Mast Cell Morphometry and Densitometry in Experimental Skin Wounds Treated With a Gel Containing Adelmidrol: A Placebo Controlle

Disclosure: This work was supported by the Royal Veterinary College (London, UK) research funds and by Innovet Italia Srl (Milan, Italy). The study was conducted at the Department of Animal Pathology, University of Pisa (Pisa, Italy). Alda Miolo is employed by Innovet Italia Srl and is in charge of the Documentation and Information Centre for the company.

Wound healing is a highly complex biological process that results from the interplay of different tissue structures and large numbers of infiltrating and resident cells.^1,2 Among these, mast cells (MCs) appear to be pivotal.^3,4 They are located near blood and lymphatic vessels and nerve fibers, the latter having functional interaction with MCs.^5–8 Mast cells are particularly numerous in places where there is interaction with the external environment (ie, the skin) because of their involvement in the immune response, inflammatory reaction, and the tissue regeneration; their numbers increase in lesional skin.^9,10 Recent data have demonstrated that MCs play important roles in the 3 phases of wound healing: the initial clot formation and early leukocyte recruitment (inflammatory phase), the subsequent development of granulation tissue, angiogenesis, and re-epithelialization (proliferating phase), and finally matrix deposition and remodeling (remodeling phase).^3,11 MCs are activated during early wound healing and release a plethora of biologically active mediators (eg, cytokines, growth factors, neurotrophins, chemotactic factors, proteases) that are able to control the key events of wound healing.¹² Most scar reactions produce an appropriate amount of connective tissue to fill the damaged dermis; however, in certain instances, excessive fibrous tissue may be deposited leading to the formation of pathological scars, in which MCs are known to be involved.¹³ It has also been reported that deregulation of MC activity can lead to a delay in wound healing.¹⁴ Therefore, it seems that in physiological and controlled degranulating conditions, MCs act as coordinators of skin wound healing, whilst in cases of uncontrolled massive degranulation (hyper-degranulation) they may behave as dysregulators of the healing process, leading to chronic disturbances and altered proliferative dynamics.^11,15 MC regulation, therefore, seems to be a critical issue and hence the development of new wound management approaches aimed at modulating MC degranulation could be of great benefit in optimizing the wound healing process. Adelmidrol is the International Non-proprietary Name (INN) of the diamide derivative of azelaic acid, azeloyldiethanolamide, and is a synthetic N-acylethanolamine (NAE). NAEs are endogenous compounds, occurring at low levels in virtually all-mammalian cells, tissues, and certain body fluid.¹⁶ Endogenous NAEs are synthesized and released by neuronal, immuno-inflammatory, and cutaneous cells.^17–18 NAEs are not stored in vesicles like other mediators, but are produced “on demand”¹⁹ and are known to increase in peripheral tissues exposed to various stressors.^20–22 Such increases are believed to protect cells and tissues against excessive propagation of the inflammatory response.^23,24 Synthetic analogues of N-acylethanolamines, whose parent compound is palmidrol (N-palmitoylethanolamine, PEA, N-(2-hydroxyethyl) hexadecanamide), act by a mechanism that has been referred to with the acronym ALIA (Autacoid Local Injury Antagonism).^25,26 They are also called aliamides for this reason.^27,28 In particular, they have been shown to down-modulate MC degranulation,²⁹ probably by means of the activation of a peripheral cannabinoid receptor functionally expressed both on MC lines and in human skin MCs.^30–32 Adelmidrol is an aliamide particularly suitable for topical application due to its amphipathic properties and it has been included in a gel coded ADL 20, currently under development (by Innovet Italia Srl, Milan) as a topical aid to wound healing. This study was designed to examine the effect of the tested gel on morphometric and densitometric features of MCs and MC counts during the healing of experimental skin wounds.

Materials and Methods
Ten healthy experimental beagles, 8 male and 2 female, aged between 2 and 9 years, were included in the study. The animals were housed in individual kennels and monitored regularly. Ethical and regulatory approval was obtained from the Royal Veterinary College Ethics Committee and United Kingdom Home Office. Under general anesthesia, 2 rows of 6 full-thickness skin wounds were created in the clipped and aseptically prepared skin of the 2 dorsal paramedial thoracolumbar areas using 5-mm biopsy punches. The wounds were not sutured, but excess blood was removed with sterile cotton swabs. Immediately after obtaining the skin biopsy specimens, wounds were allocated randomly into pairs (treatment or control groups) and were allowed to heal by secondary intention. On the treated wounds, the gel containing the active ingredient (adelmidrol 2%) was smeared 3 times daily (8:00 am, 1:00 pm, and 7:00 pm) until the end of the study. The control wounds were treated at the same times with the vehicle only. Two 8-mm biopsy punch samples were taken at the sites of the healing wounds (1 treated and 1 control) on days 1, 2, 4, 8, and 14 (yielding 10 biopsies per dog) for histological evaluation, while the last 2 wounds were left for clinical evaluation. The wound sites in each group were randomly selected at each sampling time to allow for accurate analysis in light of the fact that wounds at the cranial aspect of the dogs’ trunk heal faster than caudal wounds.³³
All biopsies, a central 5-mm wound core and 2 lateral parts of 1-mm wide adjacent uninjured skin, were vertically excised through the middle, fixed in 4% buffered formalin solution (pH 7.4), paraffin embedded, and routinely processed for light microscopy. The 5-mm tissue punches harvested at day 0 were processed and utilized as normal skin controls.
Mast cell counts. Sections were stained with the metachromatic dye toluidine blue (TB) to detect the intracytoplasmic MC granules. Toluidine blue has been used previously and was shown to be a reliable stain for MC identification, morphometry, and densitometry.34 Morphometric analysis was performed with a Quantimet 500 computerized analyser (Leica Microsystems S.P.A., Milan, Italy). MCs are commonly found in close apposition to appendages.¹¹ Recently it has been found that they accumulate at the wound edges, but not in the granulation tissue in the late phase of wound healing.^35,36 Therefore, 3 different anatomical locations were considered for each section during morphometry (the dermal MCs in the lateral wall of the ulcer bed, the perifollicular MCs in the same location, and the MCs located in the granulation tissue/panniculus [Figure 1]).

MCs were counted in consecutive fields through a 40x objective lens for each of the locations. Counted fields ranged from 5 to 30 for each anatomical location according to the biopsy dimension, each field corresponding to 0.03 mm². The mean number of MCs was calculated and expressed as TB positive cells/mm² for each anatomical location.
Mast cell densitometry. Densitometric evaluation of intracytoplasmic granule content was performed using essentially the same analyser and technique, as previously reported.³⁴ Color images were derived with a videocamera from the microscope through a 40x objective lens and displayed in black and white on a computer screen. MCs in 2 anatomical locations were considered, those in the lateral wall of the ulcer bed and those in the granulation tissue/panniculus. Five MCs were evaluated for each location. MCs in the perifollicular location were not taken into account because of the technical difficulty in retrieving 5 MCs with a broad enough cytoplasm to allow for densitometric evaluation. Only granule content of MCs in the dermis was assessed in the control skin. The video signal for each pixel ranged from 0 (black) to 257 (white). The digitalized black and white images were inverted in order to give higher numeric values to higher granule contents. The mean value of gray level intensity was calculated in 5 intracytoplasmatic fields for each MC. Mean values expressed as pixels obtained were thus considered an estimate of the granule content.
Statistical analysis. All values are expressed as least square mean ± SE (standard error). Data were transformed to square roots, with Bartlett’s correction for morphometry, and analyzed using a factorial model. Differences were evaluated by the Tukey-Kramer HSD test.

Results

MCs were readily identified in TB stained sections by their metachromatic granules. MC numbers/mm2 in normal skin (5-mm punch wounds created at day 0) were 10.4 ± 3.24 and 6.2 ± 2.78 in the lateral dermis and in the perifollicular areas, respectively. The mean numbers of MCs/mm2 obtained in the different anatomical locations in control and treated wounds are shown in Table 1. No statistically significant differences in MC counts were observed between control and treated wounds in each of the 3 anatomical locations, either when compared on the same day, or when the overall mean of MC number was considered. Although not statistically significant, the lateral dermis trended toward decreased values of MC numbers in both control and treated wounds throughout days 1, 2, and 4 followed by an increase in subsequent days (Table 1). In the perifollicular area of treated wounds, the number of MCs at days 1, 2, and 4 tended to be lower than in normal skin (Table 1). Both in the lateral dermis and in the perifollicular area, the number of cells on day 14 was higher than in previous days. Although the data were not significantly different, both control and treated wounds followed this trend. At days 1, 2, 4, and 8 MCs in the granulation tissue of both treated and control wounds tended to be less numerous than in normal skin (both lateral and perifollicular areas) and almost reached normal values at day 14 (Table 1).
In normal skin, mean densitometric MC granule content was 126.3 ± 7.01 (pixel). The results of densitometric analysis of MC content during wound healing at days 1, 2, 4, 8, and 14 in treated and control wounds are reported in Table 2. The overall mean granule content was significantly greater in treated compared to control wounds in both lateral dermis (P < 0.01) and granulation tissue (P < 0.01). Moreover, significantly higher values were observed at days 2 and 14 in the lateral dermis (P < 0.05). In treated wounds, a steady level of granule content was observed throughout the wound healing process in both lateral dermis and granulation tissue. Conversely, in control wounds, a trend toward decreased values was observed starting from day 1 throughout the healing process.

Discussion
In the present study, morphometric and densitometric methods were used in an experimental model of skin wounds to investigate the effect of a topical NAE (the aliamide adelmidrol) on MCs during wound healing. Computerized image analysis has been reported as a useful and reliable method to assess changes in the number of MCs and to estimate their granule content.^34,37 Although most of the literature dealing with MC counts carries data that are not comparable because of the methods or the staining techniques used,^38,39 MC numbers in normal skin reported in the present study are consistent with previously published reports, both in animals and humans.^40,41 The results of the morphometric study on MCs in the authors’ experimental model reveal a temporary decrease of MC numbers during the initial phase of wound healing that is followed by an increase. This was especially true in the lateral dermis, and is in accordance with results of previous studies.^42,43 Several hypotheses have been proposed to explain the numerical changes of these cells during wound healing. The most reliable of these is based on studies performed on toluidine blue-stained ultrathin sections.⁴⁴ The early decrease of toluidine blue-stained cells suggests that they are massively degranulated during the early phase of wound healing and, therefore, not detectable subsequently by microscopic examination. Ultrastructural studies document the presence of the so-called phantom cells^45,46 (ie, completely degranulated MCs) surviving throughout the early phases of wound healing. It has been suggested that the MC number increase during the later phases might be due to re-granulation of resident cells.^47,48 However, based on the densitometric results, an influx of newly recruited immature cells might also have accounted for the increase in MC numbers after the initial decrease. Further studies are needed to test this hypothesis. As for the granulation tissue, it may be speculated that the differences observed in MC counts in comparison with the other two selected locations (ie, lateral dermis and perifollicular area) depend on a gradual recruitment of MCs from circulation and from nearby tissues.⁴⁹ MC numbers did not significantly differ between treated and control wounds, suggesting that adelmidrol does not influence viability of skin MCs. The result is consistent with the authors’ previous work examining the effect of the orally active adelmidrol congener (palmidrol) on MCs in cats with eosinophilic skin conditions.³⁴ The aliamide treatment had no effect on MC number. Taken together, the previous and present results suggest that the ALIA mechanism differs markedly from other drugs known to decrease MC viability, such as glucocorticoids.⁵⁰ It is also interesting to note that one adverse effect of glucocorticoids is delayed wound healing.⁵¹ Moreover, it is known that wound closure is significantly impaired when MCs are absent.^36,52
This study is the first to report the densitometric values for MCs normally present in the skin of the canine dorsum. The comparison between the overall mean densitometric values in treated versus control wounds showed a statistically significant increase of intracytoplasmatic granular content of MCs in treated wounds compared to control, both in lateral dermis and granulation tissue. These data demonstrate for the first time that, in the present model, adelmidrol is effectively able to down-modulate skin MC degranulation in dogs during wound healing, further substantiating in this species, the experimentally proven mechanism of action of aliamides (ie, the down-modulation of mediator release by immune-inflammatory cells).^30,53,54 Previous reports have shown that palmidrol acts in the same way on skin MCs in eosinophilic lesions of cats.³⁴ MC degranulation of preformed mediators (eg, histamine, IL-4, and NGF) is known to influence both the immediate-early events at the wound site (eg, neutrophil recruitment) and granulation tissue formation by increasing fibroblast migration/proliferation.^12,55,56 Additionally, it has also been suggested that MC chymase may play an important role in the formation of granulation tissue.¹² Furthermore, an imbalance between MC subpopulations (chymase- and tryptase-positive cells) was thought to participate in wound-healing defects.^14,57

Wound healing is impaired both by diminishing the inflammatory response (eg, via corticosteroids) and by an excessive inflammatory response.⁵⁸ Since MCs play a pivotal role throughout the entire wound healing process, it is suggested that the down-modulation of MC degranulation exerted by adelmidrol may stringently regulate responses involved in tissue repair, particularly in those cases in which the process is pathological (Figure 2).³ The hypothesis is sustained by a preliminary report that showed a greater percentage area of peri-lesional skin to be occupied by elastic fibers in adelmidrol-treated wounds compared to control.⁵⁹ Moreover, the positive clinical effect of adelmidrol has recently been reported both in terms of reduction of wound area and wound volume and in terms of functional/aesthetic outcome of complicated skin wounds in animals.^60–62 Conversely, it has been shown that blocking MC degranulation (eg, by the MC stabilizer disodium cromoglycate) retards wound healing and decreases collagen content in healing wounds.⁶³ Finally, the tested aliamide has also given good clinical results in a pilot study on pediatric atopic dermatitis,⁶⁴ a disease strictly associated with MC degranulation.⁶⁵ Interestingly, a cream with the adelmidrol congener palmidrol as the active ingredient was FDA approved for the treatment of inflammatory dermatoses, and is now included among the topical alternatives to corticosteroids of value to the dermatology practice.⁶⁶ The cream has been successfully tested on pruritus, a sign greatly dependent on MC activation.^18,67–72

Conclusion
The results of this present study and other presented data suggest the potential use of aliamides in wound healing, and more generally, use in all skin diseases in which MCs play a pivotal role. One can speculate that the effects of adelmidrol observed in the present study may reflect the consequences of supplying the skin with a sufficient quantity of physiological regulators of cellular homeostasis, as already suggested by Levi-Montalcini et al,²⁶ for the adelmidrol congener, palmidrol. Although further studies are clearly needed to evaluate the effect of adelmidrol in wound healing disturbances, the results observed in this animal model further suggest the contribution of MCs in the pathophysiological wound healing process, and substantiate the mechanism of action of adelmidrol (ie, the down-modulation of MC degranulation), namely the ALIA mechanism.

Acknowledgments
The authors thank Vincenzo Miragliotta for his excellent help with artwork and Anthony Carelse for his valuable language revision.