Mouse Model of Pressure Ulcers After Spinal Cord Injury

Summary

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.

Abstract

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.

Introduction

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 necrosis^2,3.

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 income^4,5.

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 system^5,6,7,8,9,10.

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.

Protocol

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

1. Sterilize the surgical and non-surgical instruments in an autoclave. 
2. Clean the surgical operating table with 70% ethanol and warm a heating pad to 37 °C.
3. Place the heating pad on the operating table and cover it with sterile surgical drapes.
   NOTE: In all survival procedures, the "No Touch" technique is used here to maintain sterility.

2. Preparation of Animals and Performing the T9-T10 Spinal Laminectomy

1. 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.
2. Confirm complete anesthesia by eliciting no response to a tail/paw pinch induced nociception stimulation.
3. 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
4. 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.
5. 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 vertebra^11,12.
6. 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.
7. 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 control group, only the laminectomy is performed.

3. Performing the T9-T10 Complete Spinal Cord Injury

1. Using forceps, secure the spinal column at T8 and lift up to exaggerate the spinal curvature.
2. 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.
3. 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.
4. Finally, close the wound and suture the paravertebral muscles, superficial fascia, using continuous suture and then close the skin using suture clips¹².
5. 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 1^11,12,13.

4. Induction of Skin Pressure Ulcer after Complete SCI

1. Immediately after the SCI surgery, scrub the back of the animal with betadine and 70% alcohol.
2. For a PU below the SCI site, inject in the dorsal skin near the sacrum, a very small volume (10 µL) 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.
3. Gently lift a skin fold on the back of the mouse and sandwich it between 2 magnetic discs (5×12 mm diameter, 2.4 g each, 3800 G magnetic force) (Figure 1).^11,12
4. Immediately after magnet application, return animals to single cages placed onto a heating pad until full consciousness is regained (Figure 1).
5. 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.

5. Post-operative Animal Care, Euthanasia, and Tissue Collection for Histology

1. Immediately after surgery, inject the animal with 1 mL of 0.9% saline subcutaneously for hydration.
2. Subcutaneously inject buprenorphine-SR (1 mg/kg) immediately for analgesia.
3. Inject subcutaneously animal daily meloxicam (1 mg/kg), and cefazolin (50 mg/kg for 3-7 days), and twice daily manual bladder evacuation.
4. 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.
5. Remove surgical clips 7 days after SCI surgery.
6. 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.
7. Collect wounded skin samples, fix in 10% formalin for 24 h, and then store in 70% ethanol at 4 °C until sectioning.
8. To process tissues, embed in paraffin and generate thin sections (5 µm) on a microtome. Stain with hematoxylin and eosin (H&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 (α-SMA) as described in Kumar et al.¹²
   NOTE: Image analysis software can be used to quantify image features¹².

Results

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...

Discussion

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...

Materials

List of materials used in this article

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