ol><li> Preparare 300 ml di un liquido cerebrospinale artificiale (ACSF) basato glicerolo-costituiti (in mM): 26 NaHCO3, 11 glucosio, 250 glicerolo, 2,5 KCl, 1,2 NaH 2 PO 4, 1.2 MgCl 2, 2,5 e CaCl 2. Prima dell&#39;aggiunta di CaCl 2, gas la soluzione con CarboGen (95% O 2 e 5% CO 2) per stabilire un pH di 7,4 e di evitare la precipitazione di calcio (nuvolosità). Raffreddare la soluzione in un -80 ° C freezer per 45 minuti in modo da formare una sospensione di ghiaccio. </li><li> Preparare il tampone di lisi isolamento RNA etichettati microtubi tappo a vite (ferma il popping dei coperchi, quando le variazioni di pressione del tubo tra congelamento e scongelamento in azoto liquido) in basele istruzioni del fabbricante o usuali pratiche di laboratorio. Avere azoto liquido pronto in un matraccio dewar alluminio. NOTA: Usare indumenti protettivi e eyeware durante la manipolazione di azoto liquido. Assicurarsi che l&#39;azoto liquido viene utilizzato in una stanza ben ventilata come il volume della sua forma di gas è di circa 700 volte quella della sua forma liquida e può causare asfissia. </li><li> Profondamente anestetizzare topi con ketamina (400 mg / kg) tramite una iniezione intra-peritoneale. Lasciare che il riflesso arti posteriori a placarsi del tutto come indicazione che i topi sono completamente anestetizzati. </li><li> Decapitare topi con le forbici in acciaio inossidabile affilate e fare un&#39;incisione lungo la pelle sagittale del cranio con una lama di rasoio (arrotondato # 22). A questo punto e tutto passi 3,5-3,9, mantenere il più fresco possibile, l&#39;applicazione regolare di ACSF ghiacciato sopra il tessuto della volta cranica, cervello e apparato vestibolare sottostante. </li><li> Utilizzando il braccio punta di forbici modello standard fare un small &#39;incisione nel cranio a Lambda e tagliare lungo la sutura sagittale. </li><li> Scorrere delicatamente un braccio di un superficiale rongeurs sotto l&#39;osso parietale mantenendo la lama più vicino possibile alla superficie inferiore dell&#39;osso senza trascinare il cervello. Sicuro e tirare via l&#39;osso parietale laterale e l&#39;osso occipitale posteriormente fino a quando il cervello è esposto. </li><li> Utilizzare una piccola spatola di acciaio inox e sollevare il cervello dal anteriore e centrale fossa cranica per esporre il nervo vestibolococleare (CN VIII). Transetto il nervo per evitare inutili tensioni sui assoni afferenti primari che innervano direttamente le cellule ciliate vestibolari. </li><li> Rimuovere il cervello in toto dopo resezione del CN VIII. </li><li> Osservare il labirinto osseo contiene la coclea e gli organi vestibolari periferici nella fossa cranica media. Fare due piccole incisioni accanto a ogni labirinto osseo e asportare l&#39;intera struttura tenendo il canale semicircolare anteriore e tirare tardialleato. </li><li> Immergere immediatamente labirinti escisse in un piatto dissezione contenente soluzione ACSF ghiacciata (come descritto al punto 3.1), mentre continua perfusione con CarboGen. </li><li> Sotto uno stereomicroscopio, tenere premuto il labirinto della coclea e fissarla al fondo del piatto con una pinza. <ol><li> Utilizzare pinze dritto fini graffiare una piccola apertura nel midollo sopra anteriore canale semicircolare (SSC) ampolla. </li><li> Allargare delicatamente questa apertura raggiungendo immediatamente sotto l&#39;osso e sfogliando verso l&#39;esterno lontano dal ampolla. Fare attenzione qui per non spingere pinze nell&#39;apertura e danneggiare il labirinto membranoso fragile sotto. Continuare in questo modo fino a quando il utricolo, anteriore e ampolle laterale sono tutti esposti. Se possibile rimuovere anche l&#39;ampolla posteriori. </li></ol></li><li> Utilizzando una pinza sottile, sollevare delicatamente il utricolo e ampolle dal labirinto osseo fino a quando non sono completamente staccati. Ove possibile, tenerlidai canali semicircolari associati per evitare di danneggiare l&#39;epitelio sensoriale. In alcuni casi, la parte prossimale del condotto membranoso semicircolare può avere bisogno di essere tagliato con forbici iris per rilasciare le ampolle dall&#39;osso. </li><li> Saldamente cogliere gli organi vestibolari tra la punta di una pinza e posto nel buffer di lisi preparata in precedenza al punto 3.2. Mescolare delicatamente la pinza intorno nel buffer per garantire organi vestibolari hanno staccato dalla pinza. Controllare questo è il caso per portare le pinze con lo stereomicroscopio. <ol><li> Avvitare sul coperchio microtubuli e congelare il campione in azoto liquido immediatamente. </li></ol></li></ol> 4. RNA Estrazione e RT-PCR <ol><li> Seguire metodi standard di RNA messaggero (mRNA) di estrazione secondo le istruzioni del fabbricante o protocolli di laboratorio preferito. NOTA: un kit commerciale che utilizzava RNA carrier per aiutare isolare le piccole rese di mRNA è stato utilizzato in questoprotocollo. Volumi di eluizione inferiori possono essere usati per aumentare le concentrazioni finali di mRNA. NOTA: Controlli negativo &quot;no enzima&quot; (NEC) e controlli &quot;non modello DNA&quot; (NTC) devono essere completati per garantire la validità degli effetti osservati. </li><li> Applicare metodi standard di trascrizione inversa di mRNA a DNA complementare (cDNA) e l&#39;amplificazione di geni bersaglio 21-23. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

I risultati rappresentativi qui descritti mostrano che breve consegna transcranica di NIR luce (90 sec / giorno per 5 giorni) è sufficiente per aumentare i livelli di espressione antiossidante nei topi anziani rispetto ai topi sham trattati. Mentre calore emesso potrebbe rappresentare una fonte di mitocondriale e / o l&#39;attivazione neuronale, come riportato per ratto vestibolare afferenti 24 - nostra misurazione del calore emesso dal LED NIR dispositivo era &lt;0,2 ° C superiore al 90 sec, e come tale è...

Il calo delle prestazioni equilibrio e cadute successive sono comuni, e le caratteristiche, purtroppo, spesso definiscono di invecchiamento naturale 1. L&#39;impatto di questo declino può essere sia fisica che sociale, e riduce in modo significativo la qualità della vita per le persone anziane. In risposta, terapie fisiche e riabilitazione sono stati al centro della ricerca di cadute, ma non sono stati associati con la riduzione consistente della prevalenza di cadute ripetute. Allo stesso tempo, il lavoro indagando i cambiamenti nel sistema periferico o centrale vestibolare (il sistema responsabile per mantenere l&#39;equilibrio) è scarso, e potenziali strategie terapeutiche destinate a questi sistemi e le cause di squilibrio limitato. Un recente lavoro su malattie neurodegenerative legate all&#39;età, tra cui età-correlate degenerazione maculare 2-4, modelli di malattia di Alzheimer 5-8, e il morbo di Parkinson 9-12 hanno mostrato effetti neuroprotettivi di simple applicazione non invasiva del vicino infrarosso (NIR) luce. Inoltre, nel sistema vestibolare, NIR è stato usato per aumentare l&#39;attività di neuroni vestibolari afferenti primari in vitro 13. Mentre il meccanismo di Nir luce non è ben compreso,, maggior parte degli studi che utilizzano NIR hanno suggerito che Nir stimola mitocondri complesso IV (citocromo c ossidasi) 14-17 per facilitare il metabolismo cellulare. Nel epitelio sensoriale vestibolare piastra sottocuticolare delle cellule ciliate che tipo è denso in mitocondri 18 e come tale può rappresentare un luogo di azione per il trattamento terapeutico Nir. Qui, una breve, regime di trattamento non invasivo transcranially applicato NIR che può essere utilizzato per misurare il metabolismo cellulare (e di conseguenza, la funzione mitocondriale) nel topo vestibolare dell&#39;epitelio sensoriale descritto. Anche discusso è una preparazione dell&#39;epitelio sensoriale vestibolare e si dimostra che NIR aumenta l&#39;espressione di un ubiquitonoi anti-ossidante (superossido dismutasi 1) nell&#39;epitelio sensoriale - precedentemente dimostrato di essere importante per la sopravvivenza delle cellule dei capelli coclea 19.

<table><tbody><tr><td>Quantum WARP 10</td><td>Quantum Devices</td><td>2070N030-A</td><td></td></tr><tr><td>Screw top microtubules</td><td>Quality Scientific Plastics</td><td>520-GRD-Q</td><td></td></tr><tr><td>Ketamine</td><td>Parnell, Alexandria Australia</td><td></td><td></td></tr><tr><td>Standard pattern scissors</td><td>FST</td><td>14001-12</td><td></td></tr><tr><td>Carbon steel surgical blades #22</td><td>Livingstone</td><td>SBLDCL 22</td><td></td></tr><tr><td>Friedman-Pearson rongeurs</td><td>FST</td><td>16221-14</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>A60S</td><td></td></tr><tr><td>Dumont #5 SF forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Isolate II RNA Micro Kit</td><td>Bioline</td><td>BIO-52075</td><td></td></tr></tbody></table>

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

Strategie per attenuare il declino in funzione di bilanciamento con l&#39;aumentare dell&#39;età sono prevalentemente concentrati su terapie fisiche, tra cui le attività di bilancio e l&#39;esercizio fisico. Tuttavia, questi approcci non affrontano le cause alla base dell&#39;equilibrio declino. Usando topi, è stato valutato l&#39;effetto di luce vicino infrarosso (NIR) sul metabolismo di cellule dell&#39;epitelio sensoriale vestibolare. I dati raccolti mostrano che questo intervento semplice e sicuro può proteggere queste cellule vulnerabili dagli effetti deleteri di invecchiamento naturale. mRNA è stato estratto dalla isolato epitelio periferico vestibolare sensoriale (ampollare crista e macula utricular) e successivamente trascritto in una libreria di cDNA. Questa libreria è stato poi indagato per l&#39;espressione di antiossidanti onnipresente (SOD-1). Antiossidante espressione genica è stato poi utilizzato per quantificare il metabolismo cellulare. Utilizzando consegna transcranica di Nir in giovane (4 settimane) e anziani (8-9 mesi) i topi, e un regime di trattamento breve (90 sec / giorno per 5 days), questo lavoro suggerisce Nir sola può essere sufficiente per migliorare la funzione mitocondriale nell&#39;epitelio sensoriale vestibolare. Dal momento che non ci sono attualmente disponibili, a prezzi accessibili, i metodi non invasivi di terapia per migliorare la funzione delle cellule dei capelli vestibolare, l&#39;applicazione delle radiazioni NIR esterna fornisce una strategia potenziale per contrastare l&#39;impatto dell&#39;invecchiamento sul metabolismo cellulare inthe vestibolare epitelio sensoriale.

Per confrontare l&#39;impatto del trattamento Nir nei giovani (4 settimane) e anziani (8-9 mesi) topi abbiamo misurato l&#39;espressione di antiossidanti superossido dismutasi 1 (SOD-1) nei giovani (n = 16) e anziani (n = 20) i topi che erano NIR-trattata, sham-trattati, o Nir-bloccato. figura 2 mostra un aumento significativo normalizzato SOD-1 β-actina di oltre 2 volte nei giovani Nir-animali trattati rispetto ai giovani animali sham trattati (p &lt;0.01) e animali giovani NIR-bloccati (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

Disfunzione mitocondriale è una caratteristica della senescenza cellulare. Questo documento utilizza nel vicino infrarosso (NIR) trattamento non invasivo per migliorare la funzione mitocondriale nel invecchiamento del mouse vestibolare epitelio sensoriale.

Vicino infrarosso (NIR) Luce aumenta l&#39;espressione di un marcatore di mitocondriale Funzione nel mouse vestibolare sensoriale Epithelium

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

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Research

JoVE Journal

Biology

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

9.9K Views.  University of Sydney.  Disfunzione mitocondriale è una caratteristica della senescenza cellulare. Questo documento utilizza nel vicino infrarosso (NIR) trattamento non invasivo per migliorare la funzione mitocondriale nel invecchiamento del mouse vestibolare epitelio sensoriale. 

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

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Behavioral Valutazione del sistema vestibolare invecchiamento mouse

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Ricerca

Comportamento

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Un protocollo per valutare e quantificare le patologie dell'epitelio pigmentato retinico in modelli murini di degenerazione maculare legata all'età

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetica

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.

Un isolato semi-intatte Preparazione del mouse vestibolare epitelio sensoriale di Elettrofisiologia e ad alta risoluzione microscopia a due fotoni

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

Neuroscienze

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).

In vivo imaging di Optic Nerve Fiber Integrity by MRI Contrast-Enhanced nei topi

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

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Studiare il comportamento simile all'emicrania usando l'avversione alla luce nei topi

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Valutazione della funzione mitocondriale nel nervo sciatico mediante respirometria ad alta risoluzione

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Biochimica

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.

Un protocollo per la terapia di fotobiomodulazione Transcranial in topi

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

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Determinazione della respirazione mitocondriale e della glicolisi in campioni di tessuto retinico Ex Vivo

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Metodo per la valutazione degli effetti di un intervallo di lunghezze d&#39;onda e intensità di / vicino-infrarosso di terapia della luce rossa sullo stress ossidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Biologia

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-Lapse Video Microscopia per la valutazione di EYFP-Parkin aggregazione come marcatore per cellulare Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

Vicino infrarosso (NIR) Luce aumenta l'espressione di un marcatore di mitocondriale Funzione nel mouse vestibolare sensoriale Epithelium

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