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J Anat. Dec 2007; 211(6): 810–818.
PMCID: PMC2375850

Intravital insights in skin wound healing using the mouse dorsal skin fold chamber

Abstract

The skin fold chamber is one of the most accepted animal models for studying the microcirculation both in health and disease. Here we describe for the first time the alternative use of the skin fold chamber in mice for intravital microscopic investigation of skin regeneration after creating a full dermal thickness wound. The dorsal skin fold chamber was implanted in hairless SKH1-hr mice and a full dermal thickness wound (area ~4 mm2) was created. By means of intravital fluorescence microscopy, the kinetics of wound healing were analyzed for 12 days post wounding with assessment of epithelialization and nutritive perfusion. The morphology of the regenerating skin was characterized by hematoxylin-eosin histology and immunohistochemistry for proliferation and microvessel density. The model allows the continuous visualization of wound closure with complete epithelialization at day 12. Furthermore, a sola cutis se reficientis could be described by an inner circular ring of vessels at the wound margin surrounded by outer radial passing vessels. Inner circular vessels presented initially with large diameters and matured towards diameters of less than 15 µm for conversion into radial spreading outer vessels. Furthermore, wound healing showed all diverse core issues of skin repair. In summary, we were able to establish a model for the analysis of microcirculation in the healing skin of the mouse. This versatile model allows distinct analysis of new vessel formation and maturation in regenerating skin as well as evaluation of skin healing under different pathologic conditions.

Keywords: epithelialization, microcirculation, mouse model, skin regeneration, sola cutis se reficientis, vessel maturation

Introduction

The skin is the largest organ in man. Though hardly penetrable by bacteria during physiological states, damaged skin is easily negotiated by pathogens, which may lead to severe bacterial colonization with consecutive bacteremia and sepsis (Allgower et al. 1995; Cook 1998). Therefore the early repair of wounded skin is a fundamental goal of the organism (Dyson et al. 1992), and the restoration and augmentation of cutaneous wound healing has long been an elusive target for health-care professionals. Despite advances in medical sciences, skin regeneration still represents a problem of exceptional clinical relevance.

The healing of a skin wound is a complex biological process, requiring a distinct collaboration of many different cells and tissues. For this purpose different models have been established to study skin regeneration in health and disease (Breuing et al. 1992; Rossio-Pasquier et al. 1999; Escamez et al. 2004; Geer et al. 2004; Harrison et al. 2006). While most preparations used in those studies are not well suited for observing the microcirculatory process of wound healing, the skin fold chamber seems to be an ideal model, allowing repeated analysis of the regenerating skin as well as the skin microcirculation over a prolonged period of time. It may also be worthwhile to analyze the skin repair process in chronic wounds or to evaluate topical as well as systemic administration of drugs which could accelerate dermal recovery. We therefore present the dorsal skin fold chamber as a new tool for discovering the events that drive wound repair and skin regeneration.

Materials and methods

Animals

A total of 16 male homozygous (SKH1-hr) hairless mice (12–15 weeks old), with a body weight (bw) of 35–45 g, were used for the study. The animals were housed in standard laboratories with a 12 h light–dark cycle and had free access to standard laboratory food and water ad libitum. The experiments were conducted in accordance with guidelines for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee (University of Rostock, Medical Faculty, Rostock, Germany).

Implantation of the dorsal skin fold chamber and wounding

The dorsal skin fold chamber in mice was used for intravital microscopy, as previously described by our group (Lehr et al. 1993; Amon et al. 2003; Knappe et al. 2005). Mice were anesthetized intraperitoneally with a mixture of ketamine (90 mg kg−1 bw; Ketamin™ 10%, Bela-Pharm, Vechta, Germany) and xylazine (25 mg kg−1 bw; Rompun® 2%, Bayer Health Care, Leverkusen, Germany). Two symmetrical titanium frames were implanted to sandwich the extended double layer of the skin. The creation of a full dermal thickness wound was achieved after marking the area with a standardized circular ink stamp (2.5 mm in diameter) and by removing the complete skin down to the panniculus carnosus, thus creating a wound area of 3–6 mm2 (Fig. 1C). The non-wounded skin of the opposite side still consisted of epidermis, dermis, and striated skin muscle (Fig. 1C). The wounded site was covered with a removable glass coverslip incorporated in one of the titanium frames (Fig. 1A,C). A recovery period of 3 days was allowed before intravital observation.

Fig. 1
(A,B) Observation window of the mouse dorsal skin fold chamber at day 0 after creation of a circular full-thickness wound with an area of ~5 mm2. Bars: 0.5 cm (A), 625 µm (B). Schematic illustration (C) of an implanted dorsal skin fold chamber ...

Microscopic analysis of wound repair in the dorsal skin fold chamber of the hairless mouse

Mice were anesthetized with ketamine/xylazine (90/25 mg kg−1 bw i.p.) and placed prone on a custom made plexiglas platform which was placed under an intravital fluorescence microscope equipped with a 100 W mercury lamp (Axiotech vario, Zeiss, Jena, Germany). After retrobulbar injection of 0.1 mL 2% fluorescein isothiocyanate (FITC)-labeled dextran (molecular weight 150 kD; Sigma Chemical, Deisenhofen, Germany), a blue (450–490 nm) filter set allowed the analysis of wound repair by the epi-illumination technique, using a ×20 long distance objective. Microscopic images were taken and recorded with a video system (S-VHS Panasonic AG 7350-E, Matsushita, Tokyo, Japan) for off-line evaluation via a charge-coupled device video camera (FK 6990A-IQ, Pieper, Berlin, Germany) and monitored on a television screen. The fluorescent dye was injected before each microscopic observation time point (days 3, 6, 9 and 12 post wounding). The analysis of the kinetics of the healing skin included the planimetric evaluation of wound epithelialization until complete wound closure using a stereoscopic operating microscope (Leica IC-A, Leica Microsystems GmbH, Wetzlar, Germany) in a ×40 magnification with the possibility to record the microscopic images using a video system (S-VHS ET, JVC, Tokyo, Japan) for off-line evaluation. The wound closure was considered complete when the entire surface area was covered with tissue.

Microcirculatory analysis

Within the regenerating skin we analyzed the microhemodynamic parameters of (1) microvessel diameter, (2) red blood cell velocity and (3) functional density of microvessels using FITC-labeled dextran for contrast enhancement of plasma. Quantitative off-line analysis of the videotaped images was performed by means of a computer assisted image analysis system (capimage; Dr Zeintl Software, Heidelberg, Germany). Red blood cell velocity was determined using the line-shift-diagram method (Klyscz et al. 1997). Functional density of microvessels was defined as the total length of red blood cell perfused microvessels per observation area and was given in cm cm−2. All parameters were assessed both within the re-epithelializing wound and distal of the wound, representing normal skin.

Histology and immunohistochemistry

The titanium frames were explanted at day 12 and the sandwiched skin was fixed in 4% phosphate-buffered formalin for 2–3 days and then embedded in paraffin. From the paraffin embedded tissue-blocks, 5 µm sections were serially cut and stained with hematoxylin-eosin (H&E) for routine histology. For immunohistochemical demonstration of PCNA and CD31, sections collected on poly-l-lysine-coated glass slides were treated by microwave for antigen unmasking. Rabbit polyclonal anti-PCNA (1 : 200; Novo Castra Biotechnology, Newcastle, UK) and goat-polyclonal anti-CD31 (1 : 50; Santa Cruz Biotechnology, Heidelberg, Germany) were used as primary antibodies and incubated for 18 h at 4 °C. After equilibrating to room temperature, sections were incubated with horseradish peroxidase (HRP)-conjugated goat LSAB-Kit (Dako Cytomation, Hamburg, Germany) or donkey anti-goat HRP (CD31, 1 : 200; Santa Cruz Biotechnology) for 30 min. 3,3′-diaminobenzidine was used as chromogen. Then the sections were counterstained with hemalaun and examined by light microscopy for PCNA-positive cells and CD31-positive microvessels (Axioskop 40, Zeiss, Göttingen, Germany).

Laboratory analysis

By puncture of the vena cava, blood was sampled for subsequent laboratory analysis at day 12. Blood cell counts were determined using an automatic cell counter (Sysmex KX21, Sysmex GmbH, Norderstedt, Germany).

Statistical analysis

All data are expressed as means ± sem. Differences over time were assessed by using one-way repeated measures anova, and one-way anova was used for analysis of differences between parameters at a single time point. Statistics were performed using the software package sigma-stat (Jandel Corporation, San Rafael, CA, USA) with a P value set at < 0.05.

Results

None of the animals used in the study displayed signs of discomfort or disturbances in eating or drinking habits. The chamber implantation was well tolerated by mice. Assessment of systemic blood cell count at day 12 revealed physiological values in all animals (data not shown).

Wound tissue regeneration and microvascular parameters

Wound area ranged between 2.4 and 6.0 mm2 at day 0. At the subsequent time points, planimetric analysis of the wound area on digital images showed a continuous increase in epithelialization with about 50% wound coverage at day 6 and a complete wound closure by day 12 post wounding (Fig. 2).

Fig. 2
Photomacroscopic images (A) and quantitative planimetric analysis (B) of wounds during regeneration, displaying the continuous process of wound closure with complete epithelialization at day 12. Images of the upper panel in (A) display the skin fold chamber ...

The process of wound healing revealed distinct patterns of newly formed microvascular networks with an inner and an outer ring of vessels (Fig. 3A–D). The inner ring presented with circular vessels characterized by large and irregular diameters (Fig. 3C). In contrast to the inner area, the outer area of newly formed epithelium was characterized by a more radial pattern of vessels, being smaller and less heterogeneous in diameter (Fig. 3D). All newly formed vessels showed a maturation process, as given by the decrease of their caliber over the 12-day observation period (Fig. 4). Between days 3 to 9, the diameters of the circular vessels were found to be up to 10 µm larger than the radial ones, but finally reached values of ~15 µm (Fig. 4B). In contrast, radial passing vessels started with diameters of 14.8 ± 0.3 µm and reduced their caliber towards 11 µm at day 12 (Fig. 4A). The fact that diameters of vessels distal of the wound, i.e. non-wounded skin, were found with a constant caliber of 6–7 µm indicates that vessel maturation within the wound area is not yet complete.

Fig. 4
Representative intravital fluorescence microscopic images of radial (A) and circular (B) arranged vessels in the wound re-surfacing skin. Quantitative analysis of their diameters (µm) during regeneration at days 3, 6, 9 and 12 post wounding shows ...
Fig 3
Schematic illustration (A) and representative intravital fluorescence microscopic images (B–D) of the regenerating skin. The sola cutis se reficientis (A,B) consists of an inner ring of circular arranged vessels round the wound margin (C) and ...

Functional capillary density distal to the wound ranged from 170 to 190 cm cm−2 over the whole observational period of 12 days, representing physiological values (Table 1). In contrast, the density of the newly formed vessels in the wound resurfacing skin, i.e. circular and radial proceeding vessels, was found to be significantly lower when compared with that of normal skin. In addition, the density of circular vessels was characterized by a continuous and significant decrease until day 12, implying vessel regression (Table 1). Red blood cell velocity in the newly formed microvasculature ranged between 300 and 450 µm s−1 and did not significantly differ to values in normal skin (data not shown).

Table 1
Functional density (cm cm−2) of newly formed radial and circular arranged vessels in the wound re-surfacing skin and of capillary vessels distal to the wound over 12 days of observation

Wound tissue histology and immunohistochemistry

The macroscopic finding of continuous wound closure was confirmed by histology and immunohistochemistry (Figs 57). On H&E-stained paraffin sections, representing the longitudinal diameter of the wound, growing epithelial tongues from the edge of the wounds were found, finally reconstituting the epidermal covering of the dermal defect (Fig. 5). In parallel, the provisional matrix, i.e. a fibrin mesh with many inflammatory cells initially filling the wound area (Fig. 5C), is found to be replaced by a cellular and highly vascularized granulation tissue (Fig. 5D).

Fig. 5
Representative histological H&E-stained sections of skin repair in the mouse dorsal skin fold chamber at day 3 (A,C) and day 12 post wounding (B,D). Whereas the day 3 wound (A,C) is incompletely filled with a fibrin-like granulocyte-rich layer ...
Fig. 7
Representative CD31-immunohistochemistry of the mouse dorsal skin fold chamber at day 12 post wounding (A). High magnification images of the wound granulation tissue display a high density of microvessels, presenting with CD31-positive endothelial lining ...

To further analyze dermal repair, we determined proliferation and angiogenesis in wound tissue of the mice studied (Figs 6, ,7).7). Immunohistochemistry of PCNA demonstrated high proliferative activity in the basal layers of the epithelial tongue (Fig. 6C) as well as in the granulation/connective tissue, demarcating the defect towards subcutaneous tissue of non-wounded skin (Fig. 6D).

Fig. 6
Representative PCNA-immunohistochemistry in sections of skin repair in the mouse dorsal skin fold chamber at day 3 (A,C) and day 12 post wounding (B,D). PCNA staining demonstrated high proliferative activity in the basal layers of the epithelial tongue ...

Staining of expression of the endothelial cell marker CD31 served as a read-out for angiogenesis at the wound site and revealed high microvessel density within the area of granulation tissue (Fig. 7A–C).

Discussion

During skin wound regeneration, cells recreate functional structures to repair the injured tissue. Understanding the healing process is crucial for the development of new concepts and the design of novel approaches for delivery of cells, mutation of genes and understanding the cross-talk of growth factors to accelerate tissue regeneration. For this purpose, realistic experimental models are compulsory to understand the mechanisms of tissue repair and/or regeneration.

Over the course of almost seven decades, the implantation of chambers on animals for microscopic observation and microcirculatory analysis has been one of the most established and widespread models in medical research (Algire, 1943; Goodall et al. 1965; Nims & Irwin, 1973; Menger et al. 2002; Knappe et al. 2005). More recently, a variety of in vivo (Breuing et al. 1992; Rossio-Pasquier et al. 1999; Escamez et al. 2004; Geer et al. 2004) and in vitro models (Kamamoto et al. 2003; Harrison et al. 2006) have been introduced to evaluate the process of wound healing and skin regeneration. However, only a few include direct microcirculatory analysis during the process of dermal restoration (Roesken et al. 2000; Vollmar et al. 2002; Uhl et al. 2003; Langer et al. 2006). Progress in understanding the physiology of wound regeneration and in developing new therapeutic strategies depends on the availability of suitable animal models. In general, animal models aim at mimicking human wound healing problems, with dehiscence, ischemia, ulceration, infection, and scarring (Davidson, 1998). However, differences in the architecture of the tissue, functions in the immune system and general physiology among animals in contrast to humans must be taken into consideration.

Here we present a versatile new model of skin wound healing in mice using the dorsal skin fold chamber, which incorporates some major advances. First, it allows us to visualize directly the process of revascularization, angiogenesis and vessel regression in the skin by means of intravital fluorescence microscopy with analysis of the vessel diameters, blood flow velocity and functional capillary density. Furthermore, this technique allows repetitive analysis of the ongoing process of skin repair over a long period of 2–4 weeks (Lehr et al. 1993). Moreover, identical microvascular segments can be observed repeatedly at later follow-up time points. By implantation of a skin fold chamber to the back of the mouse we could avoid wound contraction by positioning the skin in between two titanium frames, which was covered by a removable glass coverslip. In addition, intravital microscopy could be performed on a plane at tissue level. In comparison with an also regularly used wound healing model in the ear of the hairless mouse (Bondar et al. 1991) the model described here allows the assessment of different ways of wound healing by wet (with coverslip) as well as dry (without coverslip) conditions, whereas in the ear model only dry wound healing can be observed. The skin fold chamber further allows us to analyze the topical application of different substances, which is itself associated with many difficulties at the ear. Furthermore, scratch artifacts with consecutive wound healing interferences and loss of data, which regularly appears after wounding at the ear, could not be observed within dermal wound healing in the dorsal skin fold chamber. Therefore wound healing can be performed in a highly standardized fashion using the skin fold chamber technique.

Skin wound healing involves two main phenomena, which can easily be investigated with this model: (1) re-epithelialization, which involves the replication and movement of epidermal cells to reconstitute tissue continuity, and (2) novel formation of granulation tissue, which is essentially composed of small vessels, fibroblasts, myofibroblasts and inflammatory cells (Martin, 1997). With the technique of epi-illuminating microscopy we could determine an almost (99%) complete wound closure within the observation period of 12 days. While the development of wound contraction represents a conditio sine qua non in the healing of an open wound, this could not be observed with the present model, although contraction is the main factor of skin closure in rodents due to a mobile integument (Yannas, 2005). In particular the contractive function of myofibroblasts, developing tension to the connective tissue, which then shrinks in size to bring the wound margins towards one another (Tomasek et al. 2002; Hinz, 2007), could be prevented by spanning the skin double layer in between the titanium frames, which makes it wound contraction nearly impossible. Thus the model allows the study of direct wound healing processes without the influence of wound contraction by myofibroblasts.

During epidermal wound closure, the wound margin cells (mostly keratinocytes) begin to shuffle (Jacinto et al. 2001). By shuffling, keratinocytes migrate from the leading edge in the direction of the center of the defect. This is achieved by unilateral contact-inhibition with dislocation of intact epidermal cells (Laplante et al. 2001) and cell-shape changes with lamelliopodial crawling forwards over the wound matrix (Jacinto et al. 2001). Activated keratinocytes, which initiate epidermal wound repair, do not only reside along the lateral margins of a wound. Residual appendages like hair follicles as well as the sweat glands apparatus are considered to be one of the primary sources of re-epithelializing keratinocytes (Miller et al. 1998; Levy et al. 2007). After complete re-surfacing of the wound area as confirmed by histological sections, we could not observe the restoration of hair or sweat glands, proving the creation of a full dermal thickness wound, which is per se not able to regenerate hair follicles or sweat glands at the wound site (Martin, 1997) and differs by the absence of skin appendages from physiological skin (Yannas, 2005). Furthermore the repaired skin of the present study did not show formations of rete ridges for strengthening of the dermal–epidermal junction, as described by van Zuijlen and coworkers (2002). The reason for this might be the short observation time period of only 12 days, as it has been described that rete ridge formation could only be seen after more than 3 weeks (Compton et al. 1998).

Intact blood supply by microcirculation to a wounded site is an indispensable prerequisite for normal tissue regeneration (Braverman, 2000; Winter, 2006). Besides the use of the skin fold chamber model in experimental studies of inflammation and sepsis, ischemia–reperfusion, angiogenesis, and transplantation (Menger et al. 2002), the present study demonstrates that the skin fold chamber is also an ideal tool for direct analysis of microhemodynamic parameters in the healing skin. By repeated measurements over the entire healing period, a distinct pattern of microvascular changes could be observed: the regenerating skin in the chamber showed an outer radial pattern of vessels which supply an inner circular ring of vessels round the wound margin, forming a sola cutis se reficientis (Fig. 3). The newly microvascular network underwent a continuous change throughout the observational period of 12 days. It was associated with a reduction of functional microvessel density as well as microvessel diameters at the different sites round the wound margin up to day 12. Both the circular inner and the radial outer vessels presented with highest values of microvessel diameters at day 3 after wound creation, and remained higher than vessels of non-traumatized skin after complete re-epithelialization of the wounds. This maturation of vessels could also be seen after revascularization of freely transplanted ovarian follicles into the skin fold chamber (Laschke et al. 2004), whereas the inhibition of the pro-apoptotic p53 protein delayed vessel maturation in this setting (Bordel et al. 2005). Furthermore, it is speculated that microvessel density is reduced by apoptosis, caused by the replacement process of provisional wound matrix, i.e. granulation tissue, by collagen-rich scar tissue (Tonnesen et al. 2000). Besides the superficial wound coverage with keratinocytes, cutaneous microcirculation seems to support the epithelialization process by closing the horizontal organized plexuses from the inner ring of circular vessels of the initial cutting edge by continuous centripetal growth with final vessel regression. The outer radial vessels supply the circular frontline of skin regeneration by equally incessant growth to the wound center until complete wound closure.

Wound repair is influenced by many factors, making it vulnerable to disruption at many levels. The healing environment of skin wounds plays an important role and can be accelerated in a wet milieu (Svensjo et al. 2000), whereas only moist or even dry wound healing is associated with a further loss of tissue and undesirable scar formation (Breuing et al. 1992; Vogt et al. 1995). The model presented here also allows observation of wet vs. dry conditions of wound healing, by putting on a glass coverslip, as shown here, or by open and thus dry wound regeneration.

Future perspectives

Our new model for skin regeneration in the skin fold chamber in mice allows distinct analysis of new vessel formation and vessel maturation in wounded respectively regenerating skin. This implies the evaluation of the impact of different angiogenic substances to the regenerating skin as well as the influences of cytokines which are necessary to stimulate fibroplasia and angiogenesis (Tonnesen et al. 2000). In addition, intravital microscopy allows evaluation of the function of (newly formed) blood vessels as a marker for oxygenation, nutrition and cell metabolism (Braverman, 2000). The availability of different fluorescent dyes, such as rhodamine 6G or sodium bisbenzimide, for in vivo staining of diverse cells, amplifies the possibilities of intravital fluorescent microscopic analysis, from morphological analysis to the study of complex pathophysiological states of action, such as features of the essential inflammatory response during skin recovery.

In conclusion, our results demonstrate distinct changes of the microvasculature of the skin to injury by using a well established model to view the microcirculation in the mouse, in accordance with the multifaceted process of skin repair. This model was developed to facilitate the understanding of wound healing, now providing a versatile tool for prolonged quantitative analysis of normal physiological and pathologically altered wound healing.

Acknowledgments

The authors thank Mrs Dorothea Frenz for her excellent technical assistance in histology and immunohistochemistry.

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