In a previous study we found that cutaneous second-intention wound healing in healthy turtles (Trachemys scripta elegans), exposed to daily variations in ambient temperature and with free access to water, progresses slowly and with an indolent behaviour . Under the same experimental conditions topical insulin significantly modified the inflammatory response and improved second-intention wound healing of skin in turtles. One week administration of daily topical insulin resulted clinically in higher mean wound contraction and microscopically increased number of heterophils (the reptile equivalents of mammalian neutrophils) , macrophages and fibroblasts at different times along the cicatrisation process. Also, crust formation and macroscopic aspects of the wound were improved, although this observation has to be considered subjective.
Insulin is a polypeptide highly conserved phylogenetically in all vertebrates including reptiles. Antiserum specific for porcine insulin has been used to identify turtle β cells by immunohistochemistry  and to measure insulin concentrations in turtle pancreas extracts by radioimmunoassay . This homology between vertebrate insulin allowed us to choose porcine insulin for the present study. Insulin regulates a variety of biological processes including protein turnover, glucose transport, hemodynamics, and fatty acid metabolism. The result is a beneficial profile of anabolic and anticatabolic effects which are important in second intention healing [15, 25,26,27]. Cutaneous wound healing is a dynamic and complex physiologic process, as tissue repairing involves pleiotropic molecular and cellular events. The initial clot stops the bleeding and facilitates the migration of inflammatory cells, which are attracted by growth factors, cytokines and chemokines released into the area. Subsequent inflammation is essential in the early phases of wound healing; first neutrophils and later macrophages infiltrate the margins of the incision and release proteolytic enzymes than serve to clean out debris and proliferating bacteria . However, the persistence of inflammation leads to delayed healing in ulterior phases . In the current study, topical insulin induced morphological changes that resulted in statistically significant higher mean numbers of heterophils, macrophages and fibroblasts at several time-points, and was associated with a significantly lower density of lymphocytes at 21 DPW in the inflammatory infiltrate. These findings demonstrate that topical insulin has the capacity to modify the inflammatory response of turtle skin after injury, in accordance with previous studies in non-diabetic mammals [15, 16, 18, 25, 26, 30].
In this experimental study topical insulin significantly increased the number of heterophils until 7 DPW. Few studies have investigated the influence of topical insulin on neutrophils during wound healing. Chen et al.  reported suppressed infiltration of neutrophils and decreased healing time in mice wounds treated with topical insulin. These results can be considered contradictory since early infiltration of neutrophils is an essential first step of the healing process and the improved healing was attributed to an insulin-induced increased function of mice neutrophils. In contrast, we found a higher number of heterophils during the first 7 DPW coinciding with insulin application, and this increase in heterophils was associated with higher numbers of macrophages and with faster wound contraction at 28 DPW. Although reptile heterophils are considered equivalent to mammals’ neutrophils , our discrepancies with the report of Chen et al.  could be explained by a different cellular response of reptile heterophils to topical insulin as well as by differences in study design as in mice, neutrophil counts were determined just during the first 3 days after injury.
Previous reports in mammals have demonstrated that macrophages produce several growth factors and cytokines that stimulate fibroblasts and keratinocytes resulting in enhanced granulation tissue formation and keratinocyte migration (re-epithelisation) . Our results, both for macrophages and fibroblasts, are consistent with these studies. Chen et al.  reported that insulin induced a noticeable increased infiltration of macrophages in the first 3 days after wounding, as macrophage number in the insulin-treated wounds was the same at day 2 as at day 3 in the control wounds. A similar response was seen in the present study on turtles. The enhanced macrophage infiltration into the wound area indicates that insulin promotes wound healing by up-regulating wound inflammatory response, specifically the quantity and function of macrophages. The effect of topical insulin would be comparable with cytokines with the ability to activate macrophages, such as interleukin 1β and macrophage-activating lipopeptide-2 that have been successfully used to promote wound closure [32, 33].
The dynamics of lymphocyte mean counts was similar to that of macrophages with higher means at 7 and 14 DPW but differences were not statistically significant due to the high standard deviation of the data. Probably, a larger sample might have achieved statistical significance. Nevertheless, a particular feature of the lymphocytic response was the pronounced drop in lymphocyte counts from day 14 to day 21 DPW that, coinciding with an increase of the mean lymphocyte count in the control group, resulted in a significant lower mean count of lymphocytes in the treated group. In our opinion, the faster and higher increase of lymphocytes and the earlier reduction of the lymphocytic infiltrate in the treated wounds are another indication of the positive effect topical insulin exerted on the inflammatory response.
The significant increase in fibroblast mean counts found in turtle insulin-treated wounds is in agreement with previous findings in mammals [16,17,18, 26]. Faster wound healing and increased wound tensile strength was observed in rats treated with topical insulin, and histological analysis of wound tissue revealed an earlier appearance of collagen fibres with more compact, dense and well-oriented morphology compared with control animals . In amphibians (Ambystoma mexicanum) it was shown that insulin can stimulate sulphate uptake and growth of cartilage as in mammals . The mechanisms by which insulin exerts these effects are not fully understood but it is known that insulin can stimulate a variety of cellular functions important in tissue repair including collagen synthesis in skin fibroblasts . In general, growth factors such as insulin can act as chemoattractants to recruit important cells such as leukocytes and fibroblasts into the wound area, stimulating angiogenesis, ECM formation and degradation, and cytokine release [18, 36, 37]. On the other hand, re-epithelisation was quite similar in both groups, although since 7 DPW insulin-treated wounds showed a better differentiated and defined new stratified epithelium and BMZ respectively. Previous research in mammals has demonstrated that topical insulin improves tissue repair through stimulation of keratinocyte migration and insulin signalling pathways [38, 39]. In their study, Liu et al. , observed that the skin wounds of rats treated topically with insulin healed faster, the surface cells in the epidermis covered the wound more quickly, and the cells in the dermis rebuilt blood vessels more rapidly. It was also shown that topical insulin stimulated the proliferation and migration of keratinocytes and the migration of microvascular endothelial cells.
The ability of topical insulin in turtles to increase the number of fibroblasts would be a major contribution to explain the faster wound contraction observed in treated animals. Wound contraction is a major component of second-intention wound healing and the pivotal feature for contraction is granulation tissue formation and remodelling [7, 8, 28]. As has been described in snakes , turtles form a persistent dried crust over the wound bed and healing is characterized by epithelialization under the crust which decreases in thickness as the dermis fills the skin defect, meanwhile contraction is very limited . Thus, the significantly higher wound contraction at day 28 in the insulin-treated wounds is a remarkable finding that could be explained by the ability of topical insulin to modulate the inflammatory response increasing macrophage infiltration, granulation tissue and collagen deposition in turtles as has been described in mammals [16, 30]. At day 28 mean wound contraction in the insulin-treated group was 24.26% higher than in the control group. A greater difference after 28 days would have been desirable but in our opinion is large enough to consider topical insulin potentially useful, alone or combined with other growth factors, in the treatment of non-healing wounds of reptiles. One weakness of the present study was its preclinical condition; for practical reasons we could not include diseased animals or animals with non-healing wounds. However, as the potential benefit of insulin or other growth factors has not been investigated before in skin wounds of reptiles, a preclinical study was necessary before undertaking clinical studies to determine the real usefulness of topical insulin.
Depending on its concentration, the amount of topical insulin may have systemic effects. Previous studies have revealed the benefits of topical insulin treatment for injuries to healthy and diabetic mammals [11, 16, 18, 26], but the concentrations used have been variable. In rats, a recent study assayed different concentrations of insulin and determined that the doses that induced the best effect in wound healing were 0.5 IU and 1.0 IU/100 g. The dose of 1.0 IU/100 g, in some animals, induced alterations in plasma glucose. Therefore, a cream with a concentration of 0.5 IU/100 g was used for all experiments . However, another study also in rats used topically regular Humulin® (Eli Lilly Turkey, Istanbul, Turkey) at a concentration of 100 IU/mL without any systemic effect . The use of vehicles that enhance insulin absorption and provide longer contact times may explain the striking differences in insulin doses between studies. In the present study, we chose glycerol as vehicle because it is soluble with aqueous insulin, hygroscopic, cosmetically acceptable and has a high viscosity index. In preliminary experiments, we tested different concentrations of insulin and found that, compared with non-treated animals, a concentration of 5 IU/ml administered for 1 week improved wound contraction without influencing plasma glucose concentrations. As a topical solution of this concentration provided a sufficient local effect, higher concentrations were not assayed. A daily application during the first week of cicatrization was considered practical and appropriate since the inflammatory reaction occurs rapidly after the injury and because turtle skin wound healing is characterized by epithelialization under a heavy crust that prevents a longer insulin application.
Second intention cutaneous healing in mammals is characterized by fibrosis, and lack of hair follicles and glands in the regenerated dermis . The ability to heal without scarification has been studied in several species of reptiles with different outcomes. Recently, in leopard geckos, 3 mm full thickness skin wounds to the tail and body resulted in a scar free healing characterized by a delay in blood vessels formation and no fibrosis; the authors concluded that a proportional vascular response was a key factor to avoid scar formation . In the present report, 6 mm full thickness skin wounds healed by granulation tissue formation resulting in fibrosis still present after 28 days, both in insulin treated and control wounds. This finding was in agreement with our previous study describing normal skin wound healing in the same turtle species. Although the epidermis and pigmentation of the healed area was indistinguishable from non-damaged skin, variable degrees of fibrosis were still present in the restored dermis after 135 days post-wounding . In other reptiles such as common garter snakes, skin wounds also form scar tissue and scale regeneration is incomplete , as has been reported in a more recent research on the regeneration of scales in tail and body skin of lizards . In this study, using a similar full thickness model of skin wounds, either in lizards able to tail regeneration (A. carolinensis) or in lizards poorly capable of tail regeneration (I. iguana), the new scales were irregular and smaller than normal and showed a different shape and pigmentation compared with normal scales .