The authors wish to acknowledge Dr. Paul Witting and Ms. Genevieve Fong for their assistance with mRNA extraction and PCR, and the Garnett Passe and Rodney Williams Memorial Foundation for support.

Ethics Statement: All procedures outlined below were approved by the University of Sydney Animal Ethics Committee.
1. Animals
NOTE: 1 and 8 - 9 month old mice (C57/BL6) were obtained from the Animal Resources Centre (Perth, Australia). Mice were housed in the Bosch Rodent Facility at the University of Sydney.
<ol>
	<li>House mice in standard mouse cages on a 12/12 hr light/dark cycle with access to food and water ad libitum.</li>
	<li>Divide mice in each age group into near infrared (NIr) treated, or sham treated (control) groups for comparison.</li>
</ol>
2. Near Infrared (NIr) Irradiation and Sham Treatment
<ol>
	<li>Shave the fur on the head and neck region of the mouse with an electric razor as closely as possible to prevent quick regrowth of hair before the completion of the treatment regime. In order to maintain the pathogen-free status of the mice, use 70% ethanol to clean instruments between shaving animals and F10 veterinary disinfectant between animals from separate cages. Do this 2 - 3 days prior to the beginning of treatment so animals are not over-handled.</li>
	<li>Restrain mouse by holding the proximal end of the tail and allow it to relax in the palm of one hand or bench top so as to minimize stress which could lead to false results.</li>
	<li>Hold the NIr (670 nm) LED (light emitting diode) device 1 - 2 cm away from the exposed (shaved) area and switch on the device for 90 sec (Figure 1A). 
	NOTE: The temperature change as a result of 90 sec exposure was measured as &#60;0.2 &#176;C in 100 ml of water.</li>
	<li>Repeat steps 2.2 - 2.3 for the sham treatment group of animals but leave the device switched off (Figure 1B).</li>
	<li>Repeat steps 2.2 - 2.3 for the NIr-blocked treatment group of animals but cover the device with aluminium foil.</li>
	<li>Repeat steps 2.2 - 2.5&#160;daily at approximate 24 hr intervals for 5 consecutive days.</li>
	<li>Extract the vestibular sensory epithelium (3 cristae and 1 utricular macula) from both ears on the fifth day post-treatment (See Section 3 below).</li>
</ol>
3. Tissue Extraction20
<ol>
	<li>Prepare 300 ml of a glycerol-based artificial cerebrospinal fluid (ACSF) consisting of (in mM): 26 NaHCO3, 11 glucose, 250 glycerol, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, and 2.5 CaCl2. Prior to the addition of CaCl2, gas the solution with carbogen (95% O2 and 5% CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness). Chill the solution in a -80 &#176;C freezer for 45 min so that an ice slurry is formed.</li>
	<li>Prepare the RNA isolation lysis buffer in labelled screw top microtubes (stops the popping of lids when tube pressure changes between freeze and thawing in liquid nitrogen) according to the instructions of the manufacturer or usual lab practices. Have liquid nitrogen ready in an aluminium dewar flask. 
	NOTE: Use protective clothing and eyeware when handling liquid nitrogen. Ensure that liquid nitrogen is used in a well-ventilated room as the volume of its gas form is approximately 700 times that of its liquid form and can cause asphyxiation.</li>
	<li>Deeply anaesthetize mice with ketamine (400 mg/kg) via an intra-peritoneal injection. Allow the hind-limb reflex to completely subside as indication that mice are fully anaesthetised.</li>
	<li>Decapitate mice with sharp stainless steel scissors and make an incision along the sagittal skin of the skull using a razor blade (rounded #22). At this point and throughout steps 3.5 - 3.9, keep the cranial vault, brain, and underlying vestibular apparatus as cool as possible by regular application of ice-cold ACSF over the tissue.</li>
	<li>Using the pointed arm of standard pattern scissors make a small incision in the skull at Lambda and cut along the sagittal suture.</li>
	<li>Gently slide one arm of a shallow rongeurs beneath the parietal bone keeping the blade as close as possible to the inferior surface of the bone without dragging the brain. Secure and pull away the parietal bone laterally and the occipital bone posteriorly until the brain is exposed.</li>
	<li>Use a small stainless steel spatula and lift the brain away from the anterior and middle cranial fossa to expose the vestibulocochlear nerve (CN VIII). Transect the nerve to prevent unnecessary tension on the primary afferent axons that directly innervate the vestibular hair cells.</li>
	<li>Remove the brain in toto after transection of CN VIII.</li>
	<li>Observe the bony labyrinth containing the cochlea and the peripheral vestibular organs in the middle cranial fossa. Make two small incisions beside each bony labyrinth and excise the entire structure by holding the anterior semicircular canal and pulling laterally.</li>
	<li>Immediately immerse excised labyrinths in a dissecting dish containing ice-cold ACSF solution (as described in step 3.1) while continuously perfusing with carbogen.</li>
	<li>Under a stereomicroscope, hold down the labyrinth by the cochlea and secure it to the bottom of the dish with forceps.
	<ol>
		<li>Use straight fine forceps to scratch a small opening in the bone above the anterior semicircular canal (SSC) ampulla.</li>
		<li>Gently enlarge this opening by reaching immediately below the bone and flicking outwards away from the ampulla. Exercise caution here to not push forceps into the opening and damage the fragile membranous labyrinth below. Continue in this fashion until the utricle, anterior and lateral ampullae are all exposed. If possible remove the posterior ampulla also.</li>
	</ol>
	</li>
	<li>Using fine forceps, gently lift the utricle and ampullae away from the bony labyrinth until they are completely detached. Where possible, hold them by their associated semicircular canals to avoid damaging the sensory epithelium. In some cases, the proximal part of the semicircular membranous duct may need to be cut with iris scissors to release the ampullae from the bone.</li>
	<li>Securely grasp the vestibular organs between the tip of forceps and place into the lysis buffer prepared earlier in step 3.2. Gently swirl the forceps around in the buffer to ensure vestibular organs have detached from the forceps. Check this is the case by bringing the forceps under the stereomicroscope.
	<ol>
		<li>Screw on the microtubule lid and freeze the sample in liquid nitrogen immediately.</li>
	</ol>
	</li>
</ol>
4. RNA Extraction and RT-PCR

<ol>
	<li>Follow standard methods of messenger RNA (mRNA) extraction according to manufacturer&#8217;s instructions or preferred lab protocols. 
	NOTE: A commercial kit that utilized carrier RNAs to help isolate small yields of mRNA was used in this protocol. Lower elution volumes can be used to increase final concentrations of mRNA. 
	NOTE: Negative &#8220;no enzyme&#8221; controls (NECs) and &#8220;no DNA template&#8221; controls (NTCs) must also be completed to ensure validity of observed effects.</li>
	<li>Apply standard methods of reverse transcription of mRNA to complementary DNA (cDNA) and amplification of target genes21-23.</li>
</ol>

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The authors declare they have no competing financial interests.

The representative results described here show that brief transcranial delivery of NIr light (90 sec/day for 5 days) is sufficient to raise the levels of antioxidant expression in older mice when compared with sham-treated mice. While emitted heat could represent a source of mitochondrial and/or neuronal activation, as reported for rat vestibular afferents24 &#8211; our measurement of heat emitted by the NIr LED device was &#60;0.2&#176;C over 90 sec, and as such is unlikely to cause the changes described here...

Declining balance performance and subsequent falls are common, and unfortunately often defining features of natural aging1. The impact of this decline can be both physical and social, and significantly reduces quality of life for older people. In response, physical therapies and rehabilitation have been the focus of research into falls but have not been associated with consistent reduction in the prevalence of repeated falls. At the same time, work investigating changes in the peripheral or central vestibular system (the system responsible for maintain balance) is scarce, and potential therapeutic strategies targeting these systems and the underlying causes of imbalance limited.
Recent work on age-associated neurodegenerative disorders including age-related macular degeneration2-4, Alzheimer&#8217;s disease models5-8, and Parkinson&#8217;s disease9-12 have shown neuroprotective effects of simple non-invasive application of near infrared (NIr) light. Further, in the vestibular system, NIr has been used to increase the activity of vestibular primary afferent neurons in vitro13. While the mechanism of NIr light is not well-understood, most studies using NIr have suggested that NIr stimulates mitochondria complex IV (cytochrome c oxidase)14-17 to facilitate cellular metabolism. In the vestibular sensory epithelium the subcuticular plate of type I hair cells is dense in mitochondria18 and as such may represent a site of action for therapeutic NIr treatment.
Here, a brief, non-invasive treatment regime of transcranially applied NIr that can be used to measure cellular metabolism (and by implication, mitochondrial function) in the mouse vestibular sensory epithelium is described. Also discussed is a preparation of the vestibular sensory epithelium and it is shown that NIr increases the expression of a ubiquitous anti-oxidant (superoxide dismutase 1) in the sensory epithelium &#8211; previously shown to be important for cochlea hair cell survival19.

<table border="0" cellpadding="0" cellspacing="0" width="603">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Quantum WARP 10</td>
			<td style="width:101px;">Quantum Devices</td>
			<td style="width:119px;">2070N030-A</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Screw top microtubules</td>
			<td style="width:101px;">Quality Scientific Plastics</td>
			<td style="width:119px;">520-GRD-Q</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ketamine</td>
			<td style="width:101px;">Parnell, Alexandria Australia</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Pattern Scissors</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">14001-12</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Carbon steel Surgical Blades #22</td>
			<td style="width:101px;">Livingstone</td>
			<td style="width:119px;">SBLDCL 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Friedman-Pearson Rongeurs</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">16221-14</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stereo microscope</td>
			<td style="width:101px;">Leica Microsystems</td>
			<td style="width:119px;">A60S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont #5 SF Forceps</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">11252-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
			<td style="width:101px;">Bioline</td>
			<td style="width:119px;">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

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

To compare the impact of NIr treatment in young (4 weeks) and older (8 - 9 months) mice we measured the expression of antioxidant superoxide dismutase 1 (SOD-1) in young (n = 16) and older (n = 20) mice that were NIr-treated, sham-treated, or NIr-blocked. Figure 2 shows a significant increase in &#946;-actin normalized SOD-1 expression of more than 2-fold in young NIr-treated animals compared to young sham-treated animals (p &#60; 0.01) and young NIr-blocked animals (p &#60; 0.01). Older NIr-treated anim...

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

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

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

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

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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.

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 ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...