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 de...

<ol>
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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 rel...

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 environ...

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 ...

Our protocol offers a significant advantage by creating the possibility to expose animals to successive unloading levels. In the past, ground-based models have used either hindlimb unloading or partial weight-bearing separately. The main advantage of this technique is the use of a single apparatus and environment to provide numerous possibilities such as mimicking a trip to, and short stay on Mars.This model will help understand the physiological state upon landing on Mars and the physiological challenges for astronauts exposed to several unloading levels during their mission. This model could help researchers explore a wide range of physiological changes including but not limited to behavior, memory, performance or stress. Demonstrating the procedure will be Daniela Riveros, a Research Fellow in our laboratory.To perform hindlimb unloading, place the fully anesthetized rat on the bench with anesthetic gas coming from a nose cone with 2%isoflurane and an oxygen flow of 1.5 liters per minute. Place the rat in a prone position and put the pelvic harness on in a rostrocaudal movement. Gently bend the pelvic harness to provide a snug fit while being careful not to squeeze the hind limbs to prevent abrasions and discomfort.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. Remove the rat from anesthesia and place the animal in a custom cage with the chain extended at its maximum. 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.Convert the hindlimb unloading suspension device into a partial weight-bearing suspension device by adding the triangle-shaped part, composed of stainless steel chains and a back rod. Place a tether jacket of the appropriate size on the fore limbs of the rat, and close it using the back bra extender. Attach one clasp of the triangle-shaped part of 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.Place the rat on top of the scale to record the loaded body weight including the weight of the entire apparatus without shortening the chain. Shorten the chain until the scale displays 40%of the loaded body weight and record the achieved gravity level. With the rod, remove the entire apparatus from the scale and place the rat back in its cage.To assess the hindlimb grip force, hold the rat with the traditional restraint by placing one hand underneath the fore limbs. Gently hold the tail with the second hand. Approach the grip bar with the rear paws, and make sure that both paws are fully resting on the bar.Finally, gently pull the rat straight back until it releases its grip. Repeat three times and record the maximal force displayed on the transducer. Calculate the average of the three measurements to account for fatigue.One week of HLU followed by one week of PWB40 resulted in a significant body weight loss similar to what was observed in rats exposed at only PWB40 for the same duration. However, these rats continued losing weight over time while subsequently being exposed to PWB40. One week of total unloading led to a decrease in hindlimb grip force compared to age-matched controls.However, after one subsequent week of PWB40, no further change regarding grip force was observed. Additionally, rats that underwent total unloading followed by partial weight-bearing displayed a significantly greater grip force loss compared to the PWB40 group. PWB40 in HLU-PWB40 groups had significantly lower wet mass of soleus, gastrocnemius, and tibialis anterior muscles than age-matched controls.However, no significant difference between the wet mass of the PWB40 and HLU-PWB40 groups were observed. The success of this method relies on the accuracy of the partial weight-bearing level. Frequent monitoring is crucial to assess animal's wellness and unloading daily.This method is non-invasive and therefore doesn't prevent the utilization of additional testing procedures regarding all systems. Longitudinal measures are also easy to implement if the loading time remains controlled. This method was entirely developed in the laboratory and the utilization of custom equipment can be challenging for new teams.This technique opens the way for extensive studies in which partial weight-bearing is at play. This could include clinical situations such as use of assistive equipment, for example crutches, canes or casts, as well as aqua-therapy.

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10.2K Views.  Harvard Medical School - Beth Israel Deaconess Medical Center. Our protocol offers a significant advantage by creating the possibility to expose animals to successive unloading levels. In the past, ground-based models have used either hindlimb unloading or partial weight-bearing separately. The main advantage of this technique is the use of a single apparatus and environment to provide numerous possibilities such as mimicking a trip to, and short stay on Mars.This model will help understand the physiological state upon landing on Mars and the phys...