Anesthesia

Weigh the pig or piglet directly or by subtraction method (weight of pig and researcher minus researcher's weight). 2. Initially anesthetize the pig or piglet by im injection of ketamine and xylazine (20 mg kg of ketamine + 2 mg kg of xylazine) mixed in one injection. 3. After the animal is manageable, transfer it to an operating table. 4. Maintain anesthesia with inhalation of 1.5 isoflurane at a flow rate of 1 L O2 min (see Notes 6 and 7). 5. Give a prophylactic im injection of penicillin G...

Embedding Isolated Wound Tissue for Histology

Tissue Tek cryomold intermediate, disposable vinyl specimen molds (15 x 15 x 5 mm). (Miles, Diagnostic Division, Elkhart, IN) (see Fig. 1). 2. Tissue Tek , O.C.T. compound, embedding medium for frozen tissue specimens (Sakura Finetek, Torrance, CA) (see Fig. 1). 3. Polyvinyl difluoride (PVDF) membrane (Immobilon-P). (Millipore, Bedford, MA) (see Fig. 1). 4. Single-use, disposable scalpel (see Fig. 1).

Notes

This step is critical in the procedure it is important that the ethanol does not absorb any water. The bottle should be kept closed between uses, or use a fresh bottle each time. 2. Our experimentation with the gene gun has used a wound design that accommodates the skin field that is targeted for transfection. This occupies a circular area (if the gun is perpendicular to the target) of about 2 cm in diameter. There is a reasonably uniform distribution of particles within this field, with some...

Introduction

The development of strategies for the treatment of angiogenesis-dependent diseases has been greatly aided by the development of in vivo models of angiogenesis (1). These bioassays provide investigators with tools to visualize vessel architecture and function and to analyze and manipulate various steps in the angiogenic response. Some of the more widely used model systems include the iris and avascular cornea of the rodent eye (2-4), the chick chorioallantoic membrane (5), the hamster cheek...

Animals Anesthesia and Euthanasia see Note

For anesthesia, mix an 8 24 dilution of halothane in olive oil in a 50-mL conical tube (see Note 3). 2. Introduce the agent into the lower chamber of a glass desiccant jar, and allow the fumes to saturate the chamber for a few minutes (see Note 4). 3. Put the mouse into the top chamber and monitor its response to the anesthesia (see Note 5). Check the animal's response to the anesthesia and proceed with wound tissue isolation as outlined in Subheading 3.2. 4. For euthanasia, either a desiccant...

References

Ellis, H. (1982) The causes and prevention of intestinal adhesions. Br. J. Surg. 69, 241-243. 2. Kovacs, E. J. and DiPietro, L. A. (1994) Fibrogenic cytokines and connective tissue production. FASEB J. 8, 854-861. 3. Myllarniemi, H., Frilander, M., Turunen, M., and Saxon, L. (1966) The effect of glove powders and their constituents on adhesion and granuloma formation in the abdominal cavity of the rabbit. Acta Chir. Scand. 131, 312-318. 4. Frazier-Jessen, M. R. and Kovacs, E. J. (1993)...

Casting Free Floating FPCL

Release the cultured cells from monolayer culture by trypsinization, suspend in culture medium, and count. 2. Mix the appropriate number of cells, tissue culture medium, and serum as outlined in Table 1. The tissue culture medium should be a room temperature. The final concentration of FBS in the FPCL will be 10 (v v). Add the indicated amount of collagen solution, mix rapidly, and pour into the bacterial culture dish (see Note 15). For the original free-floating FPCL, the optimal cell density...

Lucigenin Dependent Chemiluminescence Assay

This assay has the advantage of being unaffected by secreted molecules with the ability to oxidize cytochrome-c such as H2O2, NO, and ONOO- (8). Lucigenin-dependent chemiluminescence is not produced by the myeloperoxi-dase reaction. Data generated can be expressed either as instantaneous light intensity (mV), total light within a given time period (mVs), or as a rate of light intensity (mVs-1) (Fig. 3). 1. Suspend 5 x 106 cells in 0.25 mL of HBSS containing FBS, add 0.25 mL of lucigenin, and...

Animal Handling and Injection of Talc

Pick up a mouse by the base of the tail with the right hand. Using the thumb and index finger of the left hand, grasp the skin at the nape of the mouse's neck, near the shoulder blades. Pull the skin so that it is taught and the mouse cannot move its head or front legs. 2. To minimize the chance of injecting into peritoneal contents, tilt the mouse's head downward to shift the bowel superiorly and inject in the inferior aspect of the peritoneal cavity. Wipe the surface of the skin at the...

Tissue Repair in Models of Diabetes Mellitus

Diabetes mellitus is a major cause of impaired tissue repair. Patients with this disease not only have a propensity to develop wounds, but when they do, they tend to have difficulty healing those wounds. Simple wounds often become chronic and infectious wound complications are not uncommon. Unfortunately, the amputation rate for diabetics is much higher than for the nondiabetic population. Because healing problems are so common and devastating, several models of tissue repair have been...

Hair Removal

To ensure uniform contact of the metal bar with the skin, the hair is removed prior to creating the burns. Kaufman et al. (5) recommend removing the hair with clippers and a depilatory cream at least 24 h before inflicting burns, since clipping and depilation may produce skin edema. However, after experimenting with a variety of depilatory creams, hair removal waxes, and clippers, we have noted that hair removal with electric clippers immediately before the burns is most simple and does not...

Adam J Singer and Steve A McClain 1 Introduction

Not only are burns a leading cause of accidental death, but they also result in considerable morbidity and disfigurement leading to significant functional and social impairment. Fortunately, most burns are minor and relatively small. As a result, research efforts have largely focused on the local care of burn wounds. Because of moral and ethical considerations, the use of human skin for in vivo experiments is limited. Over the years researchers have used a variety of animal models as well as in...

Retrieval and Analysis of Sponge Samples

When excising the pelt great care must be taken so that the sponge granuloma is not penetrated, which could lead to a loss of wound fluid (see Note 1). Furthermore, excessive bleeding into the sponges should be avoided because this alters the cellularity of the sponges and the characteristics of the wound fluid. After the animal is euthanized, harvest the sponges. There are several ways to harvest the sponges. All sponges should be retrieved with minimal handling and trauma so that there is...

Data Analysis see Note

Mean intensities are calculated for each gene in each group (e.g., hypertrophic scar, scar, and skin). Ratios of the mean intensities of hypertrophic scar to scar, hypertrophic scar to skin, and scar to skin can be generated such that a ratio > 1 indicates upregulation and a ratio < 1 indicates downregulation of any particular gene, respectively. Since the distribution of ratios is positively skewed, ratios can be log transformed to generate a Gaussian distribution. Determination of...

Method A Radiolabeling Tissue Biopsies

Obtain samples of tissues to be analyzed by standard surgical biopsy technique (11,16,17). Basically, clean the area to be biopsied of any foreign material, or if hair is present, shave the area. Then use appropriate anesthetics if needed, clean the area with a skin cleanser, dry and use a skin biopsy device (trephine) to obtain a sample of tissue ranging from 3 to 6 mm in diameter. 2. Place the biopsy in incubation medium on ice (minus the isotope) and rinse to remove excessive blood. 3....

Introduction of Sepsis

Use either the burn wound seeding with P. aeruginosa or CLP, but not both. 3.3.1. Burn Wound Seeding with P. aeruginosa 3.3.1.1. Preparation of P. aeruginosa This entire portion of the protocol should be done prior to wounding of the animal. Fig. 2. Anesthesia is administered intraperitoneal into the left lower quadrant of the mouse's abdomen, but high enough to avoid injecting into the bladder. The needle should be inserted only as far as is required to enter the peritoneal cavity. Fig. 2....

Superoxide Release

This is a well-accepted and straightforward protocol for quantification of superoxide release from viable neutrophils or macrophages as determined by reduction of ferricytochrome-c (6). Inclusion of SOD lends specificity to this assay regarding demonstrating that reduction of ferricytochrome-c is owing to superoxide production and not spurious reductants. SOD catalyzes the conversion of superoxide to H2O2 and water. Fig. 4. O2 consumption by peritoneal lavage or wound-derived macrophages. Cells...

Sample Preparation

Weigh 50 mg of gold microcarriers into a 1.5-mL microcentrifuge tube. 2. Add 100-200 L of 0.1 M spermidine to suspend the particles. 3. Briefly vortex and sonicate the mixture at room temperature to break up any gold clumps. 4. Add the required volume of plasmid DNA to produce a loading ratio of 2 1 (e.g., 100 mg DNA 50 mg of gold). 5. Mix the DNA (2 g mg of gold), spermidine, and gold by vortexing for 5 s. 6. While vortexing, add 2.5 M CaCl2 dropwise to the mixture to equal two times the...

Screening Putative Antiangiogenic Compounds

Thaw Matrigel on ice overnight at 4 C. 2. Mix Matrigel on a vortex place on ice (see Note 1). 3. Pipet equal amounts of Matrigel for each test condition in separate 14-mL tubes on ice. Each mouse injection requires 0.5-1 mL, three or four mice for each condition (see Notes 2 and 3). 4. Add 150 ng mL of bFGF to all tubes except the one used for the negative control containing Matrigel alone. 5. Equalize the volumes with cold PBS so that after putative antiangiogenic ogenic substances have been...

And Reduced Inflammation

TGF-Ps and activin, both of which regulate key cellular functions during cutaneous wound repair, are known to require the nuclear transcriptional activators Smad2 and Smad3 for their intracellular signaling functions (48-50). Smad2 and Smad3 proteins are recruited to ligand-bound TGF-P and activin receptor complexes, where they are phosphorylated by the type I receptor. The phosphorylated Smads 2 and 3 undergo a conformational change, which allows them to bind to cytoplasmic Smad4, after which...

David T Efron and Adrian Barbul 1 Introduction

All wounds progress through a well-described series of events that ultimately result in healing. Cellular, biochemical, and molecular changes occur, each characterizing unique phases of the healing process. Scientists have long desired to study both the global process of healing and its individual phases or steps. Experimental models of healing have been used extensively to better understand the process of wound healing. There are models that preferentially reflect the fibroplastic or the...

General Materials

Three or four mice are needed for each test group. 2. Additional cages and supplies, one for each test group. 3. Matrigel (Collaborative Biomedical, Bedford, MA) store at -20 C. Enough will be needed to inject 0.5 or 1 mL per mouse. 5. 25-gauge Needles (Monoject). 6. 14-mL Falcon round-bottomed tubes ( 2059) (Becton Dickinson, Rutherford, NJ). 7. 10X Phosphate-buffered saline (PBS) (Biofluids, Rockville, MD) diluted to 1X in sterile, distilled H2O. 8. Endothelial cell...

Embryo Studies Address Roles for Intermediate Filaments in Wound Repair

In a further study into the mechanisms behind the perfect healing that occurs in the embryo, Eckes et al. (78) wounded midgestational mouse embryos lacking the intermediate filament protein vimentin. Embryonic day 11.5 mouse embryos are capable of healing an excisional hind leg amputation wound within 24 h (79). Another study, using the same model, had revealed that one of the major intermediate filaments in early embryonic skin, keratin 8, was not essential for normal embryonic repair (80)....

Preparation of Conditioned Medium

Place the collected wounds into culture dishes filled with sterile PBS to prevent drying. 2. Mince the wound tissue in a sterile 100-mm tissue culture plate with a scalpel. 3. Transfer the minced wound tissue with the scalpel into the wells of a 12-well plate filled with 1 mL of serum-free DMEM. 4. Incubate the minced tissue in the media in a humidified incubator at 37 C in 5 CO2 for 24 h. 5. Transfer the conditioned medium with a sterile pipet tip into a microtube. 6....

Info

Use of gene gun to validate growth factor action. The gene gun was used to propel 0.5 mg of plasmid DNA from an expression vector for P-galactosidase (control) or a constitutively active form of TGF-P1 (32) into separate sites on the ventral surface of a transgenic mouse bearing the firefly luciferase gene under control of the COL1A2 enhancer promoter (53,54). At the indicated time points, transgene activity was visualized as described in the legend to Fig. 2. The images confirm that...

Determination of Amino Acids see Note

Redissolve the dry residues from the hydrolysates in 240 L of 0.1 M HCl. Close the Pyrex tubes with Parafilm and stand at 4 C for 6 h with occasional vortex mixing for complete dissolution. Centrifuge the hydrolysates at 4000g for 10 min. Pipet triplicate 12- L aliquots and triplicate 60 L aliquots from the supernatant of each hydrolysate into polypropylene autosampler tubes, and add 20 L of the 2.25 M solution of 3-methoxy-DL-tyrosine or L-citrulline. Completely dry the polypropylene...

Kirwin M Providence Lisa Staiano Coico and Paul J Higgins 1 Introduction

Gene knockout studies and analysis of physiologically relevant in vitro models have clarified basic mechanisms in the tissue response to injury (1-5). Fundamental to this process is the genetic reprogramming required for conversion of normally sedentary cells to an actively motile, invasive phenotype (5,6). It is difficult, however, to identify important events among the diverse changes associated with the initiation and maintenance of migration or the acquisition of plasticity in a specific...

Detection of Reactive Oxygen Intermediate Production by Macrophages

Reichner 1. Introduction The reparative phenomena that follow sterile tissue injury are an ordered sequence of partially overlapping events and include the macrophage as a key cell type in orchestrating the process of repair. Several studies suggest that the wound-derived macrophage acquires a functional phenotype that is congruent with its specialized role in repair, and that this phenotype is determined by factors within the wound milieu. This phenotype...

Measurement of Dried ePTFE Tubes

Engrave the Pyrex 50-mm glase tubes 6 x with sample identification, heat the tubes in a gas flame heat, and then cool in a desiccator before use. Spike each ePTFE tube section with a thin cannula, hold close to the jaws of a slide caliper, and measure its length with a 0.1-mm working tolerance (see Note 9). Place each ePTFE tube section on a clean objective slide and divide into four pieces. Tare weigh each Pyrex glass tube on a microbalance and then weigh it with the ePTFE tube pieces...

Incorporation of Endothelial Cells into Scaffolds and Transplantation into SCID Mice

Just prior to transplantation, resuspend 1 x 106 naive HDMECs, HDMECs over-expressing Flag-tagged Bcl-2, HDMECs overexpressing AP, or empty vector controls in 36 L of a 1 1 mixture of EGM-MV growth factor-reduced Matrigel and allow to adsorb into the scaffolds (see Notes 1 and 2). 2. Incubate the loaded scaffolds for 30 min at 37 C to allow the gelation of the Matrigel (see Note 3). 3. Anesthetize male SCID mice with ketamine and xylazine and implant two scaffolds subcutaneously in the dorsal...

Immunostaining

Coplin or other staining racks and jars. 3. Whatman 1 qualitative filter papers. 4. Modified Harris hematoxylin solution (Sigma, St. Louis, MO). Store at room temperature and filter through filter paper prior to counterstaining. 5. 10X Phosphate-buffered saline (PBS) 18.6 mM NaH2PO4, 84.1 mM Na2HPO4, 1.5 M NaCl pH 7.4 in dH2O. Store at room temperature. 6. 1X PBS Dilute 10X stock with dH2O. Store at room temperature. 7. PAP Pen (Kiyota International, Elk Grove Village, IL). 8. Kim wipes...

Procedure B Analysis of Procollagen Secretion by Slab Gel Electrophoresis of Radiolabeled Proteins in Culture Medium

Louis, MO) Make fresh in DMEM at 100 M. 6. 3,4-3H -Proline (Amersham, Piscataway, NJ). 11. Molecular weight marker (cat. no. RPN756 Amersham). 12. 1 M Sodium salicylate enhancing solution. 13. X-ray film (Hyperfilm MP, cat. no. RPN1677L Amersham).

Angiogenic Assay

Grow SVEC4-10 cells to near confluence in DMEM. 2. Thaw Matrigel on ice at 4 C overnight (see Note 5). 3. Place a 48-well plate on ice. 4. Coat the wells of a 48-well plate with Matrigel (150 L well). Use chilled pipet tips (see Note 6). 5. Place the Matrigel-coated plate in a tissue culture incubator for 30-60 min to polymerize the gel. 6. Collect the cells by trypsin-EDTA treatment. Neutralize the trypsin with DMEM containing 10 serum. 7. Transfer the cells into a 15-mL tube and spin at 700g...

Induction of Burn Wound

Once the mice are adequately anesthetized, shave the dorsum with the electric clippers to ensure even burn wounding (Fig. 3). 2. Place the mouse on its back in a template constructed of plastic and a metal screen (Fig. 1A,B), so that its back is directly over the screen. Immerse mouse and template together into a 100 C water bath for 8 s (Fig. 1C). The mouse and template should be far enough in the water so that the portion of the mouse's back that is exposed by the screen is entirely in...

Preparation and Sterilization of ePTFE Tubes

Wear gloves and work on a clean table. 2. Cut the ePTFE material into pieces 4-10 cm long (see Note 2). 3. Tie a 3-0 nylon suture to one end of the ePTFE tube after perforating the material with the suture approx 2 to 3 mm from the end. Leave at least a 16-cm length of suture from the ePTFE (Fig. 3). 4. Double-pack the ePTFE tubes in sterilization pouches containing the appropriate number to be used for each subject. 5. Sterilize at 120 C for 20 min.

Preparation of Sponges

PVA sponge material is purchased in sheets. Some companies precut the material to a desired thickness for rodents 2 to 3 mm works best, but up to a 5-mm thickness can be used in rats. The porosity of the material is variable and can influence the ingrowth of granulation tissue. Pore sizes of 90-120 permit optimal granulation tissue harvest. Different sheet lots vary greatly in thickness and porosity, and these parameters can have significant consequences on the results and reproducibility of...

Ill

2Hr 4Hr 6Hr 12 Hr 24 Hr 28 Hr Time post wound Fig. 3. Kinetics of monolayer wound repair in rat keratinocyte system. Scrape injury to confluent cultures of rat keratinocytes, maintained in serum-free DMEM for 3 d prior to creation of wound with a P1000 pipet tip (see Subheading 3.1.), initiates a repair response that culminates in complete trauma site healing within 28 h. Data plotted are the mean SD of measurements from three independent experiments. 5. Place the culture dish on the microscope...

Quentin E H Low and Luisa A DiPietro 1 Introduction

Angiogenesis occurs during embryonic development and in adult tissues (1). In developmental angiogenesis, the primitive vascular network formed during neovasculogenesis matures to form the circulatory system. In the adult animal, physiological angiogenesis occurs in the ovaries during the menstrual cycle and in tissue repair. Pathological angiogenesis is observed in many diseases, including inflammatory diseases, diabetic retinopathy, and cancer (2,3). Because angiogenesis plays such crucial...

Fibroblast Growth Factors

FGFs comprise a growing family of structurally related polypeptide growth factors, currently consisting of 22 members (reviewed in ref. 2). During embryogenesis, FGFs play key roles in regulating cell proliferation, migration, and differentiation. In adult tissues, FGFs have a diverse range of effects, including mediating angiogenesis, and neuroprotection, in addition to their stimulatory effects during wound repair (reviewed in ref. 3). FGFs transduce their signals through four high-affinity...

Creation of Burn Injury

Medical Documentation Thermal Burns

Trim dorsal skin hair with an electric clipper. Remove hair shafts using a commercially available hair depilatory. We use Nair applied in a thick layer evenly covering the hair stubble (see Note 8). 2. After 30 min, completely remove the depilatory agent from the skin by several washings and scrubbing with coarse gauze (see Notes 9 and 10). 3. Dry the skin thoroughly with gauze. 4. While the depilatory is working, prepare the template block by heating to 70 C in a constant temperature water...

Rationale for PVA Sponge Model

The PVA sponge model belongs to the class of dead-space models used for studying granulation and reparative tissue ingrowth. As opposed to the steel wire mesh cylinder model, it does not have a lag phase and tissue grows within its interstices shortly after implantation. The model is best used for acute studies because it begins to elicit foreign body reaction with giant cell accumulation and fibrosis after about 2 wk in the rat and 4 wk in the mouse. Within these time parameters, it remains a...

Full Thickness Excisional Wound Model

Shave the anesthetized mice on the dorsal side. 2. Lift up the skin longitudinally along the middle of the back of the mouse by grasping it between two fingers below the neck and just above the base of the tail. The skin will naturally form a double layer as it is lifted up and away from the body. 3. Place the mouse on its side so that the folded skin rests on a firm surface (see Note 6). 4. Using a 3-mm dermal punch, create one full-thickness punch wound on either side of the midline by...

Modifications of Bells Model

Experimentation over the years has generated numerous modifications of the FPCL from that first introduced by Bell and his colleagues (1). Bell's dermal equivalent was a free-floating FPCL that contained 50,000 human dermal fibroblasts in 1.25 mg of native soluble collagen and culture medium supplemented with fetal bovine serum (FBS) per milliliter of lattice. The entire mixture was contained in a bacteriology dish, and FPCL contraction was determined by following the reduction in the diameter...

Screening Putative Angiogenic Compounds

Thaw Matrigel on ice overnight at 4 C. At no time should Matrigel be warmed to room temperature during handling. 2. Mix Matrigel on a vortex place on ice (see Notes 1 and 2). 3. Pipet equal amounts of Matrigel for each test condition in separate 14-mL tubes on ice. Each mouse injection requires 0.5-1 mL with three or four mice for each condition (see Notes 2 and 3). 4. Tubes should be included for the positive and negative controls. Negative control is Matrigel alone positive control contains...

Analysis

Light microscope for counting stained cells (e.g., Leitz Laborlux S Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany). 2. Image analysis system, to assess wound size, comprising the following components a. Microscope with camera mounting (e.g., Zeiss Axioskop 20 with phototube Carl Zeiss, Thornwood, NY). b. Charge-coupled device camera and camera control unit (e.g., DEI-750D Optronics, Goleta, CA). c. A color video monitor (e.g., Sony Trinitron PVM-14N1U Sony, San Jose, CA). 3. Computer...

Studies with Lower Mammals

Experimental studies of wound breaking strength have commonly utilized guinea pigs, rats, and mice. These animal models of healing do not totally replicate healing in humans. Animals are not as susceptible to wound infections as are humans. Scar formation is significantly less in these animals and is for all practical purposes not problematic. Histologically, a thin sheet of skeletal muscle in the dermis at its boundary with the subcutis, known as panniculus carnosus, is extensively distributed...

Preparation and Transplantation of Isolated Keratinocytes

Obtain keratinocytes from the outer root sheath of human hair follicles from freshly plucked anagen hair follicles. Autologous, but also allogenic, keratino-cytes can be used. 2. Incubate the follicles in trypsin-PBS solution for 1 h at 37 C. 3. Mechanically separate the cells into solution using sterile pipets. 4. Suspend a total of 1 to 2 x 106 keratinocytes in 5 L of PBS. 5. Apply one drop (about 200,000 cells) with a micropipet into the center of each wound of the skin organ culture 1 d...

Analysis of Tissue

When viewing the peritoneal wall under a microscope, both the mesothelium and adventitia may have very little connective tissue, making it difficult to determine which side is the mesothelium. The mesothelial side is distinguished by a smooth surface, whereas the adventitia will have very discrete wisps of sc fatty tissue present. Examination of the luminal surface of the abdominal wall musculature reveals the mesothelial surface. This is a simple squamous epithelium, which will be difficult to...

Choosing Burn Site

Our model uses a 6.25-cm2 square rigid aluminum bar applied directly to the skin. Site selection is important in order to ensure uniform burn injuries. We have found the thoracic paravertebral zone especially suitable in pigs. When created directly over the vertebral spinous processes or too far laterally over the ribs, it is likely that the burn will be nonuniform. In addition, as the ventral abdominal undersurface is approached, we have found that burns do not heal as well as those adjacent...

Aspects of Epidermal Regeneration

A partial-thickness skin injury can be simply defined as a wound that extends completely through the epidermal layer and only partially through the dermal layer. Epithelial cells that line hair follicles, sweat glands, or sebaceous glands extending into the deep dermis remain viable after a partial-thickness injury. In large partial-thickness injuries, these epithelial cells proliferate and migrate onto the surface of the wound, where they differentiate into epidermal keratinocytes (Fig. 1B)....

Choosing Thermal Insult

Two types of thermal insults can be used to create burns. In the scald burn model, heated or boiling water is circulated over the injured area. The major advantage of this method is the ability to cover uniformly an entire area of skin regardless of its uniformity and underlying surface. The major disadvantage is that this model is also technically more challenging than the contact model and exposes the research team to the risk of burning themselves. By contrast, the contact model of injury...

C

ACCELL Gene Delivery Device (Agracetus, Inc.) Supercoiled Gold Particles Teflon Tube Coated with Supercoiled Gold Particles Teflon Tube Coated with Fig. 1. Particle bombardment instrument for introduction of cDNA into skin. (A) Commercially available Helios gene gun (B) design features of Helios device (C) principles of particle-mediated gene transfer. Fig. 1. Particle bombardment instrument for introduction of cDNA into skin. (A) Commercially available Helios gene gun (B) design features of...

Analysis of Human Microvessels in SCID Mice

Immunolocalization of VCAM-1, ICAM-1, CD31, CD34, Flag, 1. Deparaffinize histological sections of polymer implants, and process for antigen retrieval by pressure cooking the sections at 120 C for 2 min in citrate buffer. 2. To prepare the sections for immunostaining with VCAM-1 and ICAM-1, first block the sections for 5 min with 10 rabbit serum in PBS, then with Sniper for an additional 5 min. 3. Incubate the samples overnight at 4 C with polyclonal goat anti-human VCAM-1 or polyclonal...

Incisional Wound Healing

A clean, uninfected incision, surgically reapproximated, causes the least amount of epithelial and connective tissue cell death and limits the extent of epithelial basement membrane disruption. As does any type of injury, incisional wounds alter the homeostatic state of the organism and trigger a sequence of events that constitutes three typical pathological phases. In the acute inflammatory phase, infiltrating phagocytes protect the wounded tissue from infection and remove necrotic debris....

Particle Mediated Gene Therapy of Wounds

Eming, and Jayasri Dasgupta 1. Introduction Advances in molecular biology and the understanding of the molecular basis of many diseases have provided tools necessary for a new approach to the treatment of both inherited and acquired diseases. This approach, called gene therapy, was initially focused on the correction of inherited diseases for which no therapeutic approaches were available (1,2). However, it is now clear that the technique of gene therapy can...

Julia M Stevenson Richard L Gamelli and Ravi Shankar 1 Introduction

Despite major advances in the treatment of critically injured burn patients, sepsis remains a major obstacle to recovery (1-4). In fact, sepsis is a leading cause of morbidity and mortality in this patient population (5-6). Those who survive the initial injury become increasingly susceptible to septic complications during the second week of hospitalization. The majority of these patients show a severe depression of their innate immunity as evidenced by T-cell suppression and functional deficits...

Cohort Specific Evaluation of mRNA Abundance

Harvest cells immediately adjacent to the wound (termed edge isolates) by pushing the wide end of a P1000 pipet tip (i.e., the end opposite that used to create the original scrape injury) along the existing wound tract (see Note 9). This displaces cells directly at, and 5 mm from, the wound edge time-lapse microscopy estimates that this procedure recovers the major fraction of the motile cohort. 2. Carefully aspirate the medium and subsequently collect the scrape-released cells by centrifuging...

Future Developments

Molecular medicine holds considerable therapeutic promise and the potential range and versatility of gene therapy in tissue repair, particularly the use of particle bombardment outlined herein. The particle-mediated acceleration strategy in tissue repair offers several advantages over other in vivo transfec-tion methods including adenoviral vectors or liposomal delivery. First, the transfection system is a simple, standardized, and reproducible technology for cutaneous applications. Second, the...

Richard Grose and Sabine Werner 1 Introduction

Wound healing is a highly ordered and well coordinated process that involves inflammation, cell proliferation, matrix deposition, and tissue remodeling (1). During the past few years, a series of candidate key players in the wound-healing scenario have been identified. These include not only a variety of different growth factors and cytokines, but also molecules that are involved in cell-cell and cell-matrix interactions, and proteins responsible for cell stability and cell migration. In most...

Hydrolysis see Note

Place the Pyrex glass tubes with their contents in the reaction vial. Place 500 L of concentrated HCl and a 1-mm3 crystal of phenol in the bottom of the reaction vial outside the Pyrex glass tubes and cap the vial (see Note 11). Connect the connecting piece of the valve cap to one branch of a three-way valve. Connect the other branches to a vacuum and nitrogen, respectively. Evacuate the reaction vial for 10 s, and then fill with nitrogen gas for 10 s. Do this evacuation flush cycle two more...

RNA Extraction

Homogenize frozen skin samples (5-10 g) without further dissection in liquid nitrogen using a mortar and pestle. It is essential to homogenize under liquid nitrogen until the sample resembles talcum powder. Homogenization on the day of tissue collection is ideal (see Notes 1 and 2). 2. Extract RNA using Tri-Reagent (5 cc g of tissue) according to the manufacturer's protocol. Repeat the final wash in 70 EtOH twice to ensure that no salt remains in the sample. 3. Precipitate RNA in 75 ethanol and...

Northern Blot

Purescript RNA isolation kit (Gentra, Minneapolis, MN). 2. RNA denaturation buffer 1X MOPS buffer, 6.5 formaldehyde, and 50 formamide. 10X MOPS buffer consists of 0.4 M 3-N-morpholino-propanesulfonic acid, pH 7.0 0.1 M sodium acetate and 0.01 M EDTA-Na2 (see Note 4). 3. Agarose formaldehyde gels 1.2 agarose 1.1 formaldehyde 1X MOPS, pH 8.0. 4. RNA gel-loading buffer 1 mM EDTA, pH 8.0, 0.25 bromophenol blue, 0.25 xylene cyanol, 50 glycerol. 5. Ethidium bromide (EtBr) (0.5 g mL in water). 6. RNA...

Procedure C Screening of Human Intestinal Smooth Muscle Cells for Smooth Muscle Specific Cytoskeletal Proteins

To ensure that the clone of cells isolated from the muscularis is phenotypi-cally true, it is important that screening for the expression of smooth muscle- specific proteins be performed with each isolate. Using the aforementioned isolation and culture techniques, we find that less than 1 in 20 isolates will not express smooth muscle-specific proteins. Those isolates are then discarded. The technique involves Western blotting of human intestinal smooth muscle (HISM) cell lysates for a-smooth...

Immunodeficient Mice Lacking TGFP 1 Show Retarded Healing

To try to dissect the TGF-P 1-dependent wound-healing defects from the effects of severe inflammation, Crowe et al. (46) crossed TGF-P 1 null mice onto the immunodeficient Scid- - background (46). Scid- - mice lack T- and B-cells and therefore do not have the machinery to mount the large inflammatory response seen in nonimmunocompromised mice lacking TGF-P1 (40). In contrast to what was predicted, the absence of inflammation in TGF-P 1- -Scid- - mice resulted in a major delay in all the primary...

Overexpression of Activin A in Basal Keratinocytes Stimulates Wound Repair

Activin A, a TGF-P superfamily member, is a homodimeric protein comprising two activin PA monomers connected by disulfide linkage. That it might play a role in the skin was first suggested by knockout mouse studies, of activin PA (56) and of the activin antagonist follistatin (57), which both showed clear phenotypes in hair follicle development. Further studies from our laboratory demonstrated activin PA to be strongly induced following wounding (58). Fig. 1. Wound-healing phenotype of...

Methods

Homogenize tissue specimens used for analysis in sterile 1X PBS in the presence of antiprotease buffer (see Note 1) at 4 C using a tissue tearor until the tissue is fully homogenized. Approximately 0.2 g of tissue can be homogenized in 1 mL of antiprotease buffer. This may require some adjustment depending on the tissue being analyzed. We routinely homogenize mouse or rat lungs in 1 and 3 mL, respectively. 2. Sonicate (microtip) the homogenized tissue for 30 s at 50 duty cycle, output set at...

Procedure A Isolation and Culture of Human Intestinal Smooth Muscle Cells

Dulbecco's modified Eagle's medium (DMEM). 3. Nystatin solution 10,000 U mL of stock. 4. Penicillin streptomycin solution (Pen Strep) 10,000 U mL of stock. 5. Supplemented DMEM-0 Each 50 mL of DMEM contains 2.5 mL Pen Strep stock and 0.25 mL of Nystatin stock. 6. Sterile scissors and forceps. 7. Sterile filter paper Whatman 1 cut in a circle to fit on the stage of a tissue slicer. 8. Tissue slicer (cat. no. 6727C10 Thomas Scientific, Swedesborough, NJ). 9. Collagenase solution 0.5 mg mL culture...

The Particle Mediated Gene Transfer Approach

A novel approach for gene transfer is a physical means of gene delivery by the bombardment of cells tissues with DNA-coated particles or microprojectiles (25-28). Originally, particle-mediated gene transfer was developed by Sanford and colleagues in 1987 to deliver genes to plant cells using gunpowder acceleration (28). Advancing the technique, an electrical discharge and then high-pressure helium replaced gunpowder as the particle propellant for most particle-mediated devices (27,29)....

Strategies for Characterizing Human Microvessels in the SCID Mouse

To confirm that the endothelial cells populating the microvessels within the implants are of human origin, several approaches can be used to verify the identity of the endothelial cells lining the microvessels. We recommend performing immunostaining with anti-human CD31 and anti-human CD34 (Fig. 4) antibodies. These antibodies only react with endothelial cells from microvessels located inside the implant and not with mouse endothelial cells that populate vessels that have infiltrated the fascia...

Microarray Methodology

Prescan DNA microarray membranes from the same lot (GF211 Research Genetics) to ensure that all cDNA data points are present to exclude false positives and negatives. 2. Boil the membrane in 0.5 SDS in DEPC H2O with for 5 min. 3. Incubate the membrane for 2 h at 42 C in a hybridization oven in a roller bottle containing 5 mL of microhybridization solution (1 g L), 5 L of Cot-1 DNA (1 g L), and 5 L of poly dA. 4. Add the entire purified sample of 33P -labeled probe to the prehybridization...

Preparation of Transfectant Grade DNA

Large-scale preparations of plasmid DNA are obtained using Qiagen kits. 1. Inoculate LB medium (10 mL) containing ampicillin with one colony of expression vector-transformed (16) E. coli bacteria and incubate overnight at 37 C with moderate shaking. 2. Add culture to 500 mL of LB medium containing ampicillin and grow to an OD600 of 0.8. 3. Add chloramphenicol to a final concentration of 1 mg mL for overnight incubation at 37 C with shaking to amplify plasmid copy number and inhibit bacterial...

David L Williams and I William Browder 1 Introduction

Wound healing is a physiological process that is essential to the reestablishment of homeostasis (1-4). It is generally accepted that wound repair is an immune-mediated event (5) that involves a number of cell types such as macrophages, neutrophils, fibroblasts, endothelial cells, and keratinocytes, along with a complex and exquisitely choreographed interaction of wound growth factors (1,2,4,6,7). While there have been significant advances in our understanding of wound repair, there are still...

Cell Culture and Microscopy

An established line of newborn rat epidermal keratinocytes is used in the monolayer denudation injury model (15) primary keratinocyte cultures derived from pup skin produce equivalent results (see Note 1). 2. Dulbecco's modified Eagle's medium DMEM 1 g of D-glucose L, from Gibco-BRL or Cellgro) supplemented with 10 (v v) fetal bovine serum (FBS), L-glutamine (20 mM), streptomycin (1 mg mL), and penicillin (1000 U mL) is used in all experiments (see Note 2). 3. Hank's balanced salt solution...

Tissue Sectioning

Prepare 7- im sections extending transversely across the central cryopreserved rabbit cornea from frozen tissue blocks maintained at -20 C with a cryostat (see Note 4) and place on Superfrost plus slides (see Note 5). These slides are important for tissue adherence during staining. 2. Freeze the slides with sections at -85 C until use. 3. Just prior to staining, fix cryosections with acetone at -20 C for 10 min and then wash with two changes of PBS. 4. Place formalin-fixed mouse eyes in molds...

Excisional Wounding

Anesthesia solution Ketavet (ketamine hydrochloride), 100 mg mL solution, stable to date as given by the manufacturer (Pharmacia & Upjohn GmbH, Erlangen, Germany) and Rompun (xylazine-hydrochloride), 20 mg mL solution, stable to date as given by the manufacturer (Bayer, Leverkusen, Germany). Immediately prior to use, add 800 L of Ketavet and 500 L of Rompun to 25 mL of sterile Dulbecco's phosphate-buffered saline (PBS) using a sterile 50-mL polypropylene conical tube. Mix carefully by...

Monitoring of Wounds and Analgesia

Assess wounds during treatment and dressing changes for signs of infection, including erythema around the wound, purulent discharge, or foul odor. The rate of infection is very low (< 1 ) in this model. If an infection does develop, treat the wound aggressively with topical antibiotics and exclude it from data analysis. 2. Second-degree partial-thickness burns can cause significant pain during the first few hours after injury, but this usually subsides within 12-24 h. The animal will,...

Macroscopic Description of Burns

Initially the deep partial-thickness burns have a pale white appearance with a surrounding rim of erythema that subsides within several minutes (Fig. 2). Within the next few days the burns become red and develop a thick scab that does not allow direct observation of the reepithelialization underneath the scab. As a result, we rely on tissue biopsies for evaluation of the degree of reepithelialization. This is in contrast to human burns, in which direct visualization of the neoepidermis is...

Wound Repair in Aging

Reed, Teruhiko Koike, and Pauli Puolakkainen The purpose of this chapter is to review the changes in wound healing that occur in the aged. Unlike pathological conditions such as infection or diabetes as a cause for impaired wound repair, aging may simply reduce the speed at which an individual heals. Thus, a goal of this review is to assess whether aging represents truly impaired or merely slowed wound healing. This chapter focuses on cutaneous wound healing in response to an acute...

Cytokines and Chemokines

Cytokines are small, secreted proteins of up to 20 kDa that affect the behavior of immune cells as well as other cells. They include the interleukins lymphokines and several related signaling molecules, such as tumor necrosis factor-a (TNF-a), TNF-P, and interferons. Chemokines are a subset of cytokines that stimulate chemotaxis and extravasation of immune cells via binding to G-protein-coupled receptors on the surface of target cells. It has long been thought that the proinflammatory...

Measurement of Chemokines at the Protein Level in Tissue

Burdick, John A. Belperio, and Michael P. Keane The recruitment of specific leukocyte subpopulations in response to injury is a fundamental mechanism of acute and chronic inflammation. The elicita-tion of leukocytes is dependent on a complex series of events, including reduced leukocyte deformability, endothelial cell activation, and expression of endothelial cell-derived leukocyte adhesion molecules leukocyte-endothelial cell adhesion and leukocyte activation and...

Oxygen Consumption

A dedicated system including a detector and a device capable of continuous recording and measuring dissolved oxygen content in the medium containing leukocytes is required. An increased rate of oxygen consumption following PMA stimulation indicates NADPH oxidase activity. 3.5.1. Chamber Preparation and Instrument Calibration 1. Rinse the oxygen chamber with at least 10 vol of dH2O. 2. Inject dH2O into the chamber and set gain to 140-150. 3. Inject sodium dithionite. If the chamber is properly...

Histological Analysis Tunel Assay and Immunocytochemistry

H& E staining is performed on either fresh frozen rabbit corneal sections or formalin-fixed mouse whole-eye sections using standard histological methods and light microscopy (Fig. 1). 3.7.2. Propidium Iodide Staining Similar to H& E staining, cell nuclei in fresh frozen rabbit cornea or formalin-fixed mouse whole-eye sections can be stained with 0.05 propidium iodide solution for 10 s. Then wash three times with PBS and once in water. Finally, mount a cover slip and examine the section...

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...