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

Summary

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.

Abstract

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’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’ health and function.

Introduction

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 astronauts^1,2,3,4,5 and in rodents^6,7,8,9, 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’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 mice^6,13, 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’ 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’ 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.

Protocol

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.

1. 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.
2. 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.
3. Place the rat in a prone position and put the pelvic harness on in a rostro-caudal movement.
4. Gently bend the pelvic harness to provide a snug fit while being careful not squeeze the hindlimbs to prevent abrasions and discomfort.
5. 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.
6. Remove the rat from anesthesia and place the animal in a custom cage with the chain extended at its maximum.
7. 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.
8. Observe the animal for a few minutes to assess its comfort and make sure that at all times, both hind limbs remain completely unloaded.

2. Partial weight bearing

NOTE: This step can be realized in both awake and anesthetized animals.

1. Convert the HLU suspension device into a PWB suspension by adding the triangle-shaped part composed of stainless steel chains and a back rod.
2. Anesthetize the animal following the same procedures as detailed for the HLU (steps 1.1 and 1.2).
3. 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.
4. 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.
5. 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.
6. Place the rat on top of the scale to record the “loaded” body weight, i.e., the weight of the animal and the entire apparatus, without shortening the chain.
7. Shorten the chain until the scale displays 40% of the “loaded” body weight and record the achieved gravity level (expressed as the ratio between unloaded weight and loaded weight).
8. Observe the animal to make sure that the unloaded weight is stable and that the rat is equally loaded on all limbs.
9. Remove the entire apparatus from the scale using the rod and place the rat back in its cage.

3. Assessment of hindlimb grip force

1. Hold the rat with a traditional restraint by placing one hand underneath the forelimbs. Gently hold the tail with the second hand.
2. 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.
3. Gently pull the rat straight back until it releases its grip. Record the maximal force displayed on the transducer.
4. Wait approximately 30 s between measurements and repeat the test 3 times.
5. Calculate the average of the three measurements for scoring, to account for fatigue.

4. Recording of muscle wet mass

1. 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.
2. 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.
3. 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.
4. 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.
5. Once isolated, cut the attachment point of the gastrocnemius and soleus muscles in the popliteal area.
6. Gently pull the soleus away from the gastrocnemius and detach them by cutting the calcaneus tendon.
7. Place the rat in a supine position. Carefully remove the fascia and peel the tibialis anterior from the ankle in an upward movement.
8. Cut the tibialis anterior at its superior attachment point.
9. Record the exact wet mass of each excised muscle using a tared precision scale and a weighing boat.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
10G Insulated Solid Copper Wire	Grainger	4WYY8	100 ft solid building wire with THHN wire type and 10 AWG wire size, black
2 Custom design plexiglass walls	P&K Custom Acrylics Inc.	N/A	2 clear plexiglass custom wall 3/16" tick, width 12 3/16", height 18 13/16", 1 rounded slot 0.25 in of diameter located at the center top of the wall
3M Transpore Surgical Tape	Fisher Scientific	18-999-380	Transpore Surgical Tape
Accessory Grasping Bar Rat	Harvard Apparatus	76-0479	Accessory grasping bar rat, front or hind paws
Analytical Scale	Fisher Scientific	01-920-251	OHAUS Adventurer Analytic Balance
Animal Scale	ZIEIS by Amazon	N/A	70 lb capacity digital scale big top 11.5" x 9.3" dura platform z-seal 110V adapter 0.5 ounce accuracy
Back Bra Extenders	Luzen by Amazon	N/A	17 pcs 2 hook 3 rows assorted random color women spacing bra clip extender strap
Digital Force Gage	Wagner Instruments	DFE2-010	50 N Capacity Digital Grip Force Meter Chatillon DFE II
Gauze	Fisher Scientific	13-761-52	Non-sterile Cotton Gauze Sponges
Key rings and swivel claps	Paxcoo Direct by Amazon	N/A	PaxCoo 100 pcs metal swivel lanyard snap hook with key rings
Lobster Claps	Panda Jewelry International Limited by Amazon	N/A	Pandahall 100 pcs grade A stainless steel lobster claw clasps 13x8mm
Rat Tether Jacket - Large	Braintree Scientific	RJ L	Rodent Jacket
Rat Tether Jacket - Medium	Braintree Scientific	RJ M	Rodent Jacket
Silicone tubing	Versilon St Gobain Ceramics and Plastics	ABX00011	SPX-50 Silicone Tubing
Stainless Steel Chains	Super Lover by Amazon	N/A	4.5m 15FT stainless steel cable chain link in bulk 6x8mm