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

摘要

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.

摘要

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.

引言

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.

方案

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.

结果

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...

讨论

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...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
10G 绝缘实心铜线	Grainger	4WYY8	100 英尺实心建筑电线，THHN 线型和 10 AWG 线径，黑色
2 定制设计有机玻璃墙	P&K Custom Acrylics 公司	N/A	2个透明有机玻璃定制墙3/16"刻度，宽度12 3/16"，高度18 13/16"，1个直径0.25英寸的圆形槽，位于墙的中心顶部
3M Transpore手术胶带	Fisher Scientific	18-999-380	Transpore手术胶带
配件抓杆大鼠	佛仪器	76-0479	配件抓杆大鼠，前爪或后爪
分析秤	Fisher Scientific	01-920-251	OHAUS Adventurer 分析平衡
动物秤	ZIEIS by Amazon	N/A	70磅容量数字秤 大顶 11.5" x 9.3" 硬脑膜平台 Z-Seal 110V 适配器 0.5 盎司精度
背部文胸延长器	Luzen by Amazon	N/A	17 件 2 钩 3 行各种随机颜色女性间距文胸夹延长带
数字测力计	瓦格纳仪器	DFE2-010	50 N 容量数字握力计 Chatillon DFE II
纱布	Fisher Scientific	13-761-52	非无菌棉纱布海绵
钥匙环和旋转扣	Paxcoo Direct by Amazon	N/A	PaxCoo 100个金属旋转挂绳弹簧钩带钥匙环
龙虾扣	Panda Jewelry International Limited by Amazon	N/A	Pandahall 100个A级不锈钢龙虾爪扣 13x8mm
鼠绳夹克 - 大	Braintree Scientific	RJ L	啮齿动物夹克
鼠绳夹克 - 中号	Braintree Scientific	RJ M	啮齿动物夹克
硅胶管	Versilon St Gobain 陶瓷和塑料	ABX00011	SPX-50 硅胶管
不锈钢链	Super Lover by Amazon	N/A	4.5m 15FT 不锈钢电缆链环散装 6x8mm