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 the strength modality to be assessed or trained: maximal strength, endurance-resistance, speed, etc. depending on the objectives of the study.</li>
	<li>Adjust the parameters carefully when functional assessment or training is framed, considering whether it focuses on the results of these tests or whether they are complementary to other types of clinical, functional, histological, or molecular determinations.</li>
	<li>Plan all issues related to training, particularly the timetable, duration of the training period, and frequency of sessions, and draw a training table.
	<ol>
		<li>Specify the warm-up steps and the inclination of the ladder, which will be the same throughout the training. Specify sets, repetitions, load (based on the results of the resistance tests done prior to the training period), and rest in between, paying attention to load increases based on the previous session.</li>
		<li>Modify the plan, as with human training, depending on the welfare of the animal. Modifications include decreasing repetitions, increasing rest time between sets or repetitions, and decreasing load to avoid overtraining and injury.</li>
	</ol>
	</li>
	<li>Upon completion, submit the design for evaluation and approval by the animal ethics research committee.</li>
</ol>
2. Devices and materials for resistance exercise
<ol>
	<li>Devices: Static and dynamic ladders 
	NOTE: Two types of ladders, so-called static and dynamic ladders (see Figure 1), can be used for resistance training and evaluation (see Table of Materials).
	<ol>
		<li>Use a vertical ladder with at least 30 steel wire steps of 1.5 mm diameter, separated by 15 mm, and a resting area of at least 20 x 20 cm on the top of the ladder. The slope of the ladder must be adjustable from 80&#176; to 110&#176; with the horizontal plane (Figure 1C). Delimit two lanes to prevent non-linear climbing.</li>
		<li>Use a dynamic ladder similar to the static ladder, with a plastic filament barrier at the top, that can be opened to control access to the resting area, and a plastic filament barrier at the bottom, to prevent the animals from climbing down. The angle of inclination of the ladder must be adjustable between 80&#176; and 100&#176;, the most common being 85&#176;. 
		&#8203;NOTE: The ladder can circulate by means of an upper and a lower shaft with a diameter of 8 cm. Lower shaft is driven by an electric motor which makes the steps descend at the front and ascend at the rear, creating an endless ladder. It is equipped with a reduction gear and a speed regulator for lowering the speed from 11.6 cm/s to 3.3 cm/s, and the most common speed is 5.6 cm/s.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63098/63098fig01.jpg" /> 
Figure 1: Resistancetraining devices: static and dynamic ladders. (A) Mouse training with external weight on a static ladder. (B) Two mice training with weight on a dynamic ladder. (C) Schematic representation of ladder angles for training and evaluation. <a href="https://www.jove.com/files/ftp_upload/63098/63098fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Materials
	<ol>
		<li>Prepare the following materials: weights, wire for holding weights, steel gator clip, and clinical adhesive tape. 
		NOTE: The weights are steel cylinders of different mass (5, 10, 15, 20, 25, and 50 g), with a 5 mm diameter hole in the center to string them on a&#160;wire (Table of Materials). The wire to hold the weights is made of steel with a diameter of 1-1.5 mm and a length of 5-10 cm, depending on the number of weights to be loaded.</li>
		<li>Cut a piece of elastic adhesive bandage (Table of Materials) of approximately 3.0-3.5 cm x 1.0-1.5 cm size and attach it around the animal&#39;s tail to hold the weights. Be sure not to over-tighten as it may lead to blood flow restriction. 
		NOTE: At first, the animals&#39; behavior will be&#160;fighting against the tape and bitting it, but after a couple of days, they will tolerate it, grooming as usual and showing no signs of stress.</li>
		<li>Insert the desired weights in the wire and hook the gator clip (Table of Materials: Steel gator clip and wire for holding weights).</li>
		<li>Clamp the gator to the clinical tape attached to the animal&#39;s tail.</li>
		<li>Immediately after climbing the required rungs, remove the clamp and allow the animal to rest with the clinical tape on the tail, but without the weight (Figure 1).</li>
	</ol>
	</li>
</ol>
3. Acclimatization
NOTE: Proper acclimatization is essential to avoid exercise rejection and to minimize stress. Acclimatization is a crucial stage before resistance evaluation tests or training protocols are performed. Adequate time should be spent to achieve behavioral signs of comfort in the animals. Details of daily acclimatization with the static and dynamic ladders are shown in Table 1 and&#160;Table 2, respectively.
<ol start="1">
	<li>Accustom the animals to stay in the resting area at the top of the ladder (static or dynamic). Leave the animals in this place in groups of four, with bedding from their cage, for 15 min every day. Usually, after 3-5 days, the animals will show no signs of stress.</li>
	<li>Teach animals to climb up, not down, the ladder. Using the static ladder, place the mice on a rung close to the top, from where they can see the resting area.&#160;They will instinctively go to it. Then, teach them progressively to climb up from five rungs (3x) the first day, to 10 rungs (3x) the following day, up to 15 rungs (3x) (Table 1). 
	Use the same procedure with the dynamic ladder, first without movement, and then with the ladder moving at 5.4 cm/s and 6.6 cm/s and the animals climbing up for 2 min, completing five series (Table 2).</li>
	<li>Adapt the animals to carry weights, starting from the third day of acclimatization. Stick a piece of clinical tape to the base of the tail which will be used to hold weights.</li>
	<li>From the seventh day of acclimatization, attach small weights (5-10 g) to the clinical tape with a gator clip. Avoid performing too many series, so the adaptation is not transformed into training. 
	NOTE: Acclimatization of the control group is mandatory in case this group performs the resistance test. After this period, perform a ladder-climbing reminder once a week, with tape but without weights.</li>
</ol>
4. Resistance evaluation
<ol start="1">
	<li>Incremental tests to assess maximal strength 
	NOTE: This test intends to determine the maximal resistance measured as the maximum weight at which the animals can climb 10 rungs on the static ladder, which defines the 10-repetition maximum (10 RM)4. This protocol was adapted from previous studies (reviewed in Kregel et al.15).
	<ol>
		<li>For warming-up perform three series of 10-repetitions, 10 steps/repetition, without external load. For the first series set the slope at 90&#176;, and thereafter at 85&#176;. Allow a rest period of 60 s between series.</li>
		<li>Set the slope at 85&#176; (to prevent the weights from grazing or hooking on the rungs of the ladder).</li>
		<li>Attach the tape around the animal&#39;s tail to hold the weights and prepare the weights as previously explained.</li>
		<li>Start the test with an external load of 10 g and perform one series of 10 steps.</li>
		<li>Remove the weight and allow a rest period of 120 s in the resting area.</li>
		<li>Perform successive series of 10 steps increasing the external load by 5 g until exhaustion. Allow the resting period (120 s) between series.</li>
		<li>If one animal fails to climb 10 steps with a particular weight load, allow for another attempt with the same load after 120 s of rest. If it succeeds to climb with the load, it continues the test with the next load. If it fails again, record the weight load of the last completed series as its&#160;maximal weight load.</li>
		<li>The test result can be expressed as absolute external weight (g), as maximum load in relation to body weight (%), or as the mass lifted per gram of body weight, as per the discretion of the researcher. 
		NOTE: The previous protocol&#160;represents a model on which numerous modifications are possible for example, to assess the maximal resistance of genetically modified mice with neuromuscular disabilities. These animals are not able to climb with external loads and have difficulties climbing 10 rungs with the ladder set at 90&#176; of slope (unpublished data). The protocol consisted of climbing five steps without external load, starting with a slope of 110&#176;. The slope decreased 5&#176; in each series until 85&#176; with 120 s rest after each series. In this case, maximal resistance was expressed as the accumulated number of steps climbed (without considering repetitions after failures). The wild-type control group, after reaching the 85&#176; slope, will&#160;continue with the test by adding external weight to the tail, following previous protocol, until exhaustion.</li>
	</ol>
	</li>
	<li>Maximal endurance-resistance test with the static ladder
	<ol>
		<li>For warming-up perform three series of 10-repetitions, 10 steps/repetition, without external load. For the first series, set the slope at 90&#176;, and thereafter at 85&#176;. Allow a rest period of 60 s between series.</li>
		<li>Set the slope at 85&#176;.</li>
		<li>Clip the weight on the clinical tape placed around the tail of the mouse. 
		NOTE: Depending on the age and the characteristics of the animals, the external load can be the maximum weight obtained in a previous incremental test, a percentage of it (e.g., 50%), or a percentage of body weight (e.g., 100%-200%). If this test is performed after a period of training, it is recommended to use the same load as in the initial test to assess the changes.</li>
		<li>Perform consecutive series of 10 steps until exhaustion. No resting time is allowed after each series.</li>
		<li>The test result is the number of climbed rungs.</li>
	</ol>
	</li>
	<li>Maximal endurance-resistance test with the dynamic ladder 
	NOTE: The use of the dynamic ladder allows the researcher to control the climbing speed.
	<ol>
		<li>Set the slope at 85&#176;.</li>
		<li>Set the speed at 4.2 cm/s.</li>
		<li>For warming-up perform three series of 100 steps, without external load. Allow a rest period of 60 s between series.</li>
		<li>Clip the weight on the clinical tape placed around the mouse tail. 
		NOTE: Depending on the age and the characteristics of the animals, the external load can be the maximum weight obtained in a previous incremental test, a percentage of it (e.g., 50%), or a percentage of body weight (e.g., 100%-200%). If this test is performed after a period of training, it is recommended to use the same load as in the initial test to assess the changes.</li>
		<li>Start at 4.2 cm/s and increase the speed by 1.2 cm/s every 60 s until exhaustion. 
		&#8203;NOTE: The test result is the exercise time, the number of rungs climbed, or the maximum speed.&#160;</li>
	</ol>
	</li>
</ol>
5. Resistance training with static ladder
NOTE: Before starting the training period, acclimatization (Table 1) and training planning are necessary. To reduce anxiety, adapt and train the mice in groups of four animals sharing the same cage.
<ol>
	<li>For everyday warming-up perform three series of 10 repetitions, 10 steps/repetition, without external load. For the first series set the slope at 90&#176;, and thereafter at 85&#176;. Allow a rest period of 60 s between series.</li>
	<li>The training session starts in the resting area. Clip the gator with the weight on the clinical tape.</li>
	<li>Gently place the mouse 10-20 rungs below the resting place. Allow the mouse to grip the rung and climb to the resting area. 
	Repeat this process&#160;until the number of rungs in this series (e.g.,10 rungs x 10 series) is completed.</li>
	<li>Remove the weight from the mouse tail and wait for 120 s until the next series.</li>
	<li>Increase the number of steps and the maximum weight loads of the series throughout the training period, while maintaining the weekly schedule. 
	&#8203;NOTE: An example of the variation of loads during a week planning is shown in Table 3. Shortly, Tuesday and Friday with high weight load (40-50 g) and a low number of steps (500-400); Monday and Thursday with intermediate weight load (25-35 g) and an intermediate number of steps (800-600); and Wednesday without weight load but a high number of steps (2,000). This design facilitates recovery from previous training sessions and avoids injuries and overtraining. Examples of 3 weeks of training with multiple designs using the static ladder are shown in Table 4 (at the beginning, in the middle, and at the end of the training period, respectively)4.</li>
</ol>
6. Resistance training with dynamic ladder
NOTE: After acclimatization, the training on the dynamic ladder is quite like the static one (Table 2). Training is performed on 2-4 mice at a time.
<ol>
	<li>Set the slope to 85&#176;, close the door to the resting area, and start the ladder at the desired speed (e.g., 5.4 cm/s).</li>
	<li>For warming-up perform three series of 100 steps, without external load. Allow a rest period of 60 s between series.</li>
	<li>Before the training sessions starts, 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 already on the ladder.</li>
	<li>Gently place the mouse at the top of the moving staircase with the weight on the tail. Allow the mice to grip the rung and climb.</li>
	<li>When the number of rungs in this series is reached (e.g., 100), remove the weights. Then the door is opened so that the animal can go to the resting area. The rest time is 120 s before the next series. 
	NOTE: The number of steps climbed is counted as a function of the climbing time at the set speed.</li>
	<li>Repeat this procedure until the training session is completed. The detailed daily training program is shown in Table 5.</li>
</ol>
7. Evaluation of the crossover effect of resistance training on endurance performance
NOTE: For this, an incremental treadmill test is performed4, after 24 h of rest.
<ol>
	<li>After a warm-up of 3 min at 10 cm/s, start the incremental test at 10 cm/s and 10&#176; angle of inclination.</li>
	<li>Increase the speed by 3.33 cm/s every 3 min until exhaustion. 
	NOTE: No electric shocks are used, so a painter&#39;s brush is placed at the back of the treadmill to prevent the mice from running off it.</li>
</ol>
8. Animal behavior during procedures
NOTE: Continuous monitoring of the adaptation of mice to training should be performed to detect extreme fatigue, overtraining, or injury.
<ol>
	<li>Observe signs of animal welfare, in particular grooming and refusal to training. The normal behavior of the mouse, after a series of intense training, is to remain inactive for about one minute due to fatigue. After that, they start grooming, exploring, or trying to remove the tape on the tail.</li>
	<li>In the case of a mouse refusing to train a series, try giving longer rests or even not performing that&#160;series to prevent inhibition.</li>
	<li>Occasionally, when carrying out lightweight exercises, gently push the animal&#39;s tail, to encourage it to finish the series. The animals stop climbing because it is not a demanding task. Conversely, when animals are carrying a heavy load, gently shift the animal&#39;s weight to ease the load and encourage it to finish the series, and then allow the animal to rest until the next training session. The animals may stop or even attempt to descend because of the heavy load.</li>
</ol>
9. Safety procedures
<ol>
	<li>Security procedures for researchers: Conduct research in the animal facility laboratory and use shoe covers, coveralls, gloves, caps, and masks. There are no additional requirements other than those specific to animal research.</li>
	<li>Security for animals: During the exercise sessions continuous attention must be paid to the animals, due to potential risks, such as falls or jumps. Place a hand under the weights to catch and hold the mice in case of fall due to exhaustion, since its ability to hold on to the rungs properly will be limited.</li>
</ol>

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W., Krusnauskas, R., Hodson-Tole, E., Venckunas, T., Degens, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endurance+exercise+plus+overload+induces+fatigue+resistance+and+similar+hypertrophy+in+mice+irrespective+of+muscle+mass.">Endurance exercise plus overload induces fatigue resistance and similar hypertrophy in mice irrespective of muscle mass.</a> Experimental Physiology. 105 (12), 2110-2122 (2020).</li><li>Knab, A. M., 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=Repeatability+of+exercise+behaviors+in+mice.">Repeatability of exercise behaviors in mice.</a> Physiology &amp; Behavior. 98 (4), 433-440 (2009).</li><li>Konhilas, J. P., 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=Loaded+wheel+running+and+muscle+adaptation+in+the+mouse.">Loaded wheel running and muscle adaptation in the mouse.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (1), 455-465 (2005).</li><li>Reiter, A., 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=Functional+measures+of+grip+strength+and+gait+remain+altered+long-term+in+a+rat+model+of+post-traumatic+elbow+contracture.">Functional measures of grip strength and gait remain altered long-term in a rat model of post-traumatic elbow contracture.</a> The Journal of Biomechanical Engineering. , (2019).</li><li>Stieglitz, T., Schuettler, M., Schneider, A., Valderrama, E., Navarro, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+measurement+of+torque+development+in+the+rat+foot:+measurement+setup+and+results+from+stimulation+of+the+sciatic+nerve+with+polyimide-based+cuff+electrodes.">Noninvasive measurement of torque development in the rat foot: measurement setup and results from stimulation of the sciatic nerve with polyimide-based cuff electrodes.</a> IEEE Transactions on Neural Systems and Rehabilitation Engineering. 11 (4), 427-437 (2003).</li><li>Seo, D. Y., 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=Humanized+animal+exercise+model+for+clinical+implication.">Humanized animal exercise model for clinical implication.</a> Pfl&#252;gers Archiv. 466 (9), 1673-1687 (2014).</li><li>Tanaka, H., Swensen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+resistance+training+on+endurance+performance.+A+new+form+of+cross-training.">Impact of resistance training on endurance performance. A new form of cross-training.</a> Sports Medicine. 25 (3), 191-200 (1998).</li><li>Hakkinen, K., Mero, A., Kauhanen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+endurance,+sprint+and+strength+training+on+physical+performance+capacity+in+young+athletes.">Specificity of endurance, sprint and strength training on physical performance capacity in young athletes.</a> The Journal of Sports Medicine and Physical Fitness. 29 (1), 27-35 (1989).</li><li>Vellers, H. L., Kleeberger, S. R., Lightfoot, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-individual+variation+in+adaptations+to+endurance+and+resistance+exercise+training:+genetic+approaches+towards+understanding+a+complex+phenotype.">Inter-individual variation in adaptations to endurance and resistance exercise training: genetic approaches towards understanding a complex phenotype.</a> Mammalian Genome. 29 (1), 48-62 (2018).</li></ol>

The corresponding author ensures that all authors have no conflicts of interest.

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...

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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 ...

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2.4K Views.  University of Oviedo. The present protocol describes resistance training and testing using static and dynamic ladders in animal models.

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

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 ...

Principles of Rodent Surgery for the New Surgeon

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform surgery on a rodent. Students, post-doctoral scholars, and others interested in performing surgery on rodents as part of a research protocol may not have had formal surgical training as part of their required coursework. Surgery itself is a technical skill, and one that will improve with practice. The principles of aseptic technique, however, often remain unexplained or untaught. For most new surgeons, this vital information is presented in piecemeal fashion or learned on the job, neither of which is ideal. It may also make learning how to perform a particular surgery difficult, as the new surgeon is learning both a surgical technique and the principles of asepsis at the same time. This article summarizes and makes recommendations for basic surgical skills and techniques necessary for successful rodent surgery. This article is designed to supplement hands-on training by the user's institution.

Before attempting surgery, a new surgeon should have training in basic surgical techniques and concepts. This article will present basic surgical considerations with an emphasis on rodents.

For both scientific and animal welfare reasons, training in basic surgical concepts and techniques should be undertaken before ever seeking to perform ...

Manual Restraint and Common Compound Administration Routes in Mice and Rats

Being able to safely and effectively restrain mice and rats is an important part of conducting research. Working confidently and humanely with mice and rats requires a basic competency in handling and restraint methods. This article will present the basic principles required to safely handle animals. One-handed, two-handed, and restraint with specially designed restraint objects will be illustrated. Often, another part of the research or testing use of animals is the effective administration of compounds to mice and rats. Although there are a large number of possible administration routes (limited only by the size and organs of the animal), most are not used regularly in research. This video will illustrate several of the more common routes, including intravenous, intramuscular, subcutaneous, and oral gavage. The goal of this article is to expose a viewer unfamiliar with these techniques to basic restraint and substance administration routes. This video does not replace required hands-on training at your facility, but is meant to augment and supplement that training.

Working safely and humanely with research rodents requires a core competency in handling and restraint methods. This article will present the basic principles required to safely handle and effectively administer compounds to mice and rats.

Being able to safely and effectively restrain mice and rats is an important part of conducting research.  Working confidently and humanely with mice and ...

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 ...

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 ...

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.