Humana Press Wound Healing Methods and Protocols Edited by Luisa A. DiPietro Aime L. Burns Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Wound Healing Methods and Protocols Edited by Luisa A. DiPietro Aime L. Burns Excisional Wound Healing 3 3 From: Methods in Molecular Medicine, vol. 78: Wound Healing: Methods and Protocols Edited by: Luisa A. DiPietro and Aime L. Burns © Humana Press Inc., Totowa, NJ 1 Excisional Wound Healing An Experimental Approach Stefan Frank and Heiko Kämpfer 1. Introduction Wound healing disorders present a serious clinical problem and are likely to increase since they are associated with diseases such as diabetes, hypertension, and obesity. Additionally, increasing life expectancies will cause more people to face such disorders and further aggravate this medical problem. Thus, several animal models have been established to serve as an experimental basis to determine molecular and cellular mechanisms underlying and controlling an undisturbed healing process. Here we describe a model of excisional skin wounding in mice that can be used to assess molecular, cellular, and tissue movements in healthy mice as well as in mouse models characterized by impaired or altered healing conditions such as genetically defi cient or transgenic animals. Moreover, we point out that the presented model of excisional skin wounding can be easily adapted from a basic experimental model to a model that deals with more detailed questions of interest. The presented method represents an animal model that provides access to investigate complex tissue movements associated with repair such as hemorrhage, granulation tissue formation, reepithelialization, and angiogenic processes (1–3). These processes are initiated by the complete removal of the skin including epidermis, dermis, sc fat and the underlying panniculus carnosus smooth muscle layer by excising skin areas (about 5 mm in diameter) from the backs of the animals. Accordingly, repair of injured skin areas now requires coordinated cellular movements to restore epidermal, dermal, and sc tissue CH01,1-16,16pgs 11/03/02, 7:04 PM3 4 Frank and Kämpfer structures. These processes can be analyzed by different techniques, namely gene expression studies, immunoblot, and histological analyses. Mice of comparable age and weight should be used for each single experi- mental setup to guarantee the comparability and reproducibility of independent animal experiments. Wounding experiments are started by anesthetizing the animals. The fur of the whole back skin area is removed from the anesthetized mice using an electric razor. This step is important to subsequently allow precise removal of skin areas from the backs of the mice and, moreover, easy handling of skin wound biopsy specimens. The wounding is done with fi ne scissors, and the cut removes the epidermal, dermal, and sc layer including the panniculus carnosus. Thus, because wounding is severe, this excisional model provides the possibility of investigating central tissue movements associated with repair, starting with hemorrhage followed by reepithelialization, granula- tion tissue formation, and angiogenesis (1–3). Experience shows that wounded mice will cope well with the injuries; mice start to climb, clean, and feed soon after the end of anesthesia. A few hours after wounding, the wounded area will fi rst be closed by a thin scab, which becomes stronger within the fi rst 2 d of repair. After wounding, mice are kept in a 12-h light/12-h dark regimen, usually four animals per cage, and are fed ad libitum. Mice are sacrifi ced at the desired experimental time points. Usually, mice should be killed at day 1, 3, 5, 7, and 13 postwounding to remove the wounded tissues, as these time points refl ect central time points of repair including infl ammation, keratinocyte migration and proliferation, and the formation of new stroma (d 1–7) (1–3) as well as the end point of the acute healing process (d 13). Thus, the abovementioned experimental time points provide access to characterize representative expressional kinetics for genes of interest during the whole process of acute wound repair. For analysis, wounds are removed from sacrifi ced animals using scissors. First, it is important to remove the wound biopsy specimens including a suffi cient but constant amount of the surrounding wound margin skin tissue. Second, cutting of wound tissue must be performed deep into the underlying tissue, because only this procedure ensures that the complete granulation tissue is isolated and not lost, at least partially, on the backs of the animals. Both points are crucial for further analysis of wound-derived gene expression or histological analysis, since the wound margins as well as the granulation tissue are central to the repair process. Excised wound tissue should be immediately snap-frozen in liquid nitrogen, or directly embedded into tissue-freezing medium for histology. Snap-frozen or embedded wound tissue should be stored at –80°C until used for isolation of total cellular RNA and protein, or sectioning. CH01,1-16,16pgs 11/03/02, 7:04 PM4 Excisional Wound Healing 5 Finally, we point out that the model of excisional wounding described in this chapter can be easily adapted to investigate more detailed aspects of skin repair. To this end, mice that are characterized by wound-healing disorders (4), or transgenic animals (5), can be used. Moreover, the presented model provides access to investigate the impact of pharmacological substances (e.g., enzymatic inhibitors) or recombinant growth factors on normal and disturbed wound-healing conditions, because it allows an accompanying treatment of wounded animals by systemic or topical application of these substances during repair (6,7). 2. Materials 2.1. Excisional Wounding 1. Anesthesia solution: Ketavet ® (2-[2-chlorphenyl]-2-methylaminocyclohexanon- hydrochloride) (ketamine hydrochloride), 100 mg/mL solution, stable to date as given by the manufacturer (Pharmacia & Upjohn GmbH, Erlangen, Germany); and Rompun ® (5,6-dihydro-2-[2,6-xylidino]-4H-1,3-thiazinhydrochloride) (xylazine-hydrochloride), 20 mg/mL solution, stable to date as given by the manu- facturer (Bayer, Leverkusen, Germany). Immediately prior to use, add 800 µL of Ketavet and 500 µL of Rompun to 25 mL of sterile Dulbecco’s phosphate- buffered saline (PBS) using a sterile 50-mL polypropylene conical tube. Mix carefully by inverting the tube (see Note 1). 2. Dulbecco’s PBS without sodium bicarbonate (Life Technologies, Karlsruhe, Germany). 3. EtOH (70% [v/v] solution in H 2 O). 4. Single-use, sterile, nontoxic, nonpyrogenic syringes (3 mL) (see Fig. 1). 5. Single-use, sterile, nontoxic, nonpyrogenic needles (0.5 × 25 mm) (see Fig. 1). 6. Paper towels, examination gloves, 50-mL polypropylene conical tubes (Falcon, Becton Dickinson, Franklin Lakes, NJ). 7. Electric razor (see Fig. 1). 8. Scissors and forceps (see Fig. 1). 2.2. Isolation of Wound Biopsy Specimens 1. Scissors and forceps (see Fig. 1). 2. Paper towels, examination gloves, polypropylene conical tubes (50 mL). 3. Liquid nitrogen. 2.3. Preparation of Total Cellular RNA from Isolated Wound Tissue 1. Paper towels, examination gloves, polypropylene conical tubes (50 mL). 2. Ultra Turrax ® , electric tissue homogenizer. 3. GSCN solution (components from Sigma, Deisenhofen, Germany): 50% (w/v) guanidinium thiocyanate, 0.5% (w/v) sodium laurylsarcosyl, 15 mM sodium citrate, 0.7% (v/v) β-mercaptoethanol; must be stored at 4°C, stable for 6 wk. CH01,1-16,16pgs 11/03/02, 7:04 PM5 6 Frank and Kämpfer 4. 2 M Sodium acetate (NaOAc), pH 4.0. 5. 3 M NaOAc, pH 5.2. 6. Acidic, nonbuffered phenol (H 2 O saturated). 7. Chloroform. 8. EtOH. 9. Diethylpyrocarbonate (DEPC)-treated H 2 O: Dissolve DEPC (Sigma) at a fi nal concentration of 0.1% (v/v) in distilled H 2 O by stirring overnight. Inactivate DEPC by autoclaving. 10. Buffered phenol/chloroform solution: Dissolve 22.5 mL of phenol in 22.5 mL of chloroform. Adjust phenol/chloroform solution to pH 8.0 by adding 5 mL of Tris-HCl (1 M, pH 9.5). Mix vigorously and store overnight to separate organic and aqueous phases. Fig. 1. Surgical instruments for wound preparation. Clockwise from upper left-hand corner: scissors, forceps, embedding media, cryomolds, single-use scalpel, syringes, electric razor, conical tubes. CH01,1-16,16pgs 11/03/02, 7:04 PM6 Excisional Wound Healing 7 2.4. Preparation of Total Cellular Protein from Isolated Wound Tissue 1. Paper towels, examination gloves, polypropylene conical tubes (50 mL). 2. Ultra Turrax, electric tissue homogenizer. 3. Proteinase inhibitor phenylmethylsulfonyl fl uoride (PMSF) (Sigma): 100 mM in 70% EtOH. Store in the dark at 4°C. 4. Proteinase inhibitor leupeptin (Sigma): 1 mg/mL in H 2 O. 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 final concentration 1 mM PMSF, 1 µg/mL of leupeptin. 2.5. Embedding Isolated Wound Tissue for Histology 1. Tissue Tek ® cryomold ® intermediate, disposable vinyl specimen molds (15 × 15 × 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 difl uoride (PVDF) membrane (Immobilon-P). (Millipore, Bedford, MA) (see Fig. 1). 4. Single-use, disposable scalpel (see Fig. 1). 5. Forceps. 6. Dry ice. 2.6. Standard Laboratory Equipment Needed 1. Centrifuge for use with 50-mL polypropylene conical tubes: Heraeus Megafuge 1.0, rotor 7570F (Heraeus, Hanau, Germany). 3. Methods 3.1. Excisional Wounding 1. Freshly prepare Ketavet (ketamine)/Rompun (xylazine) solution for anesthesia. Prepare the single-use syringe for injection. 2. For ip injection, hold the mouse at its neck directly behind the ears and grasp the tail (see Fig. 2A) while holding the mouse with its head down. 3. Inject as 0.5 mL of anesthetizing solution shown in Fig. 2B (see Note 2). 4. Put the mouse back in a cage, so that the mouse will not become agitated. Anesthesia should take effect after 5–10 min. 5. Shave the back of the anesthetized mouse using the electric razor. Carefully remove the hair from the complete back of the animal (see Fig. 2C). CH01,1-16,16pgs 11/03/02, 7:04 PM7 8 Frank and Kämpfer 6. Place the anesthetized and shaved mouse on a paper towel. 7. Wipe the shaved back of the animal with a suffi cient amount of 70% EtOH. 8. Use Fig. 2D as a guide for the fi nal localization of all six wounds before you start to excise the skin areas (see Note 3). Fig. 2. Steps in excisional wound preparation. (A) For ip injection, hold the mouse at its neck directly behind the ears and grasp the tail holding the mouse with its head down. (B) Inject anesthetizing solution as shown. (C) Remove the hair from the complete back of the animal. (D) Place a total of six wounds on the back of each mouse. CH01,1-16,16pgs 11/03/02, 7:04 PM8 Excisional Wound Healing 9 9. Lift back the skin using forceps (see Figure 3A). 10. Incise the skin with a fi rst and careful cut using the scissors (see Fig. 3B). Lifting up the skin will ensure that the incision will move through the panniculus carnosus. 11. Following the fi rst cut, hold the partially removed skin area using forceps (see Fig. 3C). 12. Complete the excision with two to three additional cuts (see Fig. 3D) (see Note 4). 13. Repeat steps 9–12 to create a total of six wounds on the back of each mouse (see Fig. 2D). 14. After completion of excisional wounding, transfer the animals into cages that are covered with two to three layers of paper towels (see Note 5). 3.2. Isolation of Wound Biopsy Specimens 1. Choose the experimental time point of interest (see Notes 6 and 7). 2. Prior to isolation of wound biopsy specimens, sacrifi ce mice painlessly using a carbon dioxide (CO 2 ) chamber followed by cervical dislocation. Cervical Fig. 3. Preparation of excisional wounds. (A) Lift the back skin using forceps. (B) Incise the skin with a fi rst and careful cut using scissors. (C) Following the fi rst cut, hold the partially removed skin area using scissors. (D) Complete the excision with two to three additional cuts. CH01,1-16,16pgs 11/03/02, 7:04 PM9 10 Frank and Kämpfer dislocation must be carried out carefully to avoid disruption of the weak wound tissue (see Note 7). A mouse that has been sacrifi ced at day 3 postwounding is shown in Fig. 4A to demonstrate wound contraction during healing. 3. Hold the sacrifi ced mouse in one hand and begin to remove wound tissue using scissors (see Fig. 4B–E). It is important to include about 2 mm of the directly adja- cent skin, which represents the wound margin tissue (see Fig. 4B,C and Note 9) when cutting out the wound from the dorsal skin surface. 4. Complete your cut, which now includes the whole wound (see Fig. 4C). 5. Lift the skin tissue with forceps (see Fig. 4D). 6. Remove the wound tissue from the body (see Fig. 4D,E). 7. Immediately snap-freeze the wounds in liquid nitrogen. 8. Repeat steps 4–8 to remove all wounds from the back of the animal. 9. Remove the same amount of normal skin from the backs of nonwounded animals for use as a control, or from the same animal to analyze for systemic effects of the wounding procedure. 3.3. Preparation of Total Cellular RNA from Isolated Wound Tissue This method has been adapted from the acid guanidinium thiocyanate– phenol–chloroform extraction protocol by Chomczynski and Sacchi (8). 1. Prepare a 50-mL polypropylene conical tube with 5 mL of GSCN solution at room temperature. 2. Add 16 wounds to the tube (Note 7). 3. Immediately homogenize the tissue for 30–45 s using the Ultra Turrax homogenizer. 4. Clear the solution of hair and insoluble debris by centrifuging at 3000g for 10 min. 5. Transfer the supernatant to a fresh 50-mL polypropylene conical tube (see Note 8). 6. Add 400 µL of 2 M NaOAc (pH 4.0) to the remaining 4 mL of GSCN wound lysate supernatant. 7. Add 4 mL of acidic phenol (H 2 O saturated). 8. Add 1.2 mL of chloroform. 9. Mix vigorously for 30 s by vortexing. 10. Incubate on ice for 15 min. 11. Separate the aqueous and organic phases by centrifuging at 3000g for 10 min. 12. Transfer the supernatant (aqueous phase) to a fresh 50-mL tube (see Note 9). 13. Precipitate total cellular RNA by adding 10 mL of EtOH. 14. Incubate for at least 1 h at –20°C. 15. Pellet the RNA (Heraeus Megafuge 1.0, 3000g for 30 min). 16. Discard the supernatant, and let the RNA pellet dry for 5 min. 17. Dissolve the RNA pellet in 4 mL of DEPC-treated H 2 O. 18. Add 4 mL of buffered phenol/chloroform (pH 8.0) and mix vigorously by vortexing (1 min). CH01,1-16,16pgs 11/03/02, 7:04 PM10 Excisional Wound Healing 11 Fig. 4. Harvesting of excisional wounds. (A) A mouse that has been sacrifi ced at d 3 postwounding demonstrates wound contraction during healing. (B) To harvest the wound, hold the sacrifi ced mouse in one hand and begin to remove wound tissue using scissors. (C) Include about 2 mm of the directly adjacent skin, which represents the wound margin tissue. (D) Lift the skin tissue with forceps. (E) Remove the wound tissue from the body. (F) For embedding, place freshly isolated wound tissue scab side up directly onto a piece of PVDF membrane. CH01,1-16,16pgs 11/03/02, 7:04 PM11 [...]... molecular differences between healing and nonhealing wounds Compared with nonhealing wounds, healing wounds characteristically have lower levels of inflammatory cytokines and proteases and higher levels of growth factor activity Furthermore, as nonhealing wounds From: Methods in Molecular Medicine, vol 78: Wound Healing: Methods and Protocols Edited by: Luisa A DiPietro and Aime L Burns © Humana Press Inc.,... corresponding group) for RNA and a total of 8 wounds (2 wounds from each mouse of the corresponding group) for protein isolation as a standard procedure can be isolated These wounds are pooled prior to isolation of total cellular RNA (n = 16 wounds) and protein (n = 8 wounds) More wounds are needed for RNA preparation than protein preparation, since the amounts of total RNA isolated from wound tissue always... fibroplasia and progressive alignment of collagen bundles From: Methods in Molecular Medicine, vol 78: Wound Healing: Methods and Protocols Edited by: Luisa A DiPietro and Aime L Burns © Humana Press Inc., Totowa, NJ 37 CH03,37-54,18pgs 37 11/03/02, 7:06 PM 38 Gamelli and He Scar modification during this phase adds further to the restoration of wound tensile strength Although the process involves intense and. .. sweat glands, which are glandular structures in the dermis of humans that contain cells that participate in regeneration of the epidermis following partialthickness injuries Both pigs and humans have hair and sebaceous glands in similar number and distribution However, the absence of apocrine sweat glands in porcine skin does not appear to appreciably alter healing of pig wounds compared with human wounds... of wounds per pig References 1 Trengove, N J., Bielefeldt-Ohmann, H., and Stacey, M C (2000) Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers Wound Repair Regen 8, 13–25 2 Trengove, N J., Stacey, M C., MacAuley, S., Bennett, N., Gibson, J., Burslem, F., Murphy, G., and Schultz, G (1999) Analysis of the acute and chronic wound environments: the role of proteases and. .. Valenzuela, P., and Schultz, G S (1986) Enhancement of epidermal regeneration by biosynthetic epidermal growth factor J Exp Med 163, 1319–1324 CH02,17-36,20pgs 36 11/03/02, 7:05 PM Incisional Wound Healing 37 3 Incisional Wound Healing Model and Analysis of Wound Breaking Strength Richard L Gamelli and Li-Ke He 1 Introduction The nature and mechanism of incisional wound healing has been and continues... that regulate wound healing In the early phases of wound healing, chemokines and cytokines regulate chemotaxis and activation of inflammatory cells, as well as synthesis of proteases and protease inhibitors Growth factors play dominant roles in regulating cell proliferation, differentiation, and synthesis of extracellular matrix The analysis of fluids and biopsies collected from nonhealing wounds has also... skin wounds, including surgical incisions, skin grafts for burns or venous stasis ulcers, and other open wounds Thus, studies that increased the understanding of the cellular and molecular regulation of epidermal regeneration have led to improvements in healing of most types of skin wounds As described previously, the healing of skin wounds involves a complex system of integrated molecular signals and. .. excisional wounds Several techniques can be used to create a partial-thickness thermal injury The shape and size of the wound can be varied, according to the experimental design In previous studies, we have created 3 × 3 cm square wounds or 2 × 3 cm rectangular wounds However, other researchers have advocated the use of circular wounds because they have a larger ratio of total wound area to migrating wound. .. carefully (see Fig 4F) The wound should remain completely flat on the membrane This is important because the wound margins tend to roll inside References 1 Clark, R A F (1996) Wound repair: overview and general considerations, in The Molecular and Cellular Biology of Wound Repair (Clark, R A F., ed.), Plenum, New York, pp 3–50 2 Singer, A J and Clark, R A F (1999) Cutaneous wound healing N Engl J Med . Humana Press Wound Healing Methods and Protocols Edited by Luisa A. DiPietro Aime L. Burns Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Wound Healing Methods and Protocols Edited. DiPietro Aime L. Burns Excisional Wound Healing 3 3 From: Methods in Molecular Medicine, vol. 78: Wound Healing: Methods and Protocols Edited by: Luisa A. DiPietro and Aime L. Burns © Humana Press. 156–159. CH01,1-16,16pgs 11/03/02, 7:04 PM15 Methods in Reepithelialization 17 17 From: Methods in Molecular Medicine, vol. 78: Wound Healing: Methods and Protocols Edited by: Luisa A. DiPietro and Aime L. Burns © Humana