Name: versatility of protocols for resistance training and assessment using static and dynamic ladders in animal models
Uploaded: 2021-12-17T14:00:00
Description: Watch this Scientific Journal Video about Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models at JoVE.com

This work was supported in part by the Ministerio de Econom&#237;a y Competitividad, Spain (DEP2012-39262 to EI-G and DEP2015-69980-P to BF-G). Thanks to Frank Mcleod Henderson Higgins from McLeod&#180;s English Centre in Asturias, Spain, for language assistance.

The methods presented in this protocol have been evaluated and approved by the animal research technical committee (reference PROAE 04/2018, Principado de Asturias, Spain).
1. Planning
<ol>
	<li>Carefully select animals for the study based on the characteristics of interest (genetically modified, pathology models, age, etc.) and apply specific adaptations to the protocol (climbing without weights, reducing the number of rungs to climb, and inclination).</li>
	<li>Identify th...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+resistance+training+on+matrix+metalloproteinase+activity+in+skeletal+muscles+and+blood+circulation+during+aging.">Effects of resistance training on matrix metalloproteinase activity in skeletal muscles and blood circulation during aging.</a> Frontiers in Physiology. 9, 190 (2018).</li><li>Ghosh, S., Golbidi, S., Werner, I., Verchere, B. 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Training is an intervention with multiple applications in research, apart from the study of exercise itself. Thus, the analysis of its effect on ageing20&#160;or certain pathological conditions and physical therapy21&#160;has received much attention in recent years. In addition, numerous authors have analyzed the effect of pharmacological22&#160;or dietary21&#160;interventions on physical fitness. In this context, interest has...

Physical exercise is a determinant lifestyle factor for promoting health and decreasing the incidence of the most prevalent chronic diseases as well as some types of cancer in humans1.
Resistance exercise has raised interest because of its overwhelming relevance for health throughout life2, especially due to its benefits in counteracting age-related diseases that affect the locomotor system, such as sarcopenia, osteoporosis, etc3. Moreover, resistance exercise also affects tissues and organs not directly involved in the execution of movement, such as the brain4. This relevance in recent years has encouraged the development of resistance exercise models in animals to study the underlying tissular and molecular mechanisms, when it is not possible in humans or when the animals provide better insight and are a more controlled model.
Unlike resistance exercise in humans, for animal models&#160;researchers usually rely on forced procedures. However, aversive stimuli must be avoided in this context, mainly to preserve animal welfare, reduce stress, and decrease the severity of the experimental procedures5. It should be noted that animals enjoy exercise even in the wild6. For these reasons, it is necessary to improve adaptation to the experiment through prolonged stepwise acclimatization.
The devices, materials, and protocols used for resistance training and assessment in experimental animals must allow the precise control and modulation of numerous variables: load, volume, speed, and frequency7. They should also allow different types of muscle contractions to be performed: concentric, eccentric, or isometric. Considering the above, the protocols used should be able to specifically evaluate or train for different applications of strength: maximal strength, hypertrophy, speed, and endurance.
There are several methods of strength training, such as jumping in water8,9, weighted swimming in water10, or muscle electrostimulation11. However, static and dynamic ladders are versatile devices that are widely used12,13,14.
Resistance assessment in experimental animal models provides valuable information for many research settings, such as describing the phenotypic characteristics of genetically modified animals, evaluating the effect of different intervention protocols (dietary components supplementation, drug treatments, microbiota transplantation, etc.), or assessing the effect of training protocols. Training models provide insight into the physiology of adaptation to strength exercise, which helps to better understand the effect of exercise on health status and pathophysiology.
Consequently, there is no universal protocol for resistance training or the functional assessment of strength in animal models, so versatile protocols are needed.
The aim of this study is to identify the most relevant factors to be considered when designing and applying a protocol for resistance training and evaluation using static and dynamic ladders in animal models, as well as provide specific examples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dynamic ladder</td>
			<td>in-house production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic adhesive bandage 6 cm x 2.5 m</td>
			<td>BSN medical</td>
			<td>4005556</td>
			<td></td>
		</tr>
		<tr>
			<td>Gator Clip Steel NON-INSUL 10A</td>
			<td>Digikey electronics</td>
			<td>BC60ANP</td>
			<td></td>
		</tr>
		<tr>
			<td>Static ladder</td>
			<td>in-house production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Weights</td>
			<td>in-house production</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wire for holding weigths</td>
			<td>in-house production</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

versatility of protocols for resistance training and assessment using static and dynamic ladders in animal models

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way to gain insight into the underlying molecular mechanisms that orchestrate these adaptations. The aim of this article is to describe exercise models and training protocols designed for strength training and evaluation of resistance in animal models and provide examples. In this article, strength training and resistance evaluation are based on ladder climbing activity, using static and dynamic ladders. These devices allow a variety of training models as well as provide precise control of the main variables which determine resistance exercise: volume, load, velocity, and frequency. Furthermore, unlike resistance exercise in humans, this is a forced exercise. Thus, aversive stimuli must be avoided in this intervention to preserve animal welfare. Prior to implementation, a detailed design is necessary, along with an acclimatization and learning period. Acclimatization to training devices, such as ladders, weights, and clinical tape, as well as to the manipulations required, is necessary to avoid exercise rejection and to minimize stress. At the same time, the animals are taught to climb up the ladder, not down, to the resting area on the top of the ladder. Resistance evaluation can characterize physical strength and permit adjusting and quantifying the training load and the response to training. Furthermore, different types of strength can be evaluated. Regarding training programs, with appropriate design and device use, they can be sufficiently versatile to modulate different types of strength. Furthermore, they should be flexible enough to be modified depending on the adaptive and behavioral response of the animals or the presence of injuries. In conclusion, resistance training and assessment using ladders and weights are versatile methods in animal research.

Results with static ladder 
The progressive resistance training protocol used and described by Codina-Martinez et al.4 (Table 4) was tested in a preliminary study consisting of 7 weeks of training on a static ladder with 6-months-old wild-type C57BL6J mice (n = 4). In this preliminary study, incremental tests to assess maximal strength were performed before and after the training period. We observed a 46.4% increase in maximal strength, meaning that at the en...

Watch this Scientific Journal Video about Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models at JoVE.com

Versatility of Protocols for Resistance Training and Assessment Using Static and Dynamic Ladders in Animal Models

The present protocol describes resistance training and testing using static and dynamic ladders in animal models.

Resistance training is a physical exercise model with profound benefits for health throughout life. The use of resistance exercise animal models is a way ...

This resistance training protocol is significant because it can be applied in animal research to elucidate the effects of strength training on health. The main advantage of this method is that it can be applied for various strength training models and strength assessments. This technique provides an insight into physical fitness, specifically strength, and can be applied to study systemic diseases and the effect of treatments on muscle performance.It allows for training and evaluating the strength of mice, but has a mandatory requirement which is the realization of sufficient acclimatization. Visual demonstration of this method is critical because the visualization of handling, training, and assessing the resistance of the mice is more meaningful than text. Demonstrating the procedure will be Doctor Cristina Tomas-Zapico and Doctor Manuel Fernandez Sanjurjo from Doctor Benjamin Fernandez-Garcia's laboratory.To begin, carefully select animals for the study based on the characteristics of interest. Plan all issues related to training, particularly, the timetable, duration of the training period, and frequency of sessions, and draw a training table. Next, accustom the mice to stay in the resting area at the top of the ladder by leaving the animals in this area in groups of four with bedding from their cage for 15 minutes every day.Teach the mice to climb up and knock down the ladder. For this, use a static ladder and place the mouse on a rung close to the top of the ladder from where they can see the resting area and instinctively go to it. Teach them progressively to climb up from 5 rungs the first day, to 10 rungs the following day, up to 15 rungs.Next, use the same procedure with a dynamic ladder, first without movement, then with the ladder moving at 5.4 and 6.6 centimeters per second, and the mice climbing up for 2 minutes, completing 5 series. Next, prepare the following materials for the resistance exercise, weights, wire for holding weights, steel gator clip, and clinical adhesive tape. On the third day of acclimatization, cut a piece of the clinical adhesive tape and attach it around the mouse's tail to adapt it to carrying weights.On the seventh day of acclimatization, insert small weights in the wire and hook the gator clip. Clamp the gator to the clinical tape attached to the mouse's tail and place the mouse on the ladder, Immediately after the mouse has climbed the required rungs, remove the clamp and allow the mouse to rest with the clinical tape on the tail but without the weight. Before performing incremental tests to assess maximal strength, perform a warmup session consisting of three series of 10 repetitions, 10 steps per repetition without external load.Set the slope at 90 degrees for the first series, and after that at 85 degrees. Allow a rest period of 60 seconds between series. After attaching an external load of 10 grams on the mouse's tail, as demonstrated earlier, start the test with the slope set at 85 degrees and perform one series of 10 steps.Remove the weight and allow a rest period of 120 seconds in the resting area. Then perform a successive series of 10 steps increasing the external load by five grams until exhaustion. Allow the 120 second resting period between series.If a mouse fails to climb 10 steps with a particular weight load, allow for another attempt with the same load after 120 seconds of rest. If it fails again, record the weight load of the last completed series as the maximal weight load. If it succeeds in climbing with the load, continue the test with the next load.To perform an incremental test with the dynamic ladder for assessing maximal strength, clip the appropriate weight on the clinical tape and start the test at 4.2 centimeters per second. Increase the speed by 1.2 centimeters per second every 60 seconds until exhaustion and record the time as result. To perform the maximal endurance resistance test with the static ladder after a warmup session with no weights, as demonstrated previously, clip the appropriate weight on the clinical tape placed around the tail of the mouse.Then, keeping the ladder at an 85 degree slope, perform consecutive series of 10 steps until exhaustion with no resting time between each series and record the number of rungs climbed. To perform the maximal endurance resistance test with the dynamic ladder, set the slope at 85 degrees and speed at 4.2 centimeters per second. For warming up, perform three series of 100 steps without external load and allow rest period of 60 seconds between series.After the warmup, allow a 120 second rest period before initiating the test. Then clip the gator with the external load, place the mouse on the running ladder, and start the stopwatch. The test ends when the mouse is no longer able to maintain the rate of ascent.For resistance training with the static ladder, start the training session in the resting area and perform the warmup session without external load. Then clip the gator with the weight on the clinical tape and gently place the mouse 10 to 20 rungs below the resting place. Allow the mouse to grip the rung and climb to the resting area.Repeat this process until the number of rungs in the series is completed. Remove the weight from the mouse tail and wait for 120 seconds until the next series. Increase the number of steps and the maximum weight load of the series throughout the training period following the training plan.For resistance training with the dynamic ladder, after setting the slope to 85 degrees, close the door to the resting area and start the ladder at the desired speed. After a warmup procedure, when the mouse is in the resting area, clip the gator with the weight on the clinical tape. Alternatively, the weight can be attached when the mouse is on the ladder.Then gently place the mouse at the top of the moving ladder with the weight on the tail and allow the mice to grip the rung and climb. When the number of rungs in this series is reached, the weights are removed and the door is opened so that the animal can go to the resting area. The rest time is 120 seconds before the next series.Repeat this procedure until the training session is completed. The effect on resistance after six weeks of strength training on a dynamic ladder is shown here. The variation in maximal strength shows a significant increase after training in the strength and endurance resistance groups, while this parameter did not change in the control group.Endurance resistance measured at the end of the training period was significantly higher in the endurance resistance group as compared to that in the strength and control groups. Proper acclimatization, including learning to climb and adapting to the clinical tape and weights is important. This procedure allows researchers to design various types of resistance training interventions.Strength is relevant to human health and aging. Researchers can use these methods to explore the effects of exercise and pharmacological interventions on resistance.

versatility-protocols-for-resistance-training-assessment-using-static

Research

JoVE Journal

Biology

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&amp;middot;Cryo Kit

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

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.