This work was partially supported by the New Jersey Commission on Spinal Cord Research (CSCR15IRG010), the U.S. Department of Defense (SC160029), and the Yale Department of Surgery Ohse Research Grant Program. We thank Sean O' Leary from the W.M. Keck Center for Collaborative Neuroscience, Rutgers for technical assistance.

All animal handling and surgical procedures were performed in accordance with a protocol approved by the Rutgers University Institutional Animal Care and Use Committee. Mice were fed standard diet and water ad libitum.
1. Preparation of Surgical and Non-surgical Instruments
<ol>
	<li>Sterilize the surgical and non-surgical instruments in an autoclave.&#160;</li>
	<li>Clean the surgical operating table with 70% ethanol and warm a heating pad to 37 &#176;C.</li>
	<li>Place the heating pad on the operating table and cover it with sterile surgical drapes. 
	NOTE: In all survival procedures, the &#34;No Touch&#34; technique is used here to maintain sterility.</li>
</ol>
2. Preparation of Animals and Performing the T9-T10 Spinal Laminectomy
<ol>
	<li>Procure adult (Balb/c) 10-week-old male mice. Induce anesthesia in each animal using an inhaler beginning with 5% isoflurane, and then decrease to 2-3% to maintain sedation for the remainder of the procedures.</li>
	<li>Confirm complete anesthesia by eliciting no response to a tail/paw pinch induced nociception stimulation.</li>
	<li>Shave the hair over the dorsum (head to tail) with an electric clipper, and then apply the depilatory cream (3 min) to remove the remaining hair. Finally, wash the dorsum with running water/wet scrub, and return the animals to their cages. 
	NOTE: This is necessary to avoid additional irritation to the skin and chemical contamination at the time of skin wounding</li>
	<li>The next day, apply ophthalmic ointment to the corneas to protect the eyes from drying during the surgical procedure and then, scrub the skin with 3 alternative preparations of betadine scrub and 70% ethanol.</li>
	<li>With a scalpel, perform a skin incision (~1.0-1.5 cm) along the midline on the back at the level of T8-T12 vertebrae. 
	NOTE: The level of vertebrae is identified by back counting the vertebra from T13 using the location of floating ribs that correspond to T13 vertebra11,12.</li>
	<li>Clear the subcutaneous adipose tissue to get access to the paraspinal muscles and then dissect them slowly to expose the spinous processes and laminae on both sides. 
	NOTE: Do this procedure very carefully to avoid excess bleeding or any injury to the spinal cord, at this point.</li>
	<li>Perform a laminectomy to expose the spinal cord (T9-T10 vertebrae) by gently peeling off the spinal lamina using microdissecting forceps. 
	NOTE: Perform the laminectomy so that an excess of the spinal cord is exposed to facilitate the creation of the injury. In the&#160;control group, only the laminectomy is performed.</li>
</ol>
3. Performing the T9-T10 Complete Spinal Cord Injury
<ol>
	<li>Using forceps, secure the spinal column at T8 and lift up to exaggerate the spinal curvature.</li>
	<li>Using fine scissors, section the spinal cord between the T9 and T10 vertebra all the way to the floor of the vertebral canal, to ensure complete transection.</li>
	<li>After observing the complete transection under a surgical microscope, apply a piece of subcutaneous fat over the laminectomy site to provide additional protection to the spinal cord prior to surgical site closure.</li>
	<li>Finally, close the wound and suture the paravertebral muscles, superficial fascia, using continuous suture and then close the skin using suture clips12.</li>
	<li>Post-SCI, observe the bowel movement on the next day; however, manage the urinary bladder by manual bladder evacuation. 
	NOTE: The Basso Mouse Scale (BMS) can be used to monitor the progress of hindlimb functional recovery post-SCI at day 2 and then weekly, see Supplementary Figure 111,12,13.</li>
</ol>
4. Induction of Skin Pressure Ulcer after Complete SCI
<ol>
	<li>Immediately after the SCI surgery, scrub the back of the animal with betadine and 70% alcohol.</li>
	<li>For a PU below the SCI site, inject in the dorsal skin near the sacrum, a very small volume (10 &#181;L) of 0.125% bupivacaine solution using a 25 G needle at equidistant places ~0.5-1.0 cm apart, in an ellipse around the magnet application site. 
	NOTE: For a PU above the SCI site, inject the dorsal skin near the cervical region.</li>
	<li>Gently lift a skin fold on the back of the mouse and sandwich it between 2 magnetic discs (5&#215;12 mm diameter, 2.4 g each, 3800 G magnetic force) (Figure 1).11,12</li>
	<li>Immediately after magnet application, return animals to single cages placed onto a heating pad until full consciousness is regained (Figure 1).</li>
	<li>After 12 h of magnet application, lightly anesthetize animal with isoflurane and remove the magnets. Take a photograph of the wound sites, to record the initial appearance of PU (day 0 time point). Cover the wound with transparent dressing film (3M) to avoid drying or contamination.</li>
</ol>
5. Post-operative Animal Care, Euthanasia, and Tissue Collection for Histology
<ol>
	<li>Immediately after surgery, inject the animal with 1 mL of 0.9% saline subcutaneously for hydration.</li>
	<li>Subcutaneously inject buprenorphine-SR (1 mg/kg) immediately for analgesia.</li>
	<li>Inject subcutaneously animal daily meloxicam (1 mg/kg), and cefazolin (50 mg/kg for 3-7 days), and twice daily manual bladder evacuation.</li>
	<li>Place animals in single cages and provide accessible food and water ad libitum. Mice with complete SCI can walk using their forelimbs and approach the food and water without any difficulties.</li>
	<li>Remove surgical clips 7 days after SCI surgery.</li>
	<li>At the desired time points after SCI and skin wounding, euthanize animals by CO2 inhalation (3-5 min), in accordance with the AVMA Guidelines on Euthanasia14.</li>
	<li>Collect wounded skin samples, fix in 10% formalin for 24 h, and then store in 70% ethanol at 4 &#176;C until sectioning.</li>
	<li>To process tissues, embed in paraffin and generate thin sections (5 &#181;m) on a microtome. Stain with hematoxylin and eosin (H&#38;E) to visualize tissue morphology (Figure 3). For immunohistochemistry studies (Figure 4), stain sections using appropriate antibodies for Ki67 (proliferation), CD31 (angiogenesis), and alpha-smooth muscle actin (&#945;-SMA) as described in Kumar et al.12 
	NOTE: Image analysis software can be used to quantify image features12.</li>
</ol>

<ol>
	<li>Rappl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+changes+in+tissues+denervated+by+spinal+cord+injury+tissues+and+possible+effects+on+wound+healing.">Physiological changes in tissues denervated by spinal cord injury tissues and possible effects on wound healing.</a> International Wound Journal. 5 (3), 435-444 (2008).</li><li>Salcido, R., Popescu, A., Ahn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+pressure+ulcer+research.">Animal models in pressure ulcer research.</a> The Journal of Spinal Cord Medicine. 30 (2), 107-116 (2007).</li><li>Mak, A. F., Zhang, M., Tam, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+pressure+ulcer+in+body+tissues+interacting+with+external+forces+during+locomotion.">Biomechanics of pressure ulcer in body tissues interacting with external forces during locomotion.</a> Annual Review of Biomedical Engineering. 12, 29-53 (2010).</li><li>National Pressure Ulcer Advisory Panel. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prevention and Treatment of Pressure Ulcers: Clinical Practice Guideline. , (2014).</li><li>Marin, J., Nixon, J., Gorecki, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+risk+factors+for+the+development+and+recurrence+of+pressure+ulcers+in+people+with+spinal+cord+injuries.">A systematic review of risk factors for the development and recurrence of pressure ulcers in people with spinal cord injuries.</a> Spinal Cord. 51 (7), 522-527 (2013).</li><li>Krause, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sores+after+spinal+cord+injury:+relationship+to+life+adjustment.">Skin sores after spinal cord injury: relationship to life adjustment.</a> Spinal Cord. 36 (1), 51-56 (1998).</li><li>Redelings, M. D., Lee, N. E., Sorvillo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+ulcers:+more+lethal+than+we+thought?.">Pressure ulcers: more lethal than we thought?.</a> Advances in Skin &amp; Wound. 18 (7), 367-372 (2005).</li><li>Kruger, E. A., Pires, M., Ngann, Y., Sterling, M., Rubayi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+management+of+pressure+ulcers+in+spinal+cord+injury:+current+concept+and+future+trends.">Comprehensive management of pressure ulcers in spinal cord injury: current concept and future trends.</a> The Journal of Spinal Cord Medicine. 36 (6), 572-585 (2013).</li><li>Lala, D., Dumont, F. S., Leblond, J., Houghton, P. E., Noreau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+pressure+ulcers+on+individuals+living+with+a+spinal+cord+injury.">Impact of pressure ulcers on individuals living with a spinal cord injury.</a> Archives of Physical Medicine and Rehabilitation. 95 (15), 2312-2319 (2014).</li><li>Li, C., DiPiro, N. D., Krause, 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+latent+structural+equation+model+of+risk+behaviors+and+pressure+ulcer+outcomes+among+people+with+spinal+cord+injury.">A latent structural equation model of risk behaviors and pressure ulcer outcomes among people with spinal cord injury.</a> Spinal Cord. 55 (6), 553-558 (2017).</li><li>Kumar, S., Yarmush, M. L., Berthiaume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+complete+spinal+cord+injury+on+neovascularization+and+tissue+granulation+in+mouse+model+of+skin+pressure+ulcers.">Impact of complete spinal cord injury on neovascularization and tissue granulation in mouse model of skin pressure ulcers.</a> Journal of Neurotrauma. 34 (13), A72-A73 (2017).</li><li>Kumar, S., Yarmush, M. L., Dash, B. C., Hsia, H. C., Berthiaume, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+complete+spinal+cord+injury+on+healing+of+skin+ulcers+in+mouse+models.">Impact of complete spinal cord injury on healing of skin ulcers in mouse models.</a> Journal of Neurotrauma. 35 (6), 815-824 (2018).</li><li>Basso, D. M., Fisher, L. C., Anderson, A. J., Jakeman, L. B., McTigue, D. M., Popovich, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains.">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> Journal of Neurotrauma. 23 (5), 635-659 (2006).</li><li>Leary, S., 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=.">.</a> AVMA Guidelines for the Euthanasia of Animals. , (2013).</li><li>Stadler, I., Zhang, R. Y., Oskoui, P., Whittaker, M. S., Lanzafame, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+simple,+noninvasive,+clinically+relevant+model+of+pressure+ulcers+in+the+mouse.">Development of a simple, noninvasive, clinically relevant model of pressure ulcers in the mouse.</a> Journal of Investigative Surgery. 17 (4), 221-227 (2004).</li><li>Peirce, S. M., Skalak, T. C., Rodeheaver, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+injury+in+chronic+pressure+ulcer+formation:+a+skin+model+in+the+rat.">Ischemia-reperfusion injury in chronic pressure ulcer formation: a skin model in the rat.</a> Wound Repair and Regeneration. 8 (1), 68-76 (2000).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> Journal of Biomedicine and Biotechnology. 2011, 969618-969625 (2011).</li></ol>

The authors have nothing to disclose.

The protocol in this study describes a novel experimental model of PUs to evaluate the impact of SCI on wound healing. The skin PUs were induced via a 12 h application of two 12 mm diameter disc magnets on a dorsal skin fold, either set above or below the SCI site. Data show that SCI slows down skin wound healing in mice. Importantly, these observations were specifically made in skin wounds below the innervation level of the SCI, as wounds made above the SCI level, and thus that remained innervated, were largely unaffect...

Pressure ulcers (PUs) are major secondary complications of traumatic SCI1. PUs are localized injuries to the skin and/or underlying tissues that usually occur over bony prominences where body weight is concentrated while the patient is sitting or lying1. The skin, fat, and muscle are exposed to this constant pressure that leads to the development of localized ischemia, tissue inflammation, mechanical damage, and necrosis2,3.
The development of PUs is affected by several local factors, including the magnitude of pressure and shear, loading duration, skin moisture and temperature, injury longevity, and general skin hygiene. There are also systemic factors that play a role, such as general physical condition, bone and muscle tissue morphology and strength4, patient age, hematological measures, gender, and even socio-economic factors including marital status, education, and income4,5.
The prevention and treatment of PUs remain significant challenges in SCI patients. SCI patients develop PUs in ~30-40% of cases, with a re-occurrence rate of 60-85%, possibly due to weak scar tissue and lack of protective sensation1. Thus, PUs often leads to re-hospitalization of SCI patients, and overall pose a significant financial burden (80% more vs. SCI only) to the health care system5,6,7,8,9,10.
To the best of our knowledge, there have been no studies in controlled experimental settings to investigate the impact of SCI on the PU healing process because of the lack of suitable animal models. Here, a reproducible and clinically relevant mouse model of PU in the skin is described. This model can be used to study the dynamics of PU onset and subsequent healing, as well as to test potential therapeutic approaches to prevent PU or improve PU healing in the context of SCI.

<table><tbody><tr><td>Magnets</td><td>Master Magnetcs, Inc., Castle Rock, CO</td><td>CD14C</td><td>3800 G Magnetic force</td></tr><tr><td>Mice standard diet</td><td>PMI Nutrition International, Brentwood, MO</td><td></td><td>Standard Food Pellet</td></tr><tr><td>Isoflurane</td><td>HENRY SCHEIN Animal Health&amp;nbsp;</td><td>SKU 029405</td><td></td></tr><tr><td>ImageJ</td><td>NIH, Bethesda, MD</td><td></td><td>Image Analysis Software</td></tr><tr><td>BETADINE Surgical Scrub</td><td>HENRY SCHEIN Animal Health&amp;nbsp;</td><td></td><td></td></tr><tr><td>Ophthalmic Ointment&amp;nbsp;</td><td>HENRY SCHEIN Animal Health&amp;nbsp;</td><td>SKU 008897</td><td></td></tr><tr><td>NAIR-Hair Remover Lotion</td><td>Church &amp;amp; Dwight Co., Inc. Princeton, NJ</td><td></td><td></td></tr><tr><td>ELOXIJECT (meloxicam) Injection</td><td>HENRY SCHEIN Animal Health&amp;nbsp;</td><td>SKU 049755</td><td>5 mg/mL, 10 mL</td></tr><tr><td>Cefazolin Sodium</td><td>HENRY SCHEIN Animal Health&amp;nbsp;</td><td>SKU 054846</td><td>1 g, 10 mL bottle</td></tr><tr><td>Buprenorphine-SR&amp;nbsp;</td><td>ZooPharm, Windsor, CO</td><td>-</td><td>-</td></tr><tr><td>0.9% Sodium Chloride Injection USP</td><td>BRAUN, Irvine, CA</td><td>S8004-5384</td><td></td></tr><tr><td>10% Neutral Buffered Formalin&amp;nbsp;</td><td>VWR, Radnor, PA</td><td>16004-130</td><td></td></tr><tr><td>BALB/C Male Mouse</td><td>Charles River Lab., Wilmington, MA</td><td>28</td><td></td></tr><tr><td>Sterile Cotton Tipped Applicator</td><td>Puritan, Guilford, ME</td><td>SKU#: 25-806</td><td></td></tr><tr><td>Michel Suture Clips</td><td>Fine Science Tools (USA) Inc., Foster City, CA</td><td>12040-01</td><td></td></tr><tr><td>Surgical Suture, U.S.P.</td><td>Henry Schein Animal Health&amp;nbsp;</td><td>101-2636</td><td></td></tr></tbody></table>

mouse model of pressure ulcers after spinal cord injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

This protocol creates a PU in the setting of complete SCI. Briefly (as illustrated in Figure 1), all mice with or without complete SCI tolerated the magnets very well, which remained in their original position for the full 12 h (Figures 1c, 1d, 1f, 1h). All the mice developed two circular wounds separated by a bridge of normal tissue (Figure 1e, 1g, 1i). The initial wounding response was similar in mice without S...

Watch this Scientific Journal Video about Mouse Model of Pressure Ulcers After Spinal Cord Injury at JoVE.com

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony ...

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Research

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9.3K Views.  Rutgers University. Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.

Video: Mouse Model of Pressure Ulcers After Spinal Cord Injury

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Medicine

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

A Tissue Displacement-based Contusive Spinal Cord Injury Model in Mice

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between experimental animals. Several contusive SCI models have been developed to produce injuries using different mechanisms. The severity of the SCI is based on the height that a given weight is dropped, the injury force, or the spinal cord displacement. In the current study, we introduce a novel mouse contusive SCI device, the Louisville Injury System Apparatus (LISA) impactor, which can create a displacement-based SCI with high injury velocity and accuracy. This system utilizes laser distance sensors combined with advanced software to produce graded and highly-reproducible injuries. We performed a contusive SCI at the 10th thoracic vertebral (T10) level in mice to demonstrate the step-by-step procedure. The model can also be applied to the cervical and lumbar spinal levels.

We introduce a tissue displacement-based contusive spinal cord injury model that can produce a consistent contusive spinal cord injury in adult mice.

Producing a consistent and reproducible contusive spinal cord injury (SCI) is critical to minimizing behavioral and histological variabilities between ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, thereby improving the reproducibility of the experiments.
These methods need two requirements to be fulfilled: clarity of the surgical anatomical pathway and simplicity and convenience of the laboratory device. Crucially for the operator, a clear anatomical pathway provides minimally invasive exposure, which avoids additional damage to the experimental animal during the surgical procedures and allows the animal to maintain a consistent and stable anatomical morphology during the experiment.
In this study, the use of a novel integrated platform called the SCI coaxial platform for spinal cord injury in small animals to expose the T9 level spinal cord in a minimally invasive way and stabilize and immobilize the vertebra of mice using a vertebral stabilizer is researched, and, finally, a coaxial gravity impactor is used to contuse the spinal cord of mice to approach different degrees of T9 spinal cord injury. Finally, histological results are provided as a reference for the readers.

Minimally invasive techniques and a simple laboratory device improve the reproducibility of the spinal cord injury model by reducing operative damage to the experimental animals and allowing anatomical morphology maintenance. The method is worthwhile because the reliable results and reproducible procedure facilitate investigations of the mechanisms of disease reparation.

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...

Neuroscience

Quantifying Locomotor Dysfunction in Spinal Cord Injury Mice Using MouseWalker

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

Author Spotlight: Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and ...

A Spinal Cord Injury Contusion Device for Accurate Mouse Models

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of contusion models of spinal cord injury by impacting the spinal cord results in similar pathologies to most spinal cord injuries in clinical practice. Accurate, reproducible, and convenient animal models of spinal cord injury are essential for studying spinal cord injury. We present a novel automated spinal cord injury contusion device for mice, the Guangzhou Jinan University smart spinal cord injury system, that can produce spinal cord injury contusion models with accuracy, reproducibility, and convenience. The system accurately produces models of varying degrees of spinal cord injury via laser distance sensors combined with an automated mobile platform and advanced software. We used this system to create three levels of spinal cord injury mice models, determined their Basso mouse scale (BMS) scores, and performed behavioral as well as staining assays to demonstrate its accuracy and reproducibility. We show each step of the development of the injury models using this device, forming a standardized procedure. This method produces reproducible spinal cord injury contusion mice models and reduces human manipulation factors via convenient handling procedures. The developed animal model is reliable for studying spinal cord injury mechanisms and associated treatment approaches.

Author Spotlight: Insight Into Innovations in Spinal Cord Injury Research

Presented here is a novel automated spinal cord injury contusion device for mice, which can accurately produce spinal cord injury contusion models with varying degrees.

Spinal cord injury (SCI) due to traumatic injuries such as car accidents and falls is associated with permanent spinal cord dysfunction. Creation of ...

 A Minimal Spinal Cord Injury Model to Study Neural Stem Cell Activation and Migration

<a href='https://app.jove.com/v/57727/a-neurosphere-assay-to-evaluate-endogenous-neural-stem-cell'>Source: Lakshman, N., et al. A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury.&#160;J. Vis. Exp. (2018).</a>This video demonstrates the activation and migration of neural stem cells (NSCs) in a mouse spinal cord injury model. To expose the spinal cord, a vertebra from the thoracic region of the spine is removed from an anesthetized mouse. A superficial injury is then created in the white matter of the spinal cord using a bent needle, which preserves the central canal. Following the injury, NSCs surrounding the canal are activated and migrate to the injury site to assist tissue repair.

Mouse Model of Pressure Ulcers After Spinal Cord Injury

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Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

Automated Impactor for Contusive Spinal Cord Injury Model in Mice

A Minimal Spinal Cord Injury Model to Study Neural Stem Cell Activation and Migration