This work was supported by the National Aeronautics and Space Administration (NASA: NNX16AL36G). Authors would like to thank Carson Semple for providing the drawings included in this manuscript.

All methods described here were approved by the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center under protocol number 067-2016.
NOTE: Male Wistar rats aged of 14 weeks at baseline (day 0) are used. Rats are housed individually in custom cages 24 h prior baseline to allow for acclimation.
1. Hindlimb unloading
NOTE: The pelvic harness can be put on either anesthetized or awake animals. Here, the description of the protocol is given on anesthetized animals. Wear proper personal protective equipment (PPE) to handle animals.
<ol>
	<li>Place the rat in an anesthesia box with 3.5% isoflurane and an oxygen flow of 2 L/min. 
	NOTE: Proper anesthetization is confirmed when a firm pinch of the rear paw does not elicit a reaction.</li>
	<li>Once the animal is fully anesthetized, place the rat on the bench with anesthetic gas coming from a nosecone with 2% isoflurane and an oxygen flow of 1.5 L/min.</li>
	<li>Place the rat in a prone position and put the pelvic harness on in a rostro-caudal movement.</li>
	<li>Gently bend the pelvic harness to provide a snug fit while being careful not squeeze the hindlimbs to prevent abrasions and discomfort.</li>
	<li>Attach the stainless steel chain with the swivel clasp to the top of the pelvic harness, where a hook is attached at the base of the tail.</li>
	<li>Remove the rat from anesthesia and place the animal in a custom cage with the chain extended at its maximum.</li>
	<li>Once the rat is fully awake and mobile, shorten the chain using the top swivel clasp until the hind limbs can no longer reach the floor.</li>
	<li>Observe the animal for a few minutes to assess its comfort and make sure that at all times, both hind limbs remain completely unloaded.</li>
</ol>
2. Partial weight bearing
NOTE: This step can be realized in both awake and anesthetized animals.
<ol>
	<li>Convert the HLU suspension device into a PWB suspension by adding the triangle-shaped part composed of stainless steel chains and a back rod.</li>
	<li>Anesthetize the animal following the same procedures as detailed for the HLU (steps 1.1 and 1.2).</li>
	<li>Place a tether jacket of the appropriate size on the forelimbs of the rat (M for rats of 400 g or lower, L for rats weighing above 400 g) and close it using the back bra extender.</li>
	<li>Attach one clasp of the triangle-shaped part to the hook located on the back bra extender and the opposite clasp on the hook located on the pelvic harness at the base of the tail.</li>
	<li>Allow the animal to recover from anesthesia in the cage. Once awake, verify that the suspension is equal on both the forelimbs and the hindlimbs by shortening the chain and modifying the location of the bottom swivel clasp if needed. 
	NOTE: This step can also be realized using a force plate to confirm the equal loading on all limbs.</li>
	<li>Place the rat on top of the scale to record the &#8220;loaded&#8221; body weight, i.e., the weight of the animal and the entire apparatus, without shortening the chain.</li>
	<li>Shorten the chain until the scale displays 40% of the &#8220;loaded&#8221; body weight and record the achieved gravity level (expressed as the ratio between unloaded weight and loaded weight).</li>
	<li>Observe the animal to make sure that the unloaded weight is stable and that the rat is equally loaded on all limbs.</li>
	<li>Remove the entire apparatus from the scale using the rod and place the rat back in its cage.</li>
</ol>
3. Assessment of hindlimb grip force
<ol>
	<li>Hold the rat with a traditional restraint by placing one hand underneath the forelimbs. Gently hold the tail with the second hand.</li>
	<li>Approach the grip bar with the rear paws and make sure that both paws are fully resting on the bar. 
	NOTE: If the rat does not fully grip the bar or does not display any evidence of voluntary gripping, slightly release the restraint. If this is unsuccessful, return the rat to its cage and retry after a few minutes.</li>
	<li>Gently pull the rat straight back until it releases its grip. Record the maximal force displayed on the transducer.</li>
	<li>Wait approximately 30 s between measurements and repeat the test 3 times.</li>
	<li>Calculate the average of the three measurements for scoring, to account for fatigue.</li>
</ol>
4. Recording of muscle wet mass
<ol>
	<li>Place the rat in a CO2 euthanasia chamber. After waiting the appropriate time according to IACUC and AVMA guidelines, confirm euthanasia by a visual observation of a lack of breathing.</li>
	<li>Place the rat on the dissection table in a prone position and remove the fur and skin by incising near the ankle using small dissection scissors. Use hands to pull the skin layer off.</li>
	<li>Using small dissection scissors, gently break the muscle fascia and isolate the calcaneus tendon. 
	NOTE: The calcaneus tendon is the attachment point of both the soleus and the gastrocnemius muscles.</li>
	<li>While holding the calcaneus tendon with a small pair of tweezers, use the dissection scissors to isolate the gastrocnemius and soleus muscles from the biceps femoris, located above.</li>
	<li>Once isolated, cut the attachment point of the gastrocnemius and soleus muscles in the popliteal area.</li>
	<li>Gently pull the soleus away from the gastrocnemius and detach them by cutting the calcaneus tendon.</li>
	<li>Place the rat in a supine position. Carefully remove the fascia and peel the tibialis anterior from the ankle in an upward movement.</li>
	<li>Cut the tibialis anterior at its superior attachment point.</li>
	<li>Record the exact wet mass of each excised muscle using a tared precision scale and a weighing boat.</li>
</ol>

<ol>
	<li>Desplanches, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Functional+Adaptations+of+Skeletal+Muscle+to+Weightlessness.">Structural and Functional Adaptations of Skeletal Muscle to Weightlessness.</a> International Journal of Sports Medicine. 18 (S4), (1997).</li><li>Fitts, R. H., Riley, D. R., Wildrick, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+microgravity+environment&#8239;:+Invited+review&#8239;:+microgravity+and+skeletal+muscle.">Physiology of a microgravity environment&#8239;: Invited review&#8239;: microgravity and skeletal muscle.</a> Journal of Applied Physiology. 89, 823-839 (2000).</li><li>Fitts, R. H., Riley, D. R., Widrick, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+adaptations+of+skeletal+muscle+to+microgravity.">Functional and structural adaptations of skeletal muscle to microgravity.</a> The Journal of Experimental Biology. 204 (Pt 18), 3201-3208 (2001).</li><li>Narici, M. V., De Boer, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disuse+of+the+musculo-skeletal+system+in+space+and+on+earth.">Disuse of the musculo-skeletal system in space and on earth.</a> European Journal of Applied Physiology. 111 (3), 403-420 (2011).</li><li>di Prampero, P. E., Narici, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscles+in+microgravity:+from+fibres+to+human+motion.">Muscles in microgravity: from fibres to human motion.</a> Journal of Biomechanics. 36 (3), 403-412 (2003).</li><li>Wagner, E. B., Granzella, N. P., Saito, H., Newman, D. J., Young, L. R., Bouxsein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+weight+suspension:+a+novel+murine+model+for+investigating+adaptation+to+reduced+musculoskeletal+loading.">Partial weight suspension: a novel murine model for investigating adaptation to reduced musculoskeletal loading.</a> Journal of Applied Physiology (Bethesda, Md.&#8239;: 1985). 109 (2), 350-357 (2010).</li><li>Sung, 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=Spaceflight+and+hind+limb+unloading+induce+similar+changes+in+electrical+impedance+characteristics+of+mouse+gastrocnemius+muscle.">Spaceflight and hind limb unloading induce similar changes in electrical impedance characteristics of mouse gastrocnemius muscle.</a> Journal of Musculoskeletal and Neuronal Interactions. 13 (4), 405-411 (2013).</li><li>Mcdonald, K. S., Blaser, C. A., Fitts, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force-velocity+and+power+characteristics+of+rat+soleus+muscle+fibers+after+hindlimb+suspension.">Force-velocity and power characteristics of rat soleus muscle fibers after hindlimb suspension.</a> Journal of Applied Physiology. 77 (4), 1609-1616 (1994).</li><li>Chowdhury, P., Long, A., Harris, G., Soulsby, M. E., Dobretsov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+of+simulated+microgravity:+a+comparative+study+of+hindlimb+unloading+via+tail+versus+pelvic+suspension.">Animal model of simulated microgravity: a comparative study of hindlimb unloading via tail versus pelvic suspension.</a> Physiological Reports. 1 (1), e00012 (2013).</li><li>Morey, E. R., Sabelman, E. E., Turner, R. T., Baylink, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rat+model+simulating+some+aspects+of+space+flight.">A new rat model simulating some aspects of space flight.</a> The Physiologist. 22 (6), (1979).</li><li>. National Space Exploration Campaign Report Available from: <a target="_blank" href="https://www.nasa.gov/sites/default/files/atoms/files/nationalspaceexplorationcampaign.pdf">https://www.nasa.gov/sites/default/files/atoms/files/nationalspaceexplorationcampaign.pdf</a> (2018)</li><li>Mortreux, M., Nagy, J. A., Ko, F. C., Bouxsein, M. L., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+partial+gravity+ground-based+analogue+for+rats+via+quadrupedal+unloading.">A novel partial gravity ground-based analogue for rats via quadrupedal unloading.</a> Journal of Applied Physiology. 125, 175-182 (2018).</li><li>Ellman, R., 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=Combined+effects+of+botulinum+toxin+injection+and+hind+limb+unloading+on+bone+and+muscle.">Combined effects of botulinum toxin injection and hind limb unloading on bone and muscle.</a> Calcified Tissue International. 94 (3), (2014).</li><li>Swift, J. 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=Partial+Weight+Bearing+Does+Not+Prevent+Musculoskeletal+Losses+Associated+with+Disuse.">Partial Weight Bearing Does Not Prevent Musculoskeletal Losses Associated with Disuse.</a> Medicine &#38; Science in Sports &#38; Exercise. 45 (11), 2052-2060 (2013).</li><li>Morey-Holton, E. R., Globus, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindlimb+unloading+rodent+model:+technical+aspects.">Hindlimb unloading rodent model: technical aspects.</a> Journal of Applied Physiology. 92 (4), 1367-1377 (2002).</li><li>Andreev-Andrievskiy, A. A., Popova, A. S., Lagereva, E. A., Vinogradova, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+shift+versus+body+size:+changes+of+hematological+parameters+and+body+fluid+volume+in+hindlimb-unloaded+mice,+rats+and+rabbits.">Fluid shift versus body size: changes of hematological parameters and body fluid volume in hindlimb-unloaded mice, rats and rabbits.</a> Journal of Experimental Biology. 221 (Pt 17), (2018).</li></ol>

The authors have nothing to disclose.

This model presents the first ground-based analogue developed to investigate successive mechanical unloading levels and aims to mimic a trip to and stay on Mars.
Many steps of this protocol are critical to ensure its success and need to be closely examined. First, it is crucial to monitor the wellbeing of the animals and ensure that they are maintaining a normal behavior (i.e., performing tasks such as eating, resting, and exploring), particularly during the PWB state where they maintain a relatively normal physiological posture. Second, despite the level of PWB being extremely steady over time and requiring minimal human intervention12,14, it is essential to record the achieved partial gravity to minimize variations amongst animals. Additionally, when animals are transitioned from total mechanical unloading to partial gravity (PWB40), they already display substantial muscle atrophy and loss of function6,9,15, which may cause transient difficulties in resuming quadrupedal weight-bearing behavior and lead to a momentary awkward gait.
Due to the variable environment, several problems can arise and should be closely recorded and addressed. For example, a fluid shift occurs during the HLU period due to the slanted position of the animal, while it is not present during PWB16. In some cases, fluid redistribution can cause subtle edema most noticeable in the face or the rear paws, and usually disappears in the hours following reloading. We advise investigators to score the severity of the edema and assess it daily. If severe edema persists for greater than 48 h, animals should be excluded from the experiment.
While the use of a pelvic harness provides comfort to the animals and increased convenience to the investigator, some animals may, occasionally, either totally or partially escape from their harness during HLU or PWB. We followed the exclusion protocol based on the previous work in mice6 in which any animal that escapes three times is removed from the study. As a side note, escapes are extremely rare; in our work less than 1% of animals had to be excluded over a 1-year period (1 animal out of 148 animals studied). The daily titration of the PWB level is a crucial moment where the experimenter can ensure the snug fit of both the jacket and the pelvic harness, therefore minimizing the risk of escape. While assessing animals&#8217; weight and wellbeing daily, special consideration should be put in the maintenance of the pelvic harness. While site-specific hair-loss is the most common consequence, abrasion can appear if the pelvic harness is damaged (i.e., chewed). We advise researchers to check daily the harness condition and replace components when damaged or the entire harness when needed to prevent the appearance of skin abrasion.&#160;Wellbeing should at least include the following steps: monitoring of body weight, porphyria, food intake, presence of urine and feces, hair loss, skin abrasion, edema.
The animals&#8217; claws can also occasionally become entrapped in the hook-and-loop fastener or cloth, therefore compromising their balance. A simple and efficient way to prevent this from happening is to gently trim the claws under anesthesia before placing the jacket. This step can be repeated when needed during the course of study.
Special attention needs to be paid during the transition from HLU to PWB. While we observed that all animals are able to walk with little difficulty immediately after being placed in PWB, the amount of time required to put the same amount of weight on both the front and the hind limbs varied among rats. If a rat does not demonstrate relatively normal gait using all limbs in 24 h, we recommend it should be excluded from the study.
This novel model designed to mimic sequential gravitational environments is reliable and sustainable over time. However, some limitations exist and are yet to be addressed. First, this combination of models is only designed to assess the alterations occurring in the hind limbs of the animals as the HLU model only creates artificial microgravity on the rear limbs. Therefore, this ground-based sequential HLU-PWB analogue is not suitable to investigate fore limbs alterations. Secondly, over the 14-day period, our animals displayed a continuous but non-life-threatening loss of whole-body mass highlighting the complex readjustment of the rats to partial unloading (Figure 4A). In our previous PWB rat model study, the animals exposed at PWB40 and PWB20 for two weeks presented a significant loss over only the first 7 days and regained weight subsequently12. This was likely due to the fact that the rats were able to adjust to the quadrupedal unloading after an initial period of adaptation. However, in this study, the rats never fully adapted to the two different unloading/partial-reloading periods of one week each, likely explaining the sustained weight loss. It would be important to further extend these periods of full and partial-unloading to confirm that animals can fully adapt and settle into each environment. Stress levels have not been evaluated in this model yet and could easily be monitored in the future using regular blood sampling using the tail that remains entirely accessible.
Our longitudinal assessments of muscle function and muscle mass showed that one week of hindlimb unloading caused a tremendous decrease in rear paw grip force (Figure 4B) with one of our rats exhibiting a 70% reduction in grip force. Unsurprisingly, after 14 days, animals displayed a significantly lower grip force than animals that had been exposed to 14 days of PWB40 in our previous study12 Whereas the average wet mass of the hindlimb muscles did not differ significantly between the PWB40 and HLU-PWB40 groups, we were able to establish a strong linear correlation among our 3 groups (PWB100, PWB40, and HLU+PWB40) regarding the average soleus mass (R2 = 0.92, p &#60; 0.0001).
These results confirm that partial loading following a total mechanical unloading compromises muscle health more than what would be observed during a continuous but stable period of partial unloading. Until now, this gap in knowledge has not been investigated. Further assessment of this phenomenon should be pursued in order to develop effective countermeasures preventing muscle deconditioning in the context of a mission to the Moon or Mars. The strength of our model also resides in its versatility as it allows for a variety of different experiments with several degrees of unloading and for varying lengths of time.

Extraterrestrial targets, including the Moon and Mars, represent the future of human space exploration, but both have considerably weaker gravity than Earth. While the consequences of weightlessness on the musculoskeletal system have been extensively studied in astronauts1,2,3,4,5 and in rodents6,7,8,9, the latter thanks to the well-established hindlimb unloading (HLU) model10, very little is known about the effects of partial gravity. Martian gravity is 38% of Earth&#8217;s and this planet has become the focus of long-term exploration11; hence, it is crucial to understand the muscular alterations that may occur in this setting. To do so, we developed a partial weight bearing (PWB) system in rats12, based on previous work done in mice6,13, which was validated using both muscle and bone outcomes. However, the exploration of Mars will be preceded by a prolonged period of microgravity, which was not addressed in our previously described model12. Therefore, in this study, we altered our model to mimic a trip to Mars, comprised of a first phase of total hindlimb unloading and immediately followed by a second phase of partial weight bearing at 40% of normal loading.
Unlike most HLU models, we chose to use a pelvic harness (based on the one described by Chowdhury et al.9) rather than a tail suspension to improve animals&#8217; comfort and to be able to move seamlessly and effortlessly from HLU to PWB in a matter of minutes. In conjunction, we used the cages and suspension devices that we previously developed and described extensively12. In addition to providing reliable/consistent data, we also previously demonstrated that the fixed attachment point of the suspension system at the center of the rod did not prevent the animals from moving, grooming, feeding, or drinking. In this article, we will describe how to unload the animals&#8217; hind limbs (both totally and partially), verify their achieved gravity levels, as well as how to functionally assess the resulting muscular alterations using grip force and wet muscle mass. This model would be extremely useful for researchers seeking to investigate the consequences of partial gravity (either artificial or extra-terrestrial) on an already compromised musculoskeletal system, thus allowing them to investigate how organisms adapt to partial reloading, and for the development of countermeasures that could be developed to maintain health during and after human spaceflight.

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mimicking a space mission to mars using hindlimb unloading and partial weight bearing in rats

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been routinely employed since 1979 and the development of hind limb unloading (HLU). However, the next steps in space exploration now include to travel to Mars where the gravity is 38% of Earth&#8217;s gravity. Since no human being has experienced this level of partial gravity, a sustainable ground-based model is necessary to investigate how the body, already impaired by the time spent in microgravity, would react to this partial load. Here, we used our innovative partial weight-bearing (PWB) model to mimic a short mission and stay on Mars to assess the physiological impairments in the hind limb muscles induced by two different levels of reduced gravity applied in sequential fashion. This could provide a safe, ground-based model to study the musculoskeletal adaptations to gravitational change and to establish effective countermeasures to preserve astronauts&#8217; health and function.

Taking advantage of the new cages that we previously designed and described in detail12, we used a stainless steel chain-based suspension device that is suitable for both hindlimb unloading (HLU, Figure 1) and partial weight-bearing (PWB, Figure 2). The critical advantage of our design is the ability to go from one type of unloading to the other in a matter of minutes while maintaining an identical environment for the animals. We used a custom-made pelvic harness (Figure 2A) that is attached to a single custom-made stainless steel chain with a swivel clasp on each side for HLU. In order to modify this suspension device and achieve PWB, the addition of one triangle-shaped piece of stainless steel chain that incorporated an inflexible back rod, designed to sit just above the spine (Figure 3) is the only requirement. These steps can be performed in awake or anesthetized animals.
With the versatile environment provided in this experiment, we could successfully unload the hindlimb of all of our animals for 7 days without any complications and quickly expose them to a partial gravity at 40% of their normal loading (PWB40, average achieved gravity level of 0.4076 g &#177; 0.0036 g). During the first week of total HLU, animals displayed a significant body weight loss (Figure 4A: -7.19% &#177; 0.87%, n = 9, p &#60; 0.001), which has been witnessed in other models14, and does not differ significantly from what we observed in rats exposed at PWB40 for the same duration (-5.53% &#177; 1.44%, n = 10, p = 0.37). However, animals continued losing weight over time while subsequently being exposed to PWB40 (-9.06% &#177; 1.35% from baseline, p &#60; 0.0001).
Hindlimb grip force is a standard measurement of muscle function that can be used longitudinally (Figure 4B). We noticed that one week of total unloading led to an average decrease in grip force of 50.16% &#177; 4.10% compared to baseline (p &#60; 0.0001). After one subsequent week of partial weight bearing at 40% of normal loading, we did not notice any further change regarding grip force (-44.29% &#177; 4.67% compared to baseline, p &#60; 0.0001). At all time-points, the percent change in rear paw grip force was significantly different from the age-matched controls (p &#60; 0.0001 for both day 7 and day 14, n = 11). Additionally, we observed that after completion of the study, animals that underwent total unloading followed by partial weight bearing (HLU-PWB40) displayed a significantly greater grip force loss compared to the PWB40 group (p = 0.03).
Muscle wet mass was recorded at the end of the experiment and compared to data obtained after either two weeks of normal loading or two weeks of PWB40 (Figure 4C) and data previously published by our group12. We found that the PWB40 and HLU-PWB40 groups have significantly lower wet mass of soleus (S), gastrocnemius (G), and tibialis anterior (TA) muscles than age-matched controls (PWB100). Indeed, we recorded an average soleus mass of 0.1681 g &#177; 0.007 g for our animals which was significantly lower than the rats exposed to PWB100 for 2 weeks in our previous experiments (-24.60% &#177; 3.18%, p &#60; 0.0001). For the gastrocnemius, we recorded an average wet mass of 2.192 g &#177; 0.096 g (-10.55% &#177; 3.93%, p = 0.038 vs PWB100) and a wet mass of 0.759 g &#177; 0.029 g for the tibialis anterior (-14.40% &#177; 3.27%, p = 0.009 vs PWB100). While our data set highlighted that the animals exposed to a Mars-mission analogue (HLU-PWB100) had a decreased wet mass of the soleus and gastrocnemius muscles compared to the animals exposed to PWB40 for 2 continuous weeks (-8.75% &#177; 3.84% and -5.85% &#177; 4.14%, respectively), we did not observe a significant difference between these two groups.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59327/59327fig1.jpg" /> 
Figure 1: Description of the suspension devices and how to convert from HLU to PWB. (A) Based on our previous design, we used an aluminum rod sitting on top of the cage to hold a steady suspension device composed of a key ring secured at the center of the rod (arrow 1), a stainless steel chain (arrow 2), and two swivel clasps (arrows 3). (B) To convert the suspension device to achieve PWB, a triangle-shape structure is attached using the bottom swivel clasp. This piece is composed of stainless steel chains and a polyvinyl chloride (PVC) back rod that sits above the rat&#8217;s spine (arrow 1). On each side of the back rod is located a clasp to attach to the harness and the jacket, respectively (arrow 2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59327/59327fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59327/59327fig2.jpg" /> 
Figure 2: Hind limb unloading using a pelvic harness. (A) Front and side view drawings of the harness structure used to support the hind limbs of the animals. (B) The pelvic harness was positioned as described to fit snuggly around the hind limbs of the rat. The stainless steel link is positioned over the base of the tail and attached to the swivel clasp. The exact location and shape of the harness can vary between animals but rats should be comfortable and it is necessary that their hind limbs never touch the ground. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59327/59327fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59327/59327fig3.jpg" /> 
Figure 3: Partial weight bearing. The partial unloading requires the addition of a jacket to the animal in order to support the front limbs. The jacket is then closed with a back bra extender and a hook is attached to the extender, lying between the scapulae. Both the jacket and the pelvic harness are attached to clasps located on each end of the back rod. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59327/59327fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59327/59327fig4.jpg" /> 
Figure 4: Examples of longitudinal follow-up in animals exposed to different unloading levels. (A) Body weight (BW) change. Animals were weighed weekly without the harness or jackets and body weight was recorded. PWB100 = Partial weight bearing at normal loading; PWB40 = Partial weight bearing at 40% of normal loading; HLU-PWB40 = One week of hindlimb unloading followed by one week of PWB40. The results of Tukey&#8217;s post hoc test following a 2-way stacked ANOVA are presented as *: p &#60; 0.05, **: p &#60; 0.01, ***: p &#60; 0.001, and ****: p &#60; 0.0001 vs PWB100. (B) Change in rear paw grip force. Weekly, rear paw grip force was measured and results were expressed in percent change from baseline for each animal. The results of Tukey&#8217;s post hoc test following a 2-way stacked ANOVA are presented as ***: p &#60; 0.001 and ****: p &#60; 0.0001 vs PWB100, &#945;: p &#60; 0.05 vs PWB40. (C) Muscle wet mass after 14 days. Muscle wet mass was recorded on a precision scale immediately after sacrifice at 14 days. Results are presented as percent of the wet mass obtained in the age-matched control group (PWB100). S= Soleus; G= Gastrocnemius; TA = Tibialis anterior. The results of Tukey&#8217;s post hoc test following a one-way ANOVA are presented as *: p &#60; 0.05, **: p &#60; 0.01, and ****: p &#60; 0.0001 vs PWB100. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59327/59327fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats at JoVE.com

Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

By using an innovative ground-based analogue model, we are able to simulate a space mission including a trip to (0 g) and a stay on Mars (0.38 g) in rats. This model allows for a longitudinal assessment of the physiological changes occurring during the two hypo-gravitational stages of the mission.

Rodent ground-based models are widely used to understand the physiological consequences of space flight on the physiological system and have been ...

mimicking-space-mission-to-mars-using-hindlimb-unloading-partial

Nanyang Technological University

Research

JoVE Journal

Behavior

10.5K Views.  Harvard Medical School - Beth Israel Deaconess Medical Center. By using an innovative ground-based analogue model, we are able to simulate a space mission including a trip to (0 g) and a stay on Mars (0.38 g) in rats. This model allows for a longitudinal assessment of the physiological changes occurring during the two hypo-gravitational stages of the mission.

Video: Mimicking a Space Mission to Mars Using Hindlimb Unloading and Partial Weight Bearing in Rats

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Immunology and Infection

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

Biology

An Alternant Method to the Traditional NASA Hindlimb Unloading Model in Mice

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying disuse-atrophy in rodents 4-6. Our study evaluated an alternant method to the gold-standard Morey-Holton HU tail-traction technique in mice. Fifty-four female mice (4-8 mo.) were HU for 14 days (n=34) or 28 days (n=20). Recovery from HU was assessed after 3 days of normal cage ambulation following HU (n=22). Aged matched mice (n=76) served as weight-bearing controls.
Prior to HU a tail ring was formed with a 2-0 sterile surgical steel wire that was passed through the 5th, 6th, or 7th inter-vertebral disc space and shaped into a ring from which the mice were suspended. Vertebral location for the tail-ring was selected to appropriately balance animal body weight without interfering with defecation.
We determined the success of this novel HU technique by assessing body weight before and after HU, degree of soleus atrophy, and adrenal mass following HU. Body weight of the mice prior to HU (24.3 &plusmn; 2.9g) did not significantly decline immediately after 14d of HU (22.7 &plusmn; 1.9g), 28d of HU (21.3 + 2.1g) or after 3 days recovery (24.0 &plusmn; 1.8g). Soleus muscle mass significantly declined (-39.1%, and -46.6%) following HU for 14 days and 28 days respectively (p&lt;0.001). Following 3 days of recovery soleus mass significantly increased to 74% of control values. Adrenal weights of HU mice were not different compared to control mice.
The success of our novel HU method is evidenced by the maintenance of animal body weight, comparable adrenal gland weights, and soleus atrophy following HU, corresponding to expected literature values 2, 7, 8. The primary advantages of this HU method include: 1) ease of tail examination during suspension; 2) decreased likelihood of cyanotic, inflamed, and/or necrotic tails frequently observed with tail-taping and HU; 3) no possibility of mice chewing the traction tape and coming out of the suspension apparatus; and 4) rapid recovery and normal cage activity immediately after HU.

We developed an alternant hindlimb unloading model in mice. The primary advantage of our hindlimb unloading tail-ring method over the conventional Morey-Holton tail-traction technique is a simple straightforward procedure that minimizes stress upon the animal.

The Morey-Holton hindlimb unloading (HU) method is a widely accepted National Aeronautics and Space Administration (NASA) ground-based model for studying ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Neuroscience

Thoracic Spinal Cord Hemisection Surgery and Open-Field Locomotor Assessment in the Rat

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are valuable tools to understand the neural mechanisms involved in locomotor recovery after SCI and to design therapies for clinical populations. There are several experimental SCI models including contusion, compression, and transection injuries that are used in a wide variety of species. A hemisection involves the unilateral transection of the spinal cord and disrupts all ascending and descending tracts on one side only. Spinal hemisection produces a highly selective and reproducible injury in comparison to contusion or compression techniques that is useful for investigating neural plasticity in spared and damaged pathways associated with functional recovery. We present a detailed step-by-step protocol for performing a thoracic hemisection at the T8 vertebral level in the rat that results in an initial paralysis of the hindlimb on the side of the lesion with graded spontaneous recovery of locomotor function over several weeks. We also provide a locomotor scoring protocol to assess functional recovery in the open-field. The locomotor assessment provides a linear recovery profile and can be performed both early and repeatedly after injury in order to accurately screen animals for appropriate time points in which to conduct more specialized behavioral testing. The hemisection technique presented can be readily adapted to other transection models and species, and the locomotor assessment can be used in a variety of SCI and other injury models to score locomotor function.

The rat thoracic spinal hemisection is a valuable and reproducible model of unilateral spinal cord injury to investigate the neural mechanisms of locomotor recovery and treatment efficacy. This article includes a detailed step-by-step guide to perform the hemisection procedure and to assess locomotor performance in an open-field arena.

Spinal cord injury (SCI) causes disturbances in motor, sensory, and autonomic function below the level of the lesion. Experimental animal models are ...

Partial Heterotopic Hindlimb Transplantation Model in Rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

Medicine

Comprehensive Understanding of Inactivity-Induced Gait Alteration in Rodents

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

The present protocol describes three-dimensional motion tracking/evaluation to depict gait motion alteration of rats after exposure to a simulated disuse environment.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics ...