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. 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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 ...

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

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

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.

Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion.

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Neuroscience

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

Genetics

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Cancer Research

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 ...

Biology

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 ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

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 ...

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 ...