Part 1: Setting up and preparing the skin
<ol>
<li>Set up the following sensors breathing belt, EMG, ECG, skin conductance, temperature, ground;</li>
<li>Prepare the skin by gently rubbing with an alcohol wipe in the following locations: forehead above the eyes for EMG, inside of both wrists for ECG, back of left hand for the ground (location of ground electrode may vary depending on equipment used);</li>
<li>Attach sensors to the skin in the following locations: breathing belt around the waist, EMG on the forehead above each eye, ECG on the inside of each wrist, skin conductance on middle finger of left hand, thermometer on the pointer of left hand, ground on the back of left hand;</li>
<li>Select session for Breathing+HRV;</li>
<li>Make sure that impendence is acceptable for all sensors and that the sensors are responsive to client&rsquo;s movement and producing reasonable readings.</li>
</ol>
Part 2: Recording
<ol>
<li>Explain the procedure to the client.</li>
<li>(min 0-2) Instruct the client to sit quietly with eyes open. Record a two-minute baseline.</li>
<li>(min 2-4) At the end of two minutes, begin a two-minute cognitive stressor &ndash; arithmetic problems gradually increasing in difficulty to be solved without use of pen &amp; paper or calculator (if arithmetic is particularly difficult for the client, ask them to repeatedly subtract seven starting from 100, i.e., &ldquo;serial sevens&rdquo;). Give the following instructions to the client: &ldquo;In the next 2 minutes, I will be asking you to solve some math problems. Please work as quickly as you can and tell me the answer&rdquo;. Do not provide feedback as to whether the client&rsquo;s answer is correct or incorrect and provide minimal encouragement (eg., &ldquo;just do the best you can&rdquo;) so as not to eliminate the mild stress inducing properties of this exercise.</li>
<li>(min 4-6) Let the client know that this part of the assessment is over and ask him/her to sit quietly with eyes open for 2 minutes.</li>
<li>(min 6-8) Begin physical stressor: turn on loud noxious noise (babies crying, car crashes, etc.) and ask the client to listen to the sounds without tuning out.</li>
<li>(min 8-10) Turn off noise, and ask the client to sit quietly with eyes open for 2 minutes.</li>
<li>(min 10-12) Begin emotional stressor: ask the client to talk about a stressful incident that they remember well, include the details of the event, what they were thinking and feeling. Provide minimal empathic response so as not to mitigate the stress response.</li>
<li>(min 12-14) Interrupt the client&rsquo;s story, even if he/she has not finished, and instruct the client to sit quietly with eyes open.</li>
<li>Stop recording, save the data, and let the client know that the assessment is done.</li>
</ol>
Part 3: Debriefing and treatment planning
<ol>
<li>Ask the client about their experience during the assessment. Which parts were particularly stressful and why? Did he/she feel any physiological changes happening?</li>
<li>Review data according to the criteria described in appendix I.</li>
<li>Chose physiological parameters that need to be addressed based on above analysis. If sensor reading at baseline is higher or lower than normal, the treatment goal is to help the client bring that physiological parameter to normal in a non-stressful environment. If sensor reading during the stressor is higher or lower than normal, the treatment goal is to help the client identify stressors and be able to maintain normal level of physiological functioning under stress. If sensor reading during down time indicates lack of recovery, the treatment goal is to help the client bring that physiological parameter to normal level quickly following a stressor. Any or all of these goals may be included in the treatment plan.</li>
</ol>
Appendix I
Stress assessment interpretation guidelines
(all &ldquo;bold&rdquo; answers indicate areas to be addressed)
<table border="0">
<tbody>
<tr>
<td colspan="3">EMG (normal reading &lt;3 microvolts)</td>
</tr>
<tr>
<td>1. Is EMG elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is EMG elevated past the baseline at any of the 3 stressors?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of EMG to baseline level during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td colspan="3">Heart rate (normal range 60-80 beats per minute)</td>
</tr>
<tr>
<td>1. Is heart rate elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is heart rate elevated past the baseline at any of the 3 stressors?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of heart rate to baseline level during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td colspan="3">Breathing rate (normal rate &lt;12 breaths per minute)</td>
</tr>
<tr>
<td>1. Is breathing rate elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is breathing rate elevated past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of breathing rate to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td colspan="3">Skin conductance</td>
</tr>
<tr>
<td>1. Is skin conductance elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is skin conductance elevated past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of skin conductance to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td colspan="3">Finger temperature (normal reading &gt;90F)</td>
</tr>
<tr>
<td>1. Is finger temperature low at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Does finger temperature decrease past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of finger temperature to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td colspan="3">Heart rate variability (norms vary by age)</td>
</tr>
<tr>
<td>1. Is heart rate variability low at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Does heart rate variability decrease past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of heart rate variability during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
</tbody>
</table>
Appendix I
Stress assessment interpretation:
<table border="0">
<tbody>
<tr>
<td colspan="3">EMG (normal reading &lt;3 microvolts)</td>
</tr>
<tr>
<td>1. Is EMG elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is EMG elevated past the baseline at any of the 3 stressors?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of EMG to baseline level during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>Heart rate (normal range 60-80 beats per minute)</td>
</tr>
<tr>
<td>1. Is heart rate elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is heart rate elevated past the baseline at any of the 3 stressors?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of heart rate to baseline level during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>Breathing rate (normal rate &lt;12 breaths per minute)</td>
</tr>
<tr>
<td>1. Is breathing rate elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is breathing rate elevated past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of breathing rate to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>Skin conductance (normal reading &lt;5 micro ohms)</td>
</tr>
<tr>
<td>1. Is skin conductance elevated at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Is skin conductance elevated past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of skin conductance to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>Finger temperature (normal reading &gt;90F)</td>
</tr>
<tr>
<td>1. Is finger temperature low at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Does finger temperature decrease past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of finger temperature to baseline during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>Heart rate variability (norms vary by age)</td>
</tr>
<tr>
<td>1. Is heart rate variability low at baseline?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>2. Does heart rate variability decrease past the baseline for any stressor?</td>
<td>yes _</td>
<td>no _</td>
</tr>
<tr>
<td>3. Is there recovery of heart rate variability during rest periods?</td>
<td>yes _</td>
<td>no _</td>
</tr>
</tbody>
</table>
Treatment planning: If a reading is higher or lower than normal at baseline, goal of treatment is to help client bring that physiological parameter to normal in all circumstances If a reading is higher or lower than normal during the stressor, goal of treatment is to help client identify stressors and be able to maintain normal level of physiological functioning under stress If a reading indicates lack of recovery during down time, goal of treatment is to help client bring that physiological parameter to normal level quickly following a stressor.

<ol>
	<li>Arena, J., Schwartz, M. S., Schwartz, M. S., Andrasik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychophysiological+assessment+and+biofeedback+baselines.">Psychophysiological assessment and biofeedback baselines.</a> A Practitioners Guide. , 128-158 (2005).</li><li>Schwartz, N. M., Schwartz, M. S., Schwartz, M. S., Andrasik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitons+of+biofeedback+and+applied+psychophysiology.">Definitons of biofeedback and applied psychophysiology.</a> A Practitioners Guide. , 27-42 (2005).</li></ol>

The authors have nothing to disclose.

Completed psychophysiological in the appendi stress assessment provides a biofeedback practitioner with information necessary for formulating a treatment plan. This assessment provides information about both the individual&rsquo;s response stereotypy, or uniformity of certain physiological responses to all stressors, and stimulus-response specificity, or individual&rsquo;s unique physiological responses to different kinds of stressors. With this information, the client is then able to learn to predict what kind of physio...

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<td>Physiolab I-330-C2</td>
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psychophysiological stress assessment using biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Watch this Scientific Journal Video about Psychophysiological Stress Assessment Using Biofeedback at JoVE.com

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

psychophysiological-stress-assessment-using-biofeedback

Research

JoVE Journal

Biology

13.3K Views.  Cambridge Health Alliance. In questo video si descrive un metodo per valutare la reazione psicofisiologica allo stress della persona utilizzando il biofeedback. Saremo anche presenti linee guida generali per la pianificazione del trattamento.

Video: Psychophysiological Stress Assessment Using Biofeedback

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important treatment for aneurysms which attempts to exclude blood from flowing into the aneurysmal segment of the vessel while preserving blood flow in a normal fashion.  Improper clip placement may result in residual aneurysm with the potential for subsequent aneurysm rupture or partial or full occlusion of distal arteries resulting in cerebral infarction.  Here we describe the use of an ultrasonic flow probe to provide quantitative evaluation of arterial flow before and after microsurgical clip placement at the base of a middle cerebral artery aneurysm.  This information helps ensure adequate aneurysm reconstruction with preservation of normal distal blood flow.

Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement.

Cerebral aneurysms are abnormal widening or ballooning of a localized segment of an intracranial blood vessel.  Surgical clipping is an important ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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 ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

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