Tracheal Injury

Anesthetize Hartley guinea pigs, 400-700 g in mass, with 40 mg kg of ketamine and 5 mg kg of xylazine intramuscularly. Fig. 2. Schematic of wand used to create tracheal injury. The wand is made from stainless steel. (A) The wand is about 20 cm long and 1.5 mm in diameter. (B) The wand is milled along its length so that the cross-section is a semicircle down to a hook. The hook is designed to catch the tracheal surface when the wand is pulled backward under torsion. The tip of the wand is...

Delipidization and Drying of Removed ePTFE Tubes

Engrave glass vials with a code number for identification of the individual section of ePTFE tube. Fill each vial with 3 mL of acetone, close with a polyethylene screw cap, and place at 4 C for a minimum of 1 h (see Note 6). 2. Divide the ePTFE tubes into sections, cut perpendicularly to the long axis. For the number of sections refer to the planned analyses. Determination of hydroxyproline, hydroxylysine and proline requires an approx 10-mm length of ePTFE tube. 3. Place the section of ePTFE...

Materials

Animals ICR Harlan Sprague Dawley (Indianapolis, IN) mice weighing 18-20 g are employed for the intestinal anastomoses. We use male mice that are age and weight matched. The mice are placed in hanging cages and allowed to acclimate for 1 wk after arrival at the Division of Laboratory Animal Resources, James H. Quillen College of Medicine, East Tennessee State University, an Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility. They are...

Surgically Induced Diabetes Mellitus

The great benefit of the use of animals in biomedical research can be exemplified by the early studies on the effects of insulin on diabetes mellitus (5). Insulin's function was discovered in dogs that had their pancreas removed surgically. Investigators were able to reverse the animals' severe diabetes by treating them with insulin isolated from pancreatic extracts, eventually revolutionizing the treatment of diabetes. These techniques of surgically removing the pancreas can be performed in...

Suction Blister Induction

Suction blisters can be induced on the body, arms, and legs. We have not induced them in the head or facial skin. Make sure that the skin is clean, and remove any hair (see Notes 2 and 3). 2. Connect the suction blister device to a vacuum pump, and place the device tightly on the skin while turning on the vacuum. The vacuum should be increased to about 50 kPa, so that the device sticks tightly to the skin. Thereafter, it is not necessary to hold the device manually (see Note 4). Fig. 2. Fully...

Wire Mesh Cylinder Model

Stainless steel mesh This can be purchased from Cambridge Wire Cloth (Cambridge, MA). The material is 316SS with 0.010-in. diameter spacing. The type of metal alloy is important. Perforated plastic tubing is not satisfactory, though thin-meshed plastic of approximately the same properties as just described might be useful. We have used silicone mesh for studies requiring magnetic resonance imaging a. For rabbit 55 x 61 mm mesh rolled around a 3-cm cork borer (steel rod). b. For rat or guinea...

Image Analysis

Choose the lowest magnification on the microscope that will include all of the area to be analyzed (see Note 13). 2. Adjust the focus so that the image through the ocular as well as the image on the monitor is in focus. 3. Move the sample off to the side so that a background (glass only) reading can be taken. 4. Move the sample back into view and adjust other components, such as contrast, exposure, and color correction, until a bright, sharp image is obtained. 5. Capture the image using...

Measurement of Transcutaneous Oxygen in Patients with Ulcers of Lower Extremity

Subcutaneous oxygen measurement is accurate and simple, and application of the technique in surgical patients has improved outcomes in acute wounds. Oxygen supply is similarly critical and often impaired in chronic wounds. However, use of an invasive technique is often contraindicated or difficult in patients with chronic wounds. Transcutaneous oximetry is a noninvasive measurement that, although less reliable than sc oximetry, has been used in a parallel fashion in chronic wounds (21-26)....

Preconditioning and Anesthesia

Animals should be given a standard diet ad libitum several days prior to the investigation to allow acclimatization to their new surroundings. The pigs should be fasted overnight before any procedures to reduce the risk of vomiting and aspiration during the procedure. Housing and care for animals should be in accordance with the National Research Council guidelines (14). 1. Sedate the pigs with 5 mg kg of Talazine intramuscularly. Generally, the onset of sedation will require 5-10 min. 2....

Genetic or Spontaneous Diabetes Mellitus Models

Diabetes mellitus develops in many, if not all, species of mammals. Just as with people, there are those animals that have a predisposition for diabetes. These genetically or spontaneously induced models have been used extensively for all kinds of research, including studies of tissue repair. The genetic models can be divided into insulin-dependent models and insulin-resistant models. Frequently, the models are divided into nonobese or obese models, since most insulin-resistant models occur in...

Wounding Methods

Each of these procedures is best performed under direct visualization using a stereo-operating microscope, although corneal epithelial scrape can be performed with the loupes or a magnifying glass. Corneal epithelial scrape injuries are most easily performed by scraping with a no. 64 Beaver blade (rabbit) or a no. 15 Beaver blade (mouse). Typically a 7-mm-diameter wound is made in the rabbit cornea and a 2-mm-diameter wound is made in the mouse cornea, but the size may vary depending on what...

Microscopic Measurement of Wound Closure Rate

Seed rat keratinocytes at high initial cell densities into serum-containing DMEM (see Note 1). 2. Immediately after attaining confluency, aspirate the growth medium and wash the monolayers twice with HBSS. Wash volumes are 2 and 5 mL for 35- and 100-mm culture dishes, respectively. Fig. 2. Rat keratinocytes are grown to confluency and maintained in serum-free DMEM for 3 d to create a contact-inhibited quiescent population. A single scrape wound is created in the monolayer using the narrow end...

Applications of Particle Bombardment in Tissue Repair

As a surface tissue, the skin makes an amenable organ for gene therapy and in particular for in vivo transfection by particle bombardment. Recently, particle-mediated DNA vaccination in mice has been successful in the immunization against a series of pathogens (39-42). Additionally, this technique has been applied to the field of immunomodulation. Particle-mediated introduction of interferon-a (43), interleukin-2 (IL-2), or IL-6 cDNA has been shown to produce local antitumor immunity in...

Additional Methods

Microdialysis is much the same technique as sc oximetry (27). In this case, however, the tonometer measures small water-soluble substances as well as gases. It is less convenient, but it has been used extensively in brain research. Strangely, few investigators have recognized its potential for wound research, and although we know of investigators who have used it, we can find no publication detailing its use in wounds. one publication refers to its use for oxygen and lactate measurement in...

IL10 Causes Scarring in a Model of Fetal Wound Repair

Fetal wound healing is characterized by rapid reepithelialization, minimal inflammation, and scar-free repair (reviewed in ref. 76). Fetal wounds also show diminished expression of the proinflammatory cytokines IL-6 and IL-8, a phenomenon that was hypothesized to be owing to their negative regulation by the antiinflammatory cytokine IL-10. To test this hypothesis, Liechty et al. (77) wounded embryonic skin from IL-10 null mice that had been grafted onto strain-matched adult mice. Wounds of...

Method for Detection and Quantitation of Leukocytes During Wound Healing

Introduction The wound repair process is a highly ordered series of events that encompasses hemostasis, inflammatory cell infiltration, tissue regrowth, and remodeling. An important component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages, and lymphocytes (1). Neutrophils are the first leukocyte in the wound, entering within hours after initial injury. The...

Formalin Fixation in Wound Healing Studies

Formalin-fixed specimens are often tested as a component of a wound study, to evaluate the status of collagen biosynthesis. Collagen is the ultimate product of fibroblast and contributes to wound strength as early as d 3 postinjury. Collagen accounts for approx 70 of the dry weight of skin. The biosynthesis and modifications of collagen involves both intracellular and extracellular processing. Following synthesis on the ribosome, the a-chains are subjected to a number of enzymatic...

Migration Assay

Remove the EGM from the HMVECs, rinse with 10 mL of PBS, and refeed the cells with EBM + 0.1 BSA overnight. 2. The next morning, pipet 26 L of PBS into the bottom of the migration chambers to briefly rinse the cells. 3. Trypsinize the HMVECs and resuspend the cells at 1 x 106 cells mL in Dulbecco's Modified Eagle Medium (DME) + 0.1 BSA (you need approx 1.4 mL of cells per chamber, and no less than 1.3 mL). 4. Place 26 L of the HMVEC suspension into each well. A slightly positive meniscus should...

Freezing of HMVECs

To ensure maximum number of passages, it is recommended that the cells be split before they reach confluence (see Note 3). 1. To freeze, wash, trypsinize, quench, and centrifuge the cells as described in Subheading 3.1. 2. Remove the media via suction and add 1 mL of Cellvation 106 cells. 3. Transfer to 2-mL cryogenic vials. 4. Store the vials at room temperature for 30 min. 5. Place ampoules in an insulated container at -20 C for 60 min. 6. Remove the vials from insulated container and place...

Katherine M Malinda 1 Introduction

Angiogenesis, the process of new blood vessels forming from preexisting vessels, is an important feature in developmental processes, wound healing, and pathologic conditions such as cancer and vascular diseases. Owing to the importance of angiogenesis, a relatively simple and rapid in vivo method to determine the angiogenic potential of compounds is desirable to augment in vitro findings. One such quantitative method is the murine Matrigel plug assay, which can measure both angiogenesis and...

Epidermal Overexpression of BMP6 Inhibits Wound Reepithelialization

BMP-6 is strongly expressed in the developing murine epidermis, with mRNA levels falling after 6 d postpartum to a low level in adult skin (60). As such, it is closely associated with the most active phases of skin proliferation. To address further the role of BMP-6 in the skin, Blessing et al. (61) engineered transgenic mouse lines overexpressing BMP-6 in the suprabasal layers of the epidermis, using the keratin 10 promoter. Different lines with varied patterns of transgene expression showed...

Epidermal Overexpression of IP10 Delays Wound Repair

Interferon-y-inducible protein-10 (IP-10) is a chemokine that is detected at high levels in several chronic inflammatory conditions, including psoriasis. It is a member of the CXC family of chemokines and acts primarily in the recruitment of neutrophils and lymphocytes (reviewed in ref. 70). It is also one of a group of several chemokines that are upregulated following wounding, with an expression pattern that correlates well with recruitment of inflammatory cells to the wound site (71). To...

Immunohistochemistry

PAP pen (Kiyota Research Products, Mount Prospect, IL). 4. Phosphate-buffered saline (PBS), pH 7.4. 6. Normal mouse serum (Harlan Bioproducts, Indianapolis, IN). 7. Monoclonal rat anti-mouse CD31 antibody (clone MEC13.3 Pharmingen, San Diego, CA). 8. Biotinylated mouse anti-rat IgG antibody (Jackson Immunoresearch Laboratories, West Grove, PA). 9. Horseradish peroxidase-linked avidin-biotin complex (ABC-HRP) (Vector, Burlingame, CA). 10. Stock solution of diaminobenzidine (25 mg mL) (DAB)...

Determination of Wound Breaking Strength

A critical outcome of the wound repair process is restoration of the mechanical properties of tissue strength. Measurement of wound strength provides highly quantifiable estimates of the efficacy of the aggregate healing process. Determination of various individual components of the phases of healing can provide important insights about events operative during repair. However, if sufficient wound strength is not attained, the net effect may be wound failure. Factors that modulate wound repair...

Methods 31 Fetal Wounds

Autoclave all of the instruments to be used during this procedure. 2. Prepare a clean area of bench space and line with absorbent pads. 3. Prepare a second clean area of bench space, line with absorbent pads, and place a heat lamp nearby. 4. In a sterile fashion, fill a 3-mL syringe with sterile normal saline. 1. Weigh the animal on a scale. 2. Mix an anesthetic cocktail as follows 0.5 mL of acepromazine maleate (10 mg mL), 1.5 mL of ketamine HCl (100 mg mL), 1.5 mL of xylazine-20 (20 mg mL)....

Recommended Outcomes to Measure Healing After Thermal Injuries

A variety of macroscopic and microscopic outcomes can be used to measure healing of burns. Early indicators include infection based on the presence of erythema, purulence, or systemic indicators of infections such as fever. Microscopically, infection is defined by the presence of intradermal neutrophils containing bacteria. While this measure has a high degree of reliability (6,8,9), it does not necessarily indicate clinically relevant infections and may be overly sensitive (16). Fig. 2....

Mechanism for FPCL Contraction

All three in vitro models require translocation of collagen fibrils. With the free-floating FPCL model, translocation of collagen fibrils is by elongated cells. With the attached FPCL model, translocation of collagen fibrils is by the spreading of round cells. With the stress-relaxed FPCL model, translocation of collagen fibrils is through a process of cell contraction. In all three models, actin-myosin filament sliding of cytoskeleton microfilaments is involved. All three FPCL contraction...

Preparation of Total Cellular Protein from Isolated Wound Tissue

Paper towels, examination gloves, polypropylene conical tubes (50 mL). 2. Ultra Turrax, electric tissue homogenizer. 3. Proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) (Sigma) 100 mM in 70 EtOH. Store in the dark at 4 C. 4. Proteinase inhibitor leupeptin (Sigma) 1 mg mL in H2O. Store in aliquots at -20 C. 5. Protein homogenization buffer (stable when stored at 4 C) 137 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, pH 8.0, 10 (v v) glycerol, 1 (v v) Triton X-100. Immediately before use, add to a...

In Vitro Angiogenesis Assays see Note

In vitro assays of angiogenesis have several distinct advantages over in vivo assays. For example, assays have been developed for many of the individual phenotypic responses that are required for endothelial cells to form new vessels. In vitro assays are also attractive because the results can be quickly and accurately quantified. Examples of this type of quantification would include endothelial cell chemotaxis assays, cell proliferation, cell motility, matrix degradation, and tube formation....

Specimen Collection

Obtain biopsies of burned and unburned skin from patients at the time of debridement and eschar excision, prior to grafting. Skin samples are best obtained from the extremities or torso because these areas provide a good dermal layer for manipulation. Normal skin should not be taken from the face or genital area, to avoid cosmetic complications. The specimen obtained from the excised burned skin should include the margin between the burned and unburned skin or zone of stasis and the normal skin...

Analysis of Collagen Synthesis

Introduction The process of wound healing consists of an orderly sequence of events characterized by the specific infiltration of specialized cells into the wound site. The platelets and inflammatory cells are the first cells to arrive, providing key functions and signals needed for the influx of connective-tissue cells and a new blood supply. These chemical signals are known as growth factors or cytokines. The fibroblast is the connective-tissue cell responsible for the...

Harvesting of Abdominal Wall Tissue

Euthanize the mice by an appropriate method. 2. Perform dissection with a fine pair of curved scissors and rat-tooth forceps see Note 3 . 3. Wet the fur of the abdomen with alcohol to keep the fur from interfering with the dissection. 4. Grasp the skin and sc tissue with forceps and make a small cut through the skin, taking care not to invade the abdominal musculature. 5. Using the scissors with the curve pointing up, separate the sc tissue from the layer of muscle. Having removed the...

Data Analysis

If noninvasive, repeated measurements are made of wounds, data may be graphed as the average percentage of the surface area of the burns that has reepithelialized for each of the treatment groups vs the time after injury. The appropriate statistical test to determine whether there is a difference in the amount of healing among any of the groups at a specific time point e.g., 7 d after injury is the repeated measures analysis of variance followed by a post-hoc multiple comparison test such as...

Subcutaneous Wound Tissue Oximetry

Licox Tissue Oxygenation Catheter

Hunt and others began to develop techniques to measure average tissue oxygen from a single site in humans. Hunt developed the silicone tonometer for this purpose Fig. 7 . Silicone is freely permeable to oxygen. When a length of silicone tubing 1 mm OD is placed subcutaneously, the PO2 within the tonometer equilibrates with that of a cylinder of surrounding tissue, thus providing a mean wound tissue oxygen tension equivalent to the mean of the microelectrode-generated...

Keratinocyte Growth Factor

Epidermal Expression of a Dominant-Negative Keratinocyte Growth Factor Impairs Wound Reepithelialization Keratinocyte growth factor KGF , FGF-7 binds exclusively to a splice variant of FGF receptor 2 FGFR2IIIb , a transmembrane protein tyrosine kinase receptor that is present only on epithelial cells 7 . In previous studies, a strong upregulation of KGF expression has been observed after skin injury in mice and humans 8,9 , with mouse studies revealing KGF mRNA levels more than 150-fold...

Cacl2granulating Wound Pictures

It is absolutely critical that the bacterial collagenase used in this assay be pure and free of other, nonspecific proteolytic activity. Assurance assays such as the one that follows should be used. These assays are reprinted with permission from Worthington. See Worthington's Web site for additional information 0.05 M TES sulfonate buffer with 0.36 mM calcium chloride, pH 7.5. 4 Ninhydrin in methyl cellosolve. 0.2 M Sodium citrate with 0.71 mM stannous chloride, pH 5.0. Ninhydrin-citric...

Notes

It is important to maintain initial population input levels of gt 103 cells cm2 to prevent premature keratinocyte differentiation. Initiation of culture with clonal Fig. 5. Genetic modification of PAI-1 protein levels in wound-stimulated rat keratinocyte RK migration. RKs were transfected with non-insert-containing expression vector Rc CMV or the PAI-1 antisense Rc CMVIAP or sense Rc CMVPAI constructs. Cells were maintained in serum-free DMEM for 3 d prior to wounding. The extent percentage...

Dermovac Blistering Device

Commercially available disposable suction blister devices Dermovac blistering device Mucel Co., Nummela, Finland . These are manufactured of butene-styrene, single packed and gamma sterilized. The device contains five holes 6 mm in diameter. The inner diameter of the whole device is 40 mm and the outer diameter, 50 mm. The device is transparent, which makes it easy to observe the blister induction during blistering. It contains a 30-cm rubber tube, which can be connected to a vacuum pump see...

Rabbit Ear Chamber Model

Rabbit Ear Chamber

Healing has six main components inflammation, epithelialization, angio-genesis, contraction, collagen deposition, and resistance to infection. All are influenced by oxygen supply. None of them occur at very low oxygen tension. Collagen synthesis rises to a peak at about a Po2 of 250 mmHg. Resistance to infection peaks at about 600 mmHg. Each of the cells involved in wound healing, including epithelial, inflammatory, endothelial, and fibroblast, has a range of oxygen tensions at which it...