Influence of Short-term, Repeated Fasting on the Skin Wound Healing of Female Mice

Abstract: Caloric restriction in mice has been found to retard the rate of aging, increase mean and maximum life spans, and inhibit the occurrence of many age-associated diseases. The following study was conducted to investigate the effect of short-term repeated fasting (4 consecutive days, every 2 weeks) before wound creation on skin wound healing. Mice were used for macroscopic study (n = 40) and hydroxyproline analysis (n = 10). Mice were also used for microscopic study (n = 36) and were divided equally into a fasting group and a control group. The results revealed that there were significant differences in epithelialization, contraction, healing, amount of collagen, and hydroxyproline between the control and fasting groups (P < 0.05). Based on the results, it can be argued that caloric restriction preceding the wound-healing period has the potential to accelerate the healing process.   Caloric restriction (CR) has been found to retard the rate of the aging process while extending the maximum life span among rodents, such as mice and rats.¹ Caloric restriction is reported to have many advantages. It leads to suppression of obesity, decreases damage from free radicals, increases available antioxidants and induction of heat shock proteins, decreases the toxicity and lethality of various toxic chemicals and drugs, and suppresses certain types of cancer.² Many studies using this manipulation have examined the key concept of “undernutrition without malnutrition.”³ Such investigations are challenging, as CR is likely to induce hundreds, if not thousands of biological changes making it difficult to identify which are causal.¹ Several researchers have studied the effects of CR on skin and its repair.^4–6   Skin wound healing is a continual challenge in rehabilitative medicine. Although some advances have been made to better understand its basic principles, problems in wound healing continue to cause significant morbidity and mortality.⁷ Wound healing begins immediately following an injury and proceeds with a complicated but well-organized interaction among various types of tissues and cells. Skin wound healing is composed of three different, but correlated, phases: inflammatory, proliferative, and maturation. In the inflammatory phase, the recruitment of leukocytes (ie, neutrophils and macrophages) to the wound site is the most notable feature. During the proliferative phase, the migration and proliferation of keratinocytes, fibroblasts, and endothelial cells result in re-epithelialization, contraction, and granulation tissue formation. In the maturation phase, several proteolytic enzymes degrade excess collagen in the wound site, which leads to eventual tissue repair. It has been shown that some biological substances, such as cytokines, chemokines, and growth factors are closely involved in every phase of the wound healing process.⁸ The present study examines the effect of repeated fasting on skin wound healing.

Materials and Methods

  Animals. All mice used in the study were 6-week-old female Suri mice weighing 25 g–30 g that were produced at Razi Vaccination Institute of Mashhad (Mashhad, Iran). Thirty-six mice were selected for microscopic study, half of which were randomly allocated to the control group and the other half to the fasting group. Forty mice were allocated for macroscopic study and were divided into two equal groups (20 in control group, 20 in fasting group). At day 14 post wounding, all mice undergoing macroscopic study were euthanized, and 5 control group mice and 5 fasting group mice were randomly allocated for hydroxyproline analysis. The mice were housed under pathogen-free and standard conditions with controlled humidity and temperature with alternating light/dark cycles. All mice were contained in separate cages during the study.   Fasting regimen. Fasting periods were 4 consecutive days, every 2 weeks. This cycle was repeated 4 times. During the fasting periods, fasting mice had access only to water.³ All mice in both the control and fasting (when they were out of fasting periods) groups were fed commercial lab diet (Javaneh, Mashhad, Iran). The composition of the diet was as follows: 9% water, 25.4% soybean meal as protein, 4.5% vegetable cooking oil (as fat), 4% dehydrated alfalfa meal (as fiber), 7.1% crude ash, 50% cereals (as carbohydrates), minerals, and various vitamins.   Wound creation. Following the 2-month fasting regimen, the mice were anesthetized via halothane inhalation. The lumbodorsal hair was shaved and the exposed region cleaned with 70% ethanol, after which full-thickness excisional skin wounds were created with a 9.2 mm biopsy punch. It must be emphasized that during the wound-healing period, all mice (control and fasting) had access to food and water ad libitum due to the critical role of proteins and other elements of healing.   Macroscopic evaluation. Digital photographs were taken from the wounds of the macroscopic group (40 mice) after the region had been carefully shaved to visualize the wound margin at days 0, 3, 5, 7, 11, and 14. Each wound’s scab was carefully removed with the aid of normal saline for better visualization of the epithelialization and granulation tissue area. Rulers were held vertically and horizontally close to the wound as a reference. The areas of epithelialization and granulation tissue were measured for each wound using Scion Image software. A blinded observer performed the macroscopic analysis of the images. Wound contraction, epithelialization, and healing percentages were calculated for each wound using the following formulae9: Wound contraction: 1. Wound size (mm2) at day (x) / wound size (mm2) at day 0 × 100 = percent wound size at day (x) compared with day 0. 2. Percentage wound size at day (x) to compared with day 0 – 100 = percentage wound contraction. Wound epithelialization: Size of epithelialization area (mm2) at day (x) / size of the wound (mm2) at day (x) × 100 = percentage epithelialization. Wound healing: 1. Granulation tissue (mm2) at day (x) / size of the wound (mm2) at day 0 × 100 = percentage of unhealed area compared to wound size at day 0. 2. Percentage of unhealed area compared to wound size at day 0 – 100 = percentage healing.   Histological examination. Six mice from both groups of the microscopic study were randomly euthanized by inhalation of CO₂ on days 7 and 14 postwounding at which point the entire wound was cut out. Specimens were fixed overnight in 10% neutral buffered formalin and were embedded in paraffin and 5-µm sections were cut. To evaluate the inflammatory cells and fibroblasts-fibrocytes density and descriptive evaluation, sections were stained with hematoxylin-eosin (H&E). Masson’s trichrome and van Gieson’s staining were used to evaluate collagen fibers. Slide labels were covered with tape to enable blinded evaluation. The number of neutrophils, macrophages, fibroblasts-fibrocytes, and the amount of collagen were evaluated. Cells were counted in 10 microscopic fields from the surface of the wound to beneath the connective tissue near the wound margin and at the center of the wound (magnification x1000). For collagen, each microscopic field was subjectively scored where 0 = lack of fibers and 5 = full of fibers.   Hydroxyproline analysis. Five mice from either the control and fasting groups of the macroscopic study were randomly chosen and euthanized by inhalation of CO₂ on day 14 postwounding and samples were excised. Analysis of the specimens was done by a blind observer. Samples of skin tissue were dried at 120˚C for 16 hours and then hydrolyzed in 2 mL of 6 N HCL at 100˚C for 8 hours. Five hundred µL of citrate/acetate buffer (5% citric acid, 7.24% sodium acetate, 3.4% NaOH, 1.2% acetic acid) and 2 mL of chloramine T solution (1.13% chloramine T, 8% 1-propanol, 64% citrate/acetate buffer) were added to each sample; the samples were incubated at room temperature for 20 minutes. Ehrlich’s solution ([2 mL] 10.13 g of p-dimethlamino-bezaldehyde, 41.85 mL of 1-propanol, and 17.55 mL of 70% perchloric acid) was then added, and the resulting mixture was incubated at 65˚C for 20 minutes. The samples were cooled with cold tap water for 10 minutes. Absorbance at 550 nm was measured, and the amount of hydroxyproline was determined by comparison with a standard curve.

Statistical Analysis

  Statistical analysis was performed using the SPSS version 15 (SPSS, Chicago, IL). Results were compared using Mann-Whitney U-test. Differences were statistically significant when P < 0.05.

Results

  Epithelialization and contraction are crucial to the wound-healing process. The authors compared the epithelialization, contraction, and wound healing progress of the wounds from the fasting and control groups (Figure 1). Contraction was improved in the fasting group on days 3, 5, 7, 11, and 14 post wounding (P < 0.05). Significant differences were not observed in the percentage of wound epithelialization between the two groups on day 3 (P > 0.05). Conversely, epithelialization notably accelerated in the fasting group on days 5, 7, 11, 14 after wound creation compared to the control group (P < 0.05). Significant differences in percentage wound healing between the fasting and control groups was noted on days 3, 5, 7, 11, and 14 (P < 0.05; Figure 2).   Histological evaluations of the number of cells (neutrophils, macrophages, and fibroblast-fibrocytes) and fibers (collagen and fibrin) were compared between aspects of wound sites, days of examination, and experimental groups (Figure 3). According to our histological examinations the number of neutrophil and macrophage cells in fasting group were statistically less than control group on day 7 (P < 0.05). But no significant differences were observed on day 14 (P > 0.05). Additionally, the number of fibroblast-fibrocyte cells in the fasting group was significantly more than control group on day 7 (P < 0.05), but no significant differences were observed on day 14 (P > 0.05). The number of collagen fibers was significantly more than the control group on days 7 and 14 (P < 0.05; Figures 4, 5).   Collagen synthesis should be induced at the wound site to heal the injury; therefore, the hydroxyproline content of each wound site was measured to assess collagen synthesis. Considering the results, there was a significant difference in the amount of hydroxyproline between the fasting and control groups on day 14 (P < 0.05; Figure 6).

Discussion

  More than 60 years ago, McCay’s laboratory demonstrated that caloric restriction has significant effects on increasing the lifespan of rats. Since then, numerous laboratories with a variety of rats and mice indicated this observation and have shown that reducing calorie intake (without malnutrition) significantly increases both the mean and maximum survival of rodents.¹⁰ Caloric restriction provides the only intervention tested to date in mammals (typically rodents) that retards the appearance of age-associated pathologic and biologic changes.¹¹ Although a mechanism whereby caloric restriction slows aging is currently unknown, much of the presented data suggest that calorie-restricted rodents live longer and age more slowly because they are more resistant to stress and have an enhanced ability to protect against damaging agents.¹⁰ From this point of view, the present study was undertaken to evaluate the effects of short-term repeated fasting on skin wound healing in mice.   Wound healing is a well-coordinated process that is initiated by infiltration of inflammatory cells into the wound tissue, including neutrophils, monocytes, and lymphocytes. Inflammatory cells build a defense against contaminating microorganisms by producing various proteinases and reactive oxygen species. Furthermore, inflammatory cells are an important source of cytokines and growth factors that become involved in wound healing. The cytokines and growth factors initiate the migration and proliferation and keratinocytes at the wound edge, followed by proliferation of dermal fibroblasts close to the wound. Then fibroblasts migrate into the matrix region and deposit extracellular matrix. Angiogenesis accompanies the process of wound healing, principally through the growth of endothelial cells from pre-existing vessels at wound migration.^12–14   Inflammatory cell infiltration into the wound plays an important role on removing the damaged tissue and pathogens. The inflammatory response is believed to be instrumental in supplying the growth factors, cytokines, and chemokines that orchestrate the cell movement necessary for wound healing. Macrophages that have infiltrated into wounded tissues produce large amounts of the factors. In the re-epithelialization process, macrophages produce transforming growth factor-a (TGF-a), which is a key regulator of keratinocyte proliferation at the wound site. Macrophages produce transforming growth factor-1 (TGF-b1) and vascular endothelial growth factor (VEGF), which are a principal fibrogenic factor and a potent angiogenic factor in adult skin wound healing, respectively. In other words, delayed tissue granulation, including delays in collagen synthesis and angiogenesis, might occur due to the reduced number of activated macrophages. Sardari et al⁹ reported that an absence or a decrease in macrophages at the wound site impairs tissue repair. Wing and Young¹⁵ reported that mice fasting for 48 or 72 hours showed more resistance to the intracellular pathogen Listeria monocytogenes. They suggested that the increased resistance was due to enhanced activity of the monocyte-macrophage cell line, which is in accordance with our results. Additionally, Wing and his colleagues¹⁶ showed that blood monocyte bactericidal activity was enhanced by fasting. Sogowa and Kubo³ did not find any significant differences in immune function between the fasting and control groups, but kidney, thymus, and adrenal gland weights of the fasting mice were significantly higher than those of the control group, which showed that some immunoendocrine system changes might have happened during and after the short-term repeated fasting.³ Kubo et al¹⁷ investigated the effects of acute starvation on the immune system function of mice, and their results demonstrated that immune function, including phagocytic activity of macrophages and T cell mitogens, was enhanced by a short-period of starvation. According to the present results, there might be a relationship between macrophage activity and accelerated wound healing seen in the fasting group.   Secondary intention wound healing repairs a skin defect via contraction and epithelialization. Contraction reduces the size of a wound by centripetal movement of dermis and epidermis that border the defect.^18–22 Epithelialization is the process by which cells from the epidermis at a wound edge proliferate and migrate to cover the surface of the defect.²² It begins soon after a skin injury and continues until it covers the wound granulation bed.²² Both contraction and epithelialization were accelerated in the repeated fasting group.

Conclusion

  Skin wound healing subsequent to a repeated fasting regimen was improved and promoted epithelialization and contraction while enhancing inflammatory cell infiltration and collagen synthesis within the wound bed.

Acknowledgment

  The authors thank Mohammad Azizzadeh, veterinary epidemiologist, and Enayat Shabani, PhD student of applied linguistics for their assistance in preparing this manuscript.

References

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