Histological and Immunohistochemical Examination of Stem Cell Proliferation and Reepithelialization in the Wounded Skin

[Abstract] The skin is the largest organ that protects our body from the external environment and it is constantly exposed to pathogenic insults and injury. Repair of damage to this organ is carried out by a complex process involving three overlapping phases of inflammation, proliferation and remodeling. Histological analysis of wounded skin is a convenient approach to examine broad alterations in tissue architecture and investigate cells in their indigenous microenvironment. In this article we present a protocol for immunohistochemical examination of wounded skin to study mechanisms involved in regulating stem cell activity, which is a vital component in the repair of the damaged tissue. Performing such histological analysis enables the understanding of the spatial relationship between cells that interact in the specialized wound microenvironment. The analytical tools described herein permit the quantitative measurement of the regenerative ability of stem cells adjacent to the wound and the extent of re-epithelialization during wound closure. These protocols can be adapted to investigate numerous cellular processes and cell types within the wounded skin.


Procedure
A. Mouse skin wounding, skin collection and embedding in OCT compound without pre-fixation 1. Anesthetize mice with 3-5% isoflurane and 1 L/min O2 inside an anesthesia chamber or as the protocol approved by Institutional Animal Ethics committee ( Figure 1C). The anesthetized mouse can be maintained with exposure to the anesthesia via a nose cone ( Figure 1D).
2. Shave the mouse back skin with a fine trimmer to remove all the back skin hair ( Figure 1E). 3. Hold the skin in the interscapular region between shoulders using blunt-ended forceps such that there is a space of about 10 mm between the two wounds ( Figure 1F). 5 mm full-excisional wounds are created on the dorsal skin using a circular sheet punch ( Figures 1G-1I). 4. The size of the wound can be uniformly measured by taking the images of the wounds with a ruler near the wound to keep the scale constant in every imaging session ( Figure 1J). For the day 0-timepoint, take the image 3-4 h post wounding. This allows the tension of the skin to be 4 www.bio-protocol.org/e3894  fully released and gives a more accurate measure of the starting wound size.
To monitor the wound-closure kinetics, calculate the wound area on day 0 post wounding and normalize the subsequent time points by dividing the wound area to that of the wound area at day 0. Express this fraction in percentage to plot the wound closure kinetics.
Wound area can be measured in ImageJ as follows: Go to ImageJ > select line tool > Draw a 1.0 cm line parallel to/on the ruler (Use ruler as reference) > Analyse > Set image scale: enter "Known distance" as "1.0" and "unit of length" as "cm" > Select OK to apply settings. (Note: The line tool basically measures the length of scale in pixels. The set scale option equates/defines how many pixels are equal to a known distance (such as 1.00 cm in our case) > Select the polygon selection tool and mark the outer edge of the wound (Start at any point on the edge of the wound and left click to add intermediate points. Once the edges are marked, right-click to complete the polygon selection) > go to Analyse > Measure. A new window will appear where the area of the polygon (i.e., the wound) will be given. 5 www.bio-protocol.org/e3894   Isoflurane outlet with nose cone; 4. Light source; 5. Spray bottles containing 70% Ethanol or H2O2 solution; 6. Heat sterilizer for forceps and scissors; 7. Supporting stand for surgery. B. Dissection tools, biopsy punch and trimmers required for the wounding procedure. C. Mouse is anesthetized using isoflurane (3-5%) mixed with oxygen within an anesthetic chamber and is maintained via a nose cone adjusted to the supporting/surgery stand (D). E. Dorsal back hairs are shaved using hair trimmers. F. Dorsal skin is held using blunt forceps such that there is a gap of about 10 mm between the two wounds. G-I. 5 mm sheet hole puncher is used to generate full excisional wounds. J. Wounds were photographed with a ruler as a scale to measure wound area. 6 www.bio-protocol.org/e3894

5.
After wounding, the skin can be harvested at desired time points of an experiment. Before proceeding for skin dissection, mice are euthanized in a CO2 chamber followed by cervical dislocation. The euthanization protocol must be approved by the institute's animal ethics committee.
6. Before beginning the skin dissection, draw a reference line on the dorsolateral side, about 1 cm away from the wound using a marker to demarcate the anterior/posterior (A/P) axis. Then cut a rectangular piece of skin approx. 2.5 cm x 1 cm around the wound. Start the incision from the posterior side of the wound and proceed on the lateral sides. Cut the skin along the A/P axis such that cut lines are parallel to the direction of the straightened tail, which enables subsequent sectioning of full hair follicle longitudinally (Figures 2A-2C).
7. When embedding the wounded skin tissue, there should be around 3 mm unwounded skin left while embedding, which can act as an internal control for comparison. 8. Place the cut skin on a piece of notebook paper, which helps to support the skin and prevent it from curling. Align the reference line on the cut skin parallel to the straight line on the paper to achieve optimal hair follicle orientation ( Figures 2D-2E).
9. For wounded skin, cut the wounds into two equal halves by cutting the wound along the midline with a sharp scissor. Maintain the A/P orientation of the skin while embedding to preserve the longitudinal profile of hair follicles while sectioning the skin ( Figure 2F). After dividing the wounded skin into equal halves, each part can be either fixed in Bouin's fixative ( Figure 2G) or processed for OCT compound embedding. 10. Submerge the cut skin in OCT compound at room temperature for 15 min ( Figure 2H). 11. Using fine forceps, move the air bubbles away from the skin surface ( Figure 2H).
Optional: Flatten the skin on aluminum foil and transfer it on the glass slides. Put OCT compound on top and freeze it on a cube of dry ice. Once OCT compound solidifies after freezing, lift the frozen skin (it would look like a thin chip of ice), then embed it in an OCT block.
This way is easier to get rid of bends and twists in the skin and also prevents excessive bubbles around the tissue.
12. Orient the skin in the mould so that the wounded edge is towards the bottom. Incubate the OCT mould with skin on dry ice to freeze and solidify the OCT compound by holding the skin tissue perpendicular to the mould with the wound side down ( Figure 2I) 13. Top up the mould with more OCT compound such that the skin tissue is entirely covered in OCT compound ( Figure 2J).
14. After freezing, the OCT blocks should be kept at -80 °C at least overnight before sectioning on a cryostat. Adjust OCT block sectioning orientation such that the full length of hair follicles is visible along the entire section ( Figure 2K

B. Quantitative data analysis
Immunostaining of Ki67 and CD34: 1. Prepare a humidifying chamber by using an empty tip box by filling the bottom chamber of the tip box with ~1 cm of water so that blocking solutions and antibodies do not dry on the section during incubation (Figures 3B and 3C). Cover the box with aluminum foil if staining is to be performed in the dark.
2. Remove the frozen slide with OCT skin section from -80 °C freezer and wait for 1 min to thaw.
Make a hydrophobic barrier surrounding the tissue section using a hydrophobic ink pen ( Figure   3A). Then add 250 µl of 4% PFA per section for fixation. Incubate the slide in the humidifying chamber for 10 min at room temperature as shown in Figure 3B

antibodies, 100% methanol stored at -20 °C is used as fixative where fixation is done at -20 °C.
Follow fixation guidelines as per antibody datasheet.

Measurement of stem cell proliferation at the site of the wound:
Stem cells resident in the bulge region of the hair follicle are quiescent under normal conditions and activated in a window of anagen growth during the hair cycle. In addition, these stem cells rapidly divide in response to injury and migrate to the wound site to restore the epidermal barrier. In our studies, we found that the damage repair activity of stem cells is restricted within a range of first 3 hair follicles adjacent to the wound site (Lee et al., 2017). We define this region as wound proximal and the area 2 cm away from the wound edge is considered distal. We did not detect any stem cell activity in response to injury at the wound distal site. To measure stem cell proliferation in the wound proximal region, bulge stem cells are detected by CD34 staining which is a surface glycoprotein widely used in combination with alpha6 integrin to detect and isolate this stem cell population (Trempus et al., 2003). The cellular interactions involving bulge resident stem cells and soluble cytokines are restricted to a region of approx. 100 μm (Lee et al., 2017). Consequently, we analyzed the proliferation as an outcome of such interactions. We identify proliferating CD34 + hair follicle stem cells as those which are double-positive for both CD34 + and Ki67 + proliferation markers ( Figures   4A-4D). The arrector pilli muscle (which can be marked by α-smooth muscle actin) attaches to the hair follicle at the bulge area and can be used as a landmark to outline ROI around this niche of hair follicle stem cells within a region of interest (ROI) that is approximately 150 μm wide, which encompasses the entire bulge region of telogen stage hair follicle, and the number of Ki67 + dividing bulge stem cells are counted within this ROI ( Figure 4C). A 150 μm box (or a box of any size) can be specified on microscopy images by following these steps in ImageJ: (1) set image scale, (2) Draw a rectangle of any size from the main toolbar, (3) Go to edit on the main menu > selection > specify > enter the desired length and width of the box. The number of Ki67 + proliferating cells were counted by using ImageJ as follows: Main menu > Plugin > Analyse > Cell counter. The number of proliferating cells in hair follicles can be compared between the wound proximal and wound distal regions as shown in Figures 4E-4G.
Likewise, epidermal cell proliferation is measured by counting Ki67 + cells in a region of 300 μm x 150 μm rectangular box covering interfollicular epidermis ( Figure 4F) by ImageJ as mentioned above. The number of proliferating cells can be compared between the wound proximal and wound distal region (Figures 4E-4F, and 4H). Since the immunohistological procedure retains the tissue architecture, the quantitative analysis described here enables comparison of the regenerative capabilities of stem cells residing in different niches. Specifically, this method can be used to examine whether hair follicle stem cells respond differently to wound-induced signals as compared to progenitor cells residing in the interfollicular epidermis.
Alternative methodology: Stem cell activity is regulated by a variety of other cell types, such as immune cells, adipocytes, fibroblasts, blood vessels, lymphatic vessels and peripheral nerves, which collectively form the stem cell niche. In order to understand how each of these cell types 11 www.bio-protocol.org/e3894  impacts the stem cells, CD34 staining of stem cells can be multiplexed with lineage marker staining of other cell types. This strategy would reveal whether the other cell types in the skin environment are within proximity to stem cells to affect their activity. In addition, using mouse genetics to knockout specific cell types, the functional role of the depleted cells in modulating stem cells can be studied. We combined this approach of using immunostaining and mutant knock-out mice to study the role of skin resident γδ T cells in regulating stem cell activity in wounded skin (Lee et al., 2017).