Name: mouse model of pressure ulcers after spinal cord injury
Uploaded: 2019-03-09T13:00:00
Description: Watch this Scientific Journal Video about Mouse Model of Pressure Ulcers After Spinal Cord Injury at JoVE.com

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 t...

<ol>
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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.

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			<td height="21" style="height:21px;width:235px;">Magnets</td>
			<td style="width:303px;">Master Magnetcs, Inc., Castle Rock, CO</td>
			<td style="width:116px;">CD14C</td>
			<td style="width:187px;">3800 G Magnetic force</td>
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			<td height="21" style="height:21px;width:235px;">Mice standard diet</td>
			<td>PMI Nutrition International, Brentwood, MO</td>
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			<td>Standard Food Pellet</td>
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			<td height="21" style="height:21px;width:235px;">Isoflurane</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
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			<td></td>
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			<td height="21" style="height:21px;width:235px;">ImageJ</td>
			<td style="width:303px;">NIH, Bethesda, MD</td>
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			<td>Image Analysis Software</td>
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			<td height="21" style="height:21px;width:235px;">BETADINE Surgical Scrub</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
			<td style="width:116px;"></td>
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			<td height="21" style="height:21px;">Ophthalmic Ointment&#160;</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
			<td style="width:116px;">SKU 008897</td>
			<td></td>
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			<td height="21" style="height:21px;">NAIR-Hair Remover Lotion</td>
			<td style="width:303px;">Church &#38; Dwight Co., Inc. Princeton, NJ</td>
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			<td height="21" style="height:21px;">ELOXIJECT (meloxicam) Injection</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
			<td style="width:116px;">SKU 049755</td>
			<td>5 mg/mL, 10 mL</td>
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			<td height="21" style="height:21px;">Cefazolin Sodium</td>
			<td>HENRY SCHEIN Animal Health&#160;</td>
			<td style="width:116px;">SKU 054846</td>
			<td>1 g, 10 mL bottle</td>
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			<td height="21" style="height:21px;">Buprenorphine-SR&#160;</td>
			<td style="width:303px;">ZooPharm, Windsor, CO</td>
			<td style="width:116px;">-</td>
			<td>-</td>
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			<td height="21" style="height:21px;">0.9% Sodium Chloride Injection USP</td>
			<td style="width:303px;">BRAUN, Irvine, CA</td>
			<td style="width:116px;">S8004-5384</td>
			<td></td>
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			<td height="21" style="height:21px;">10% Neutral Buffered Formalin&#160;</td>
			<td style="width:303px;">VWR, Radnor, PA</td>
			<td>16004-130</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">BALB/C Male Mouse</td>
			<td style="width:303px;">Charles River Lab., Wilmington, MA</td>
			<td style="width:116px;">28</td>
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			<td height="21" style="height:21px;width:235px;">Sterile Cotton Tipped Applicator</td>
			<td style="width:303px;">Puritan, Guilford, ME</td>
			<td style="width:116px;">SKU#: 25-806</td>
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			<td height="42" style="height:42px;width:235px;">Michel Suture Clips</td>
			<td style="width:303px;">Fine Science Tools (USA) Inc., Foster City, CA</td>
			<td style="width:116px;">12040-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:235px;">Surgical Suture, U.S.P.</td>
			<td>Henry Schein Animal Health&#160;</td>
			<td style="width:116px;">101-2636</td>
			<td></td>
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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 ...

The overall goal of this study is to examine the effect of spinal cord injury on the dynamics of pressure ulcer development and healing in an animal model. The main advantage of this technique is that it does not impair animal movements, and is very reproducible. This model provides a platform to study the dynamics of wound healing and test various therapeutic strategies for SCI patient.Before beginning the procedure ensure proper anesthesia by the lack of response to a tail pinch. Shave the hair over the dorsum with an electric clipper. Then apply the depilatory cream, and leave for three minutes to remove the remaining hair.After three minutes remove the depilatory cream with the wet scrub and wash the dorsum similarly. And then return the animals to their cages. The next day anesthetize the animals and apply the ophthalmic ointment to the corneas to protect the eyes from drying during the surgical procedure.Then, scrub the animal skin three times each with betadine scrub and 70%ethanol. Before incision, use a syringe with a 25 gauge needle to inject 100 microliters of 0.125%bupivacaine around the incision site as analgesia. Count back from the floating ribs that correspond to T13 to identify T8 to T12.After creating a one to 1.5 centimeter incision, along the midline, on the back, at the level of T8 to T12 vertebrae clear the subcutaneous adipose tissue to access the paraspinal muscles. And then dissect them slowly to expose the spinous processes and lamina on both sides. It is very critical to do this procedure very carefully to avoid excessive bleeding and any injury to the spinal cord.Perform a laminectomy of the T9 to T10 vertebrae to expose the spinal cord by gently peeling off the spinal lamina using micro dissecting forceps. Use forceps to secure and lift the spinal column at T8 to exaggerate the spinal curvature. Then, use fine scissors to section the spinal cord between the T9 and T10 vertebrae all the way to the floor of the vertebral canal to ensure complete transection.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. Finally, close the wound and suture the paravertebral muscles and superficial fascia. Then close the skin using suture clips.Immediately after the SCI surgery scrub the back of the animal with betadine and 70%alcohol. For a pressure ulcer below the spinal cord injury site use a 25 gauge needle to inject 10 microliters of 0.125%bupivacaine solution in an ellipse in the dorsal skin, near the sacrum, with points 0.5 to one centimeter apart. Gently lift a skin fold on the back of the mouse and sandwich it between two magnetic disks.Immediately after magnet application return the animals to single cages placed onto a heating pad until full consciousness is regained. After 12 hours of magnet application lightly anesthetize the animal with isoflurane and remove the magnets. Take a photograph of the wound sites to record the initial appearance of the pressure ulcer at the day zero time point.Cover the wound with transparent dressing film to avoid drying or contamination. Single-house the animals following the procedure. Immediately after surgery inject the animal with one milliliter of 0.9%saline for hydration and buprenorphine SR for analgesia.Perform manual bladder evacuation twice a day. Remove the surgical clips seven days after spinal cord injury surgery. Collect wounded skin samples from the euthanized mouse at the desired time point after spinal cord injury and skin pressure ulcer induction.Fix in 10%formalin for 24 hours. After 24 hours transfer to 70%ethanol and store at four degrees Celsius until sectioning. This image depicts the disappearance of the epidermis in a control animal by day three of post-magnet removal.Arrows indicate the epidermal wound edges located where the epithelial lining thins out. In the control animal the epidermis reappears by day seven. For wounds created below the level of the SCI, in SCI mice, significantly slower migration is seen.Here, the figure depicts slower wound healing and closure in the SCI mice as compared to the control mice. Immunostaining for Ki67, a marker of proliferating cells indicated by the red arrows, revealed less proliferation in the SCI group. Staining for CD31, a marker for blood vessels indicated by the red arrows, revealed lower blood vessel density in the SCI group.Finally, immunostaining for alpha smooth muscle actin revealed lower levels of this protein in the skin wounds from SCI mice. While attempting this procedure it is important to remember to place the magnets properly to develop two circular wounds separated by intact skin. This will reduce the variability in wound development and healing.Skin pressure ulcers in spinal cord injured patients impart significant costs to the U.S.healthcare system. This animal model provides a platform for testing various therapeutic strategies to aid pressure ulcer healing in spinal cord injured patients.

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