This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.
The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined “middle” of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.
Cutaneous wound healing is a complex biological process with sequentially overlapping phases. It requires the coordination of cellular and molecular processes that are temporally and spatially regulated in order to restore the barrier function of the damaged epithelium. In the first phase, inflammation, neutrophils and macrophages migrate into the wound, mobilizing local and systemic defenses1. Following and overlapping the inflammatory phase is the proliferation stage. Fibroblasts begin rapidly proliferating and migrating into the granulation tissue. Keratinocytes away from the leading edge directionally proliferate towards the wound as differentiated keratinocytes in the leading edge migrate to re-epithelialize the wound2. Finally, the remodeling and maturation phase begins, during which fibroblasts in the granulation tissue start to synthesize and deposit collagen. The remodeling and organization of the new matrix can last up to 1 year following injury3. Due to the complexity of overlapping events involving cross-talk between multiple cell types, and despite years of research, many of the cellular and molecular mechanisms underlying wound healing remain poorly understood.
The mouse model is the predominant mammalian model for investigating mechanisms of wound healing due to their ease of use, relatively low cost and genetic manipulability1,4,5. Although different types of wounds have been described in the murine model, the most common is an excisional wound (either bilateral punch or direct punch biopsy), followed by incisional wound models4. The excisional wound model has a distinct advantage over the incisional model as it inherently generates control tissue that has not undergone the healing process. The punch biopsy tissue that is excised as part of the surgical protocol can be processed in the same manner as the wounded tissue and used to establish the homeostatic conditions for a desired criterion. Excised control tissue may also be useful if assessing the effects of a skin pretreatment or confirming successful gene alteration at the time of injury4.
Healing parameters can be assessed by many different techniques, including planimetry or histology. However, planimetry can only evaluate visible characteristics of the wound, and due to the presence of a scab, often does not correlate to measurements of healing that are visualized by histology, thereby making histology the “gold standard” of analysis4. Despite histological analysis being the gold standard, it is most often performed on an arbitrary subset of the wound6,7. For instance, cutting the wound in “half” prior to embedding and sectioning the wound is currently common practice to reduce the time and resources spent on sectioning materials and data analysis. The method of morphometric analysis described in this protocol was developed to encompass the entire wound tissue, to accurately reflect the morphological characteristics of the wound, and to increase the likelihood of detecting wound healing defects with a small effect size. In this protocol, we detail a surgical method for generating the most commonly studied murine wound, the bilateral full-thickness excisional wound, as well as a detailed and rigorous method for histological analysis such is rarely used in the field.
All experiments were completed in accordance and compliance with federal regulations and University of Iowa policy and procedures have been approved by the University of Iowa IACUC.
1. Animals and husbandry
2. Surgery
NOTE: It is unnecessary to maintain sterile surgical conditions. While care should be taken to maintain sterility between animals, the punch biopsy itself is done on a clean, but nonsterile surface. The surgery duration per animal is between 10 and 15 min.
3. Post wound monitoring
4. Harvesting wounds
5. Wound fixation and embedding
6. Day 0 wound area analysis
7. Serial sectioning
8. Mounting of paraffin sections
9. Histological staining
10. Microscopic imaging
11. Morphometric analysis
NOTE: When the wound spans multiple pictures, sum the measurements taken from the individual pictures to obtain one value per metric per wound section to record in the spreadsheet.
Figure 5 depicts the range in measured and calculated values obtained by performing morphometric analysis on wild-type wounds generated in different mouse strains by multiple surgeons and analyzed by different individuals. Wild-type mice from different strains can display statistical differences as described both in our studies and in the literature9,10. Based on these representative results, we recommend that, within one study, mice from only one strain be used. Although we recommend that the same individual perform all the wounds within a particular study, multiple individuals could act as surgeons as long as the area of the wounds at day 0 is not statistically different between individual’s work. Finally, because the morphometric analysis described in this protocol can be extensive, multiple individuals could analyze parts of the same experiment, but only if the results of their analysis of two samples are within 5% of each other. However, it is preferable to have a single individual analyzing the wounds in a blinded manner to avoid bias.
Figure 6 displays a meta-analysis comparing wound measurements obtained by following the protocol described in this study11 with measurements obtained from the “middle” of the wound and 40 surrounding sections. In Figure 6A, the epidermal area and wound area were calculated from the measurement of the epidermal and wound lengths on wound sections followed by the calculation of the percentage of the epidermal area among wound area (sometimes referred to as “percentage of closure” or “percentage of epithelialization”). Similarly, in Figure 6B, the percentage of the epidermis in the wound was obtained as the ratio of the epidermal volume (calculated from the measured epidermal area) over the wound volume (calculated from the measured wound area). For both parameters, the analysis of the whole wound showed strong statistical differences between groups (up to P < 0.001 following One-way Anova). However, the significance was decreased (up to P < 0.01 following One-way Anova) when only 40 sections in the middle of the would were analyzed. These results demonstrate a decrease in the level of significance when only a subset of the wound is analyzed. These data suggest that defects with a small effect size will likely only be detected when performing morphometric analysis on the entire wound, and that more mild wound healing phenotypes will be missed from a “middle of the wound” type of analysis.
Common practice for analyzing in vivo wound healing involves measuring the area of the wound on histological sections chosen somewhere in the wound12. With that in mind, the average of the measured wound area from the serial sections of entire wounds was compared with the one obtained from the middle subset sections. The results showed no significant difference between experimental groups and between method of analysis (Figure 6C). However, the current protocol uses the measured area (shown in Figure 6C) over the whole wound to calculate the wound volume. As shown in Figure 6D, the calculated wound volume (which can only be calculated using the analysis of the whole wound) is significantly different between the experimental groups. In sum, these representative results demonstrate the importance of in-depth histological analysis of wound healing parameters described in this protocol in order to detect phenotypes that would otherwise have been missed using a more traditional wound healing analysis.
Figure 1: Bilateral excisional wound procedure. (A) Representative photograph of a mouse after clipping and shaving hair from the surgical area. (B) The skin pinched between the shoulder blades along the dorsal midline. (C) The mouse positioned on its side with the skinfold laid flat. (D) Representation of the biopsy punch placement. (E) Punched skinfold. (F) The mouse with two bilateral wounds at day 0 and the excised punch biopsy control tissues as indicated by the white arrows. Please click here to view a larger version of this figure.
Figure 2: Wound harvest procedure. (A-C) Macroscopic photographs of 6 mm excisional wounds after 4 days (A), 7 days (B) and 11 days (C). (D-E) With a scalpel blade, a cutaneous incision was made in the shape of a wide rectangle that includes the wounds and surrounding unwounded tissue. (F) The skin was released from underlying tissue with forceps and scissors. (G) Representation of harvested bilateral wounds. (H) The dotted white line represents the border of the tissue that would be harvested following the use of a punch biopsy (this method allows for standardized amount of tissue harvested). (I) The dotted white line represents the border of the tissue that would be trimmed for histology (rectangular shape allows for easier embedding). Please click here to view a larger version of this figure.
Solution | Time (minutes) | Temperature (Celsius) | Pressure (kPa) |
80% ETOH | 30 | RT | N/A |
95% ETOH | 30 | RT | N/A |
100% ETOH | 30 | RT | N/A |
Xylene | 30 | RT | N/A |
Xylene | 30 | RT | N/A |
Paraffin | 30 | 60 | 20 |
Paraffin | 30 | 60 | 20 |
Table 1: Sample processing procedure for paraffin embedding. RT = room temperature. N/A = not applicable.
Figure 3: Wound embedding and sectioning. (A) Paraffin-processed wound lying flat in an embedding mold. (B) The wound was held at 90˚ (“standing”) from the horizontal surface of the mold for embedding. (C) Paraffin-embedded wound properly oriented for sectioning (D) Paraffin block mounted on the microtome was sectioned into ribbons of about 20 sections (E) Paraffin ribbons cut in 5-section increments as indicated by the white brackets. (F) Serial sections laid flat in a warm water bath (40-45 ˚C) were mounted on a microscope slide. The cartoon in (A-C) represents the wound (orange) in the skin (blue) with the proper orientation of the epidermis (e) and the dermis (d) for each step. Please click here to view a larger version of this figure.
Figure 4: Histological characteristics of wounds and illustration of morphometric parameters. (A, B) Histological features of a day 4 (A) and a day 7 (B) 6 mm wound. (C-F) Representation of the different measurements used to perform the quantitative morphometric analysis: length of the epidermis (C, D, dotted black line), measured area of the epidermis (C, D, yellow shaded area), length of the wound (E, F, dotted yellow line) and measured area of the entire wound (E, F, gray shaded area). HF = hair follicle; scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 5: Morphometric characteristics of representative 6 mm wild-type wounds at day 4, day 7 and day 11 post-wounding. Scattered plots represent data obtained from morphometric analyses of 6 mm wild-type wounds generated in different mouse strains by multiple surgeons and analyzed by different individuals. (A) wound volume, (B) epidermal volume, (C) percentage of epidermis in the wound, (D) wound area (calculated), (E) epidermal area (calculated), and (F) percentage of epidermal area among wound area demonstrate the range of variation in morphometric values. Please click here to view a larger version of this figure.
Figure 6: Meta-analysis comparing parameters obtained from whole wound with middle of the wound identifies new defects with higher significance. Wound healing morphometry was performed on day 7 wounds injected with saline (control), transforming growth factor beta 3 (Tgfb3), Tgfb3 with neutralizing antibody (Tgfb3 + NAB) and neutralizing antibody alone (NAB). Measurements were performed on serial sectioned wounds (“whole”) or on 40 sections from the middle of the wound (“middle”) and used to calculate the percentage of epidermal area over wound area (A), the percentage of epidermis in the wound (B), and the wound volume (D). (C) represents the average of the wound area measured on the whole or middle of the wound. * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant, One-way Anova). This figure has been modified using data from Le et al.11. Please click here to view a larger version of this figure.
Supplementary Table 1: An example of a spreadsheet for recording morphometric measurements. Please click here to download this table.
The bilateral excisional wound model is a highly customizable procedure which can be used to study many different aspects of wound healing. Before beginning a wound healing project, investigators should perform a power analysis to determine the number of wounds needed to detect a defect of a particular effect size. Inconsistencies exist within the literature on whether individual mice or wounds should be used as biological replicates, however, a recent study showed that there is no significant correlation between two wounds on a single animal4. This suggests that wounds from a single animal are independent from each other and can be considered as biological replicates, reducing the number of mice required to detect a defect. This is relevant when considering small effect sizes for which a high number of wounds is required to reach significance4. In addition, the results of the power calculation may influence how many wounds are performed on each animal, with 2 and 4 wounds per animal being most common.
The surgical protocol itself is also highly flexible. While we recommend anesthetization by isoflurane vaporizer due to the short procedure length and quick recovery (10-15 min per mouse), investigators without access to a vaporizer can use injectable anesthesia including ketamine/xylazine or pentobarbital. Selection of an appropriate analgesic is particularly critical, as it pertains to the inflammatory phases of wound healing. The use of nonsteroidal anti-inflammatory drugs (NSAIDS) such as Flunixin meglumine or Meloxicam should be used with caution as they can decrease inflammation. Opioids are, therefore, preferred in studies where inflammation is being investigated. We recommend analgesics (e.g., Buprenorphine Sustained-Release) which provide up to 48 h of analgesia and eliminate the need for repeated, additional doses. All surgical procedures should be executed in accordance with federal guidelines and supported by an approved animal protocol.
Wound healing is a process that encompasses several phases, each of which being characterized by different biological processes involving distinct cell types3. The inflammatory phase occurs between days 0 and 5, with the early migration of polymorphonuclear neutrophils (PMN) and macrophages to the site of injury13. The proliferative phase occurs between 3 and 14 days with re-epithelialization taking a varying amount of time based on the size of the wound14. In this protocol, we used a 6 mm biopsy punch and most wounds were re-epithelialized by 7 days. However, this time frame would need to be shortened if smaller punches were used (they are available as small as 1 mm). In combination with earlier time points, these smaller wounds may be preferable to reduce the amount of histological analyses15. Finally, the remodeling and maturation phases occur after 7 days and up to a year following the injury16. These later time points may be required to investigate the maturation of the wound or to investigate wound healing delays in experimental animals. Therefore, the investigator will need to determine the critical time points required to investigate particular phases of wound healing based on their particular hypothesis5.
The analysis of wound healing is often not restricted to histological analyses. Unstained paraffin sections can be used for additional analyses such as immunofluorescence or Masson’s trichrome for collagen deposition. The processing of control tissue (punch biopsies) and the harvesting steps of the tissue will all depend on how, in addition to histological analyses, wound healing is assessed. As part of the surgical protocol, punch biopsies are removed in order to generate the excisional wound. These punches can serve as unwounded control tissue for downstream applications such as protein (for western or cytokine profiling), DNA or RNA extraction and should be processed accordingly. It is recommended that at least one punch be saved for histological analysis, especially in cases where skin is treated prior to wounding (for example, application of tamoxifen for induction of Cre-Lox recombination17). Examination of the punch can determine the effect of the treatment on cutaneous morphology or simply allow the assessment of baseline cutaneous morphology of the particular mouse model being used. Wounds not used for histology can be harvested with a punch biopsy that encompasses the size of the wound. This procedure has a few advantages, including harvesting the same amount of tissue regardless of the wound size (larger wounds have less surrounding healthy tissue). Finally, we describe use of 4% paraformaldehyde as the fixative and paraffin as the material for preserving and embedding tissues, respectively. Other fixatives may be required for certain applications and can be substituted (for example, Carnoy’s or Bouin’s fixatives). For best immunofluorescent staining, freezing and embedding of the wound in Optimal Cutting Temperature compound followed by frozen sectioning remains the method of choice.
Sectioning of wounded tissue can present many challenges, particularly day 4 wounds because of the presence of the scab. To generate high-quality sections, it is recommended to verify that all parts of the microtome are tightly fastened, the block holder is retracted to its initial position, the blade holder is set between 0 and 10˚ and the blade is tightly fastened but not overtightened. Although the paraffin block is chilled, the tissue may still shred. If shredding continues, a piece of ice can be placed on the block for 5 minutes while still mounted. Once sectioning has begun, it is highly recommended to avoid removing the block from the microtome to prevent the loss of paraffin sections. It is imperative to record any lost paraffin sections, both their numbers and their ranking in the serial sectioning. This will affect the morphometric analysis and needs to be considered. After initiating sectioning, the identification of wounded tissue on unstained sections may be difficult, especially if not familiar with histology. When in doubt, it is recommended to be conservative and keep all the sections that contain tissue. Once the histological stain is performed, the tissue organization will be more evident and the wound more distinct. Occasionally, hair follicles may not be clearly visible or may be far from the wound edge. If this is the case, other key characteristics of the wounded tissue can be used to establish the boundaries of the wound including an abrupt increase followed by immediate decrease in epidermal thickness and sharp changes in the organization of the connective tissue (Figure 4A-B).
Digital imaging is a critical step in the morphometric analysis. The morphometric analysis should be performed on individual images using a landmark on each image to avoid repeated measurements. However, it is possible to stitch all the frames together using digital software and perform the analysis on a single image. Although this seems easier at first, manipulation of a large image may slow the analysis process down. The choice of the section is also critical for analysis. Although we recommend to digitally acquire the top section of every 8th slide, the quality of the section should be prioritized and the best of the 5 sections on that slide should be imaged. Sections with small folds could still be analyzed by estimating the area/length of the fold. The number of that particular section should be recorded both in the digital file and in the Excel spreadsheet, such that the distance between the previous and the next analyzed section can be adjusted accordingly.
Cutaneous wounds affect an estimated 6.5 million people with treatment costing over $25 billion annually18 and are an inherent component of surgical procedures in addition to being secondary to many other health concerns, including diabetes and obesity. The mouse has been used as a convenient model to study human diseases because of the ease in manipulating its genome, despite often exhibiting a subtle phenotype compared to the human disorder. The bilateral excisional wound model and subsequent morphometric analysis described in this protocol is significant because of its ability to address the difficulty in detecting minor defects in an inherently complex wound healing process19. Because of its greater sensitivity and reduced variability, this protocol provides opportunities to use fewer animals to obtain the same experimental sensitivity. The protocol is highly customizable and can be used to study all stages of tissue repair. Detailed histological morphometric analysis in combination with phenotypic characterization of the tissue have great potential to increase the knowledge needed to further the understanding of critical factors modulating tissue repair.
The authors have nothing to disclose.
We are grateful to all the members of the Dunnwald Lab who have contributed to the optimization of this protocol over the years, and to Gina Schatteman whose persistence in promoting the use of serial sectioning for wound analysis made its creation possible. This work was supported by funding from NIH/NIAMS to Martine Dunnwald (AR067739).
100% ethanol | |||
70% ethanol | |||
80% ethanol | |||
95% ethanol | |||
Alcohol Prep | NOVAPLUS | V9100 | 70% Isopropyl alcohol, sterile |
Ammonium hydroxide | |||
Biopsy pads | Cellpath | 22-222-012 | |
Black plastic sheet | Something firm yet manipulatable about the size of a sheet of paper | ||
Brightfield microscope | With digital acquisition capabilities and a 4X objective | ||
Cotton tipped applicators | |||
Coverslips | 22 x 60 #1 | ||
Dental wax sheets | |||
Digital camera | Include a ruler for scale, if applicable | ||
Dissection teasing needle (straight) | |||
Embedding molds | 22 x 22 x 12 | ||
Embedding rings | Simport Scientific Inc. | M460 | |
Eosin Y | |||
Glacial acetic acid | |||
Hair clipper | |||
Heating pad | Conair | Moist dry Heating Pad | |
Hematoxylin | |||
Microtome | |||
Microtome blades | |||
Paint brushes | |||
Paraffin Type 6 | |||
Paraformaldehyde | |||
Permount | |||
Phosphate buffer solution (PBS) | |||
Povidone-iodine | Aplicare | 82-255 | |
Processing cassette | Simport Scientific Inc. | M490-2 | |
Razor blades | ASR | .009 Regular Duty | |
Scalpel blades #10 | |||
Scalpel handle | |||
Sharp surgical scissors | sterile for surgery | ||
Skin biopsy punches | Size as determined by researcher | ||
Slide boxes | |||
Slide warmers | |||
Superfrosted microscope slides | Fisher Scientific | 22 037 246 | |
Temperature control water bath | |||
Tissue embedding station | Minimum of a paraffin dispenser and a cold plate | ||
Tissue processor | Minimum of a oven with a vacuum pump | ||
Triple antibiotic opthalmic ointment | |||
tweezers, curved tip | sterile for surgery | ||
tweezers, tapered tip | sterile for surgery | ||
WypAll X60 | Kimberly-Clark | 34865 |