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.
