An ex vivo model of human skin photoaging induced by UVA radiation compatible with summer exposure in Brazil.

Skin is the largest body organ and can be affected by several factors, such as ultraviolet (UV) radiation. UV radiation is subdivided in UVA, UVB and UVC according to the radiation wavelength. UVC radiation does not cross the ozone layer; UVB cause DNA damage and is closely related to carcinogenesis; UVA radiation penetrates deeply into the skin, reaching epidermis and dermis and is considered the main promoter of skin aging, known as photoaging. In order to understand photoaging mechanisms and propose efficient therapies, several photoaging study models have been developed, each with benefits and limitations, but most of them use very high doses of UVA radiation, which is not compatible with our daily sun exposure. The objective of this work was to develop a human ex vivo photoaging model induced by UVA exposure compatible to a summer in Brazil. For this, human skin fragments were obtained from healthy donors who underwent otoplasty surgery and skin explants were prepared and placed in plates, with the epidermis facing upwards. Skin explants were exposed to UVA at 16 J/cm2 carried out by protocols of 2 or 4 exposures. Results showed an increase of oxidative damage, inflammatory cells, collagenolytic and elastolytic MMPs expression as well as a decrease of elastin expression, suggesting that the experimental model based on skin explants is able to evaluate UVA-induced aging in human skin.

Oleic acid and hydroxytyrosol present in olive oil promote ROS and inflammatory response in normal cultures of murine dermal fibroblasts through the NF-κB and NRF2 pathways.

Few studies have evaluated the effects of olive oil on normal tissues like skin and its components. Hence, we investigated whether olive oil could increase the production of ROS and oxidative damage in murine dermal fibroblast cultures in a short-term exposition. In addition, we evaluated the role of oleic acid and hydroxytyrosol, which are the two most important components of olive oil, in the associated mechanisms of action, and the metabolism of long-chain fatty acids from olive oil. To study this, neonatal murine dermal fibroblasts (NMDF) were incubated with olive oil, oleic acid, or hydroxytyrosol for 24 or 72 h. The NMDF incubated with olive oil or oleic acid showed an increase in the production of ROS after 24 h, lipid peroxidation, and protein carbonylation after 72 h, as well as increased expression of nuclear factor-kappa B (NF-κB) p65 and cyclooxygenase-2 (COX-2) after 72 h. However, NMDF treated with olive oil or hydroxytyrosol demonstrated an increase in the expression of nuclear factor-erythroid2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) after 72 h. In addition, NMDF treated with olive oil also showed an increase in the protein expression of diacylglycerol acyltransferase1 (DGAT1), which promotes triacylglycerol synthesis, and in the levels of triacylglycerols. The microscopic analysis showed Nile red-positive lipid droplets inside olive oil-treated NMDF after 72 h. Moreover, gas chromatography-mass spectrometry demonstrated high levels of oleic acid in the olive oil-treated NMDF after 72 h. In conclusion, oleic acid present in the olive oil promotes the production of ROS and oxidative damage in murine dermal fibroblasts, which leads to NF-κB p65 and COX-2 expression, while hydroxytyrosol promotes NRF2 and HO-1 expression. In addition, NMDF area capable of absorbing long-chain fatty acids derived from olive oil, which promotes the synthesis and the accumulation of triacylglycerols into cytoplasm of NMDF through DGAT1 activation.

Topical application of a commercially available formulation of vitamin C stabilized by vitamin E and ferulic acid reduces tissue viability and protein synthesis in ex vivo human normal skin.

BACKGROUND: Aqueous formulations of vitamin C stabilized by vitamin E and ferulic acid at low pH effectively protect skin against reactive oxygen species-induced damage. However, the effects of these formulations on human skin have not clearly been described. The aim of this study was to investigate whether topical application of two commercially available formulations of vitamin C alter human skin using an ex vivo model. METHODS: Human skin explants were topically treated on alternate days with commercially available formulation 1 (15% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline or formulation 2 (20% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline. Only saline was applied to control skin explants. RESULTS: Topical formulation 1 at 100%, 50%, or 10%, but not formulation 2 at 100%, 50%, or 10%, reduced the viability of ex vivo human skin compared to the control after 7, 10, and 13 days. In addition, compared to the control, ex vivo human skin treated with formulation 1 at 50%, but not formulation 2 at 50%, also decreased mRNA levels of actin and ribosomal protein L10 and gene expression of extracellular matrix components after 10 days. Furthermore, after 10 days, topical application of formulation 1 at 50%, but not formulation 2 at 50%, decreased the protein expression of proliferating cellular nuclear antigen, lysyl oxidase, ß-actin, and glyceraldehyde-3-phosphate dehydrogenase compared to the control. CONCLUSIONS: Topical formulation 1, but not formulation 2, may reduce the viability of and protein synthesis in ex vivo human skin. Those effects might be due to action of vehicle of formulation 1 on ex vivo human skin.

Short-Term Administration of a High-Fat Diet Impairs Wound Repair in Mice.

High intake of dietary fat plays an important role in obesity development in animals and humans, and prolonged intake of high-fat diet might lead to low-grade chronic inflammation. Previous study showed that diet-induced overweight delays cutaneous wound healing in both obesity-prone and obesity-resistant animals, highlighting the importance of diet composition in the wound healing process. This study evaluated the hypothesis that a short-term administration of high-fat diet could affect cutaneous wound healing. Male mice (C57/bl6) were randomly divided into standard (10% energy from fat) or high-fat (60% energy from fat) chow groups. After 10 days of diet administration, an excisional lesion was performed and the animals were sacrificed 6 or 10 days later. There was no difference in the fasting blood glucose between groups. Ten days after wounding, high-fat chow group presented increased inflammatory infiltrate, levels of inducible nitric oxide synthase and cyclo-oxygenase-2 proteins, and lipid peroxidation. The high-fat chow group presented delayed wound closure, increased amount of myofibroblasts and vessels, and decreased deposition of type I collagen. These findings support the hypothesis that short-term administration of high-fat diet exerts negative effects on mice cutaneous wound healing, due to the interference in the inflammatory phase.

Olive oil promotes wound healing of mice pressure injuries through NOS-2 and Nrf2.

The pressure injury environment is characterized by overproduction of reactive oxygen species and exacerbated inflammation, which impair the healing of these lesions. Mediterranean-like diet may be a good intervention to improve the healing of pressure injury owing to its anti-inflammatory and antioxidant components. Thus, this study evaluated the hypothesis that olive oil, as a main source of lipid in Mediterranean diet, could improve cutaneous wound healing of pressure injury in mice. Male Swiss mice were randomly divided into standard, olive oil, or soybean oil plus olive oil groups and fat represented 10% of total calories in all groups. Four weeks after the beginning of diet administration, 2 cycles of ischemia-reperfusion (IR) by external application of 2 magnets disks were performed in the dorsal skin to induce pressure injury formation. Fourteen days after the end of the second IR cycle, olive oil-based diet reduced neutrophils cells and cyclooxygenase-2 protein expression and increased nitric oxide synthase-2 and protein and lipid oxidation. Olive oil based-diet also increased nuclear factor erythroid 2-related factor 2 protein expression and collagen type I precursor protein expression. In addition, administration of olive oil-based diet promoted wound closure at 7, 10, and 14 days after the end of the second IR cycle. These findings support the hypothesis that olive oil-based diet improves cutaneous wound healing of pressure injury in mice through the reduction of inflammation and stimulation of redox equilibrium.

Topical retinol attenuates stress-induced ageing signs in human skin ex vivo, throughEGFR activation viaEGF, but notERK andAP-1 activation.

Stress-induced oxidative damage and the inflammatory response lead to degradation of collagen and elastic fibres and wrinkle formation. Topical retinol (or vitamin A) can be a strategy to attenuate the effects of stress in skin as it promotes collagen and elastic fibre production and reduces protease synthesis. This study investigated the effect of topical retinol in stressed human skin using in vitro and ex vivo models. Human skin explants were treated with high levels of epinephrine (as observed in stressed patients) and topically with retinol for 13 days. Human dermal fibroblasts were treated with conditioned medium of ex vivo retinol-treated and non-stressed (without epinephrine) human skin for 24 hours. In ex vivo human skin, retinol reversed the epinephrine-induced reduction in epidermal proliferation and differentiation, normalizing epidermal thickness. Retinol also inhibited the epinephrine-induced reduction in elastic fibre deposition and organization, restoring dermal thickness. In addition, retinol reversed the epinephrine-induced increase in c-JUN protein expression, but it did not alter extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in ex vivo human skin. Conditioned medium of ex vivo retinol-treated and non-stressed human skin presented an increased protein expression of epidermal growth factor (EGF). In human dermal fibroblasts, conditioned medium of ex vivo retinol-treated and non-stressed human skin increased protein and gene expression of fibrillin-1 and protein expression of EGF receptor (EGFR). In conclusion, topical retinol attenuates stress-induced skin ageing signs in human skin ex vivo, probably through EGFR activation via EGF, but not by the stress-activated ERK 1/2 and c-JUN pathways.

Acute Exposure to Diesel-Biodiesel Particulate Matter Promotes Murine Lung Oxidative Stress by Nrf2/HO-1 and Inflammation Through the NF-kB/TNF-α Pathways.

Air pollution caused by fuel burning contributes to respiratory impairments that may lead to death. We aimed to investigate the effects of biodiesel (DB) burning in mouse lungs. DB particulate matter was collected from the exhaust pipes of a bus engine. Mice were treated with 250 µg or 1000 µg of DB particulate matter by intranasal instillation over 5 consecutive days. We demonstrated that DB particulate matter penetrated the lung in the 250-µg and 1000-µg groups. In addition, the DB particulate matter number in pulmonary parenchyma was 175-fold higher in the 250-µg group and 300-fold higher in the 1000-µg group compared to control mice. The instillation of DB particulate matter increased the macrophage number and protein levels of TNF-alpha in murine lungs. DB particulate matter enhanced ROS production in both exposed groups and the malondialdehyde levels compared to the control group. The protein expression levels of Nrf2, p-NF-kB, and HO-1 were higher in the 250-µg group and lower in the 1000-µg group than in control mice and the 250-µg group. In conclusion, DB particulate matter instillation promotes oxidative stress by activating the Nrf2/HO-1 and inflammation by p-NF-kB/TNF-alpha pathways.

Manual Mobilization of Subcutaneous Fibrosis in Mice.

OBJECTIVE: The aim of this study was to induce the remodeling of subcutaneous fibrosis in mice by the manual mobilization of skin and subcutaneous tissue. METHODS: Seven days after the induction of subcutaneous fibrosis, mice were divided into 3 groups: control, stretch, and manual mobilization. Stretch was achieved by elongating the trunk, and manual mobilization was achieved by using the indicator fingertip of both hands, side by side, touching the back and performing a brief stretch. Stretch or manual mobilization was performed once a day for 7 days. RESULTS: Fibrosis was present in the subcutaneous tissue of control animals, whereas brief stretch and manual mobilization were found to reduce fibrosis. CONCLUSIONS: Mechanical stimulation through manual mobilization, or brief stretching, reduced subcutaneous fibrosis after tissue injury.

Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2 and NRF2 expression.

AIMS: In pressure ulcers, the synthesis of reactive oxygen species induced by ischemia and reperfusion leads to chronic inflammation and tissue damage, which impair the closure of these lesions. Caffeic acid phenethyl ester (CAPE), found in propolis, promotes cutaneous wound healing of acute lesions and severe burns. However, the effects of CAPE on wound healing of pressure ulcers have not been investigated. This study investigated the effects of CAPE administration in a murine model of pressure ulcers. MAIN METHODS: To induce pressure ulcers, two cycles of ischemia and reperfusion by external application of two magnetic plates were performed in the skin dorsum of mice. After the last cycle, animals were treated daily with CAPE or vehicle until they were euthanized. KEY FINDINGS: The nitric oxide synthesis, lipid peroxidation, macrophage migration, protein nuclear factor kappa B and nitric-oxide synthase-2 expression were increased 3 days after ulceration but decreased 7 days later, in pressure ulcers of the CAPE group compared to that of the control group. CAPE reduced the protein expression of nuclear factor-erythroid2-related factor 2 in pressure ulcers 3 days after ulceration, but increased 7 days later. Myofibroblast density was increased in the CAPE group 7 days after ulceration, but reduced 12 days later when compared to control group. In addition, CAPE promoted collagen deposition, re-epithelialization and wound closure of mice pressure ulcers 12 days after ulceration. SIGNIFICANCE: CAPE brings forward inflammatory response and oxidative damage involved in injury by ischemia and reperfusion, promoting dermal reconstruction and closure of pressure ulcers.

Time Course of the Phenotype of Blood and Bone Marrow Monocytes and Macrophages in the Lung after Cigarette Smoke Exposure In Vivo.

Alveolar macrophages play a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Monocytes are recruited from blood during inflammation and then mature into alveolar macrophages. The aim of this study was to investigate the effect of cigarette smoke (CS) at different times in lung macrophages and monocytes from blood and bone marrow in mice. Male mice (C57BL/6, n = 45) were divided into groups: control, CS 5 days, CS 14 days and CS 30 days. Five days' CS exposure induced a pronounced influx of neutrophils and macrophages in the lung associated with increased levels of keratinocyte chemoattractant (KC), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and matrix metalloproteinase (MMP)-12. After 14 days of CS exposure, neutrophil recruitment and cytokine production were greatly reduced. Moreover, chronic CS exposure led to increased recruitment of macrophages (with high expression of CD206), transforming growth factor-ß (TGF-ß) production as well as no detection of TNF-α, interleukin (IL)-6 and KC. CS can also change the monocyte phenotype in the blood and bone marrow, with an increase in Ly6Clow cells. These results show for the first time that CS can change not only macrophage polarization but also monocyte. These results suggest that continued recruitment of Ly6Clow monocytes may help the distinct renewing macrophage M2 population required for COPD progression.

Exercise prior to, but not concomitant with, stress reverses stress-induced delayed skin wound healing.

Stress-induced prolonged inflammation impairs cutaneous wound healing. Exercise may inhibit this effect via an anti-inflammatory mechanism. Our aim was to investigate the effect of moderate exercise on skin wound healing in chronically stressed mice. Mice were trained five times per week on a treadmill or received no training. Mice underwent daily rotational stress from the 6th week until euthanasia. During the 8th week, two wounds were created in the dorsum and collected 10 days later. A control group only received wounds. Exercise was performed prior to and simultaneous with stress for 2 weeks or only prior to stress. Stress increased normetanephrine levels 10 days after wounding, resulting in an increased amount of inflammatory cells and reduced expression of inflammatory cytokines as well as angiogenesis, myofibroblast differentiation and matrix deposition. Concomitant exercise and stress potentiated these effects, intensifying the delayed wound contraction. When exercise was performed only prior to stress, however, the mice showed reduced inflammatory cells in granulation tissue 10 days after wounding and improved wound healing compared with animals with exercise and concomitant stress. Moderate exercise in association with stress potentiates the stress effect; however, when exercise was performed prior to stress, wound healing was improved.

Brazilian red propolis improves cutaneous wound healing suppressing inflammation-associated transcription factor NFκB.

The use of natural products in wound healing has been extensively studied in the context of complementary and alternative medicine. Propolis, a natural product, is a polyphenol-rich resin used for this purpose. This study aimed to investigate the effect of Brazilian Red Propolis Extract (BRPE) on inflammation and wound healing in mice, using a tissue repair model. The BRPE polyphenol content was analyzed by liquid chromatography coupled to mass spectrometry (LC/MS). A full-thickness excision lesion was created, and mice were treated orally with daily doses of vehicle solution (water-alcohol solution containing 2% of ethanol, control group) or 100mg/kg of BRPE (P100 group) during nine consecutive days. BRPE chemical composition analysis showed that this complex matrix contains several phenolic compounds such as phenolic acids, phenolic terpenes and flavonoids (especially catechins, flavonols, chalcones, isoflavones, isoflavans, pterocarpans and bioflavonoids). After BRPE administration, it was observed that, when compared to the control group, P100 group presented faster wound closure (p<0.001); less neutrophils per mm2 (p<0.05) and macrophages (p<0.01) in tissue analyses, down regulation of the inflammatory transcription factor pNF-κB protein expression, and reduced production of inflammatory cytokine, such as TGF-ß, TNF-α (p<0.0001), and IL-6 (p<0.001). These findings suggest a positive role of BRPE oral administration in the wound healing process via suppressing the inflammatory response during tissue repair.

Heat delays skin wound healing in mice.

In vivo studies have shown that the combination of infrared radiation (IR) and visible light (VIS) is responsible for the activation of metaloproteinases, causing matrix degradation and damage to healthy skin. However, the role of heat originating from the VIS spectrum on wound healing remains poorly understood. Our objective was to investigate the macroscopic, microscopic and biochemical effects of heat induced by visible light on cutaneous wound healing in mice. Male mice were anesthetized, subjected to a cutaneous excisional wound and divided into two groups ( n = 10/group) exposed to 23℃ or 43℃ in a thermal chamber for 30 min every other day, for 13 days. On day 14, the animals were sacrificed, and their lesions were processed for histochemistry, immunohistochemistry and protein expression analysis. The wound area was 42% greater 11 days ( p < 0.01) and 29% greater 14 days ( p < 0.001) after wounding in the 43℃ group than in the 23℃ group. The 43℃ group presented a lower (17%) percentage of reepithelialized wounds ( p < 0.001) 14 days after wounding. The length of the epidermal gap was greater in the 43℃ group ( p < 0.01). The volume density of myofibroblasts and the number of F4/80-positive macrophages was greater in the 43℃ group ( p < 0.05). The 43℃ group showed increased protein expression of type III collagen ( p < 0.001), decreased protein expression of type I collagen ( p < 0.05), increased MMP-1 expression ( p < 0.05), and decreased MMP-2 activity ( p < 0.001). The protein expression of fibrillin-1 ( p < 0.001), MMP-12 ( p < 0.05), TGF-ß 1/2/3 ( p < 0.01) and ERK activation ( p < 0.05) was increased in the 43℃ group. Our results suggest that heat delays the stages of wound healing in mice.

Viability of randomized skin flaps-an experimental study in rats.

BACKGROUND: Randomized skin flaps are extensively used in plastic surgery, but the possibility of necrosis has challenged their use. Several studies have been conducted aiming to find ways to reduce the occurrence of necrosis. We evaluated the effects of pentoxifylline (PTX) and hyaluronidase (HLD), each alone or combined, on randomized rat skin flaps. MATERIALS AND METHODS: Fifty male Wistar rats were divided into five groups of 10 animals each: control I, control II, PTX, HLD, PTX-HLD. Substances were administered from the first to the 14th postoperative day. The necrotic area was measured on the seventh and 14th postoperative day; the animals were killed on the 14th day, when samples were collected for histologic and immunohistochemical examination. RESULTS: On the seventh day, percentage of the necrotic area was significantly reduced in PTX, HLD, and PTX-HLD animals compared with control groups. On 14th day, percentage of the necrotic area in PTX, HDL, and PTX-HLD groups was also significantly reduced compared with control groups. PTX and PTX-HLD showed a significant reduction in dermis cellularity, VV of macrophages, and myofibroblasts compared with control groups; PTX showed a significant enhancement of LV of blood vessels compared with all other groups. CONCLUSIONS: The use of each substance alone or combined increased flap viability compared with control groups. On the seventh day, PTX exhibited lower viability than HLD, whereas on the 14th day there was no difference between treated groups. PTX alone enhanced the LV of blood vessels, whereas PTX-HLD did not. However, PTX-HLD was more effective in decreasing the dermis cellularity and macrophage VV than HLD alone.

Selective inhibition of COX-2 improves cutaneous wound healing of pressure ulcers in mice through reduction of iNOS expression.

AIMS: Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are involved in chronic inflammation observed in chronic lesions. Nonetheless, neither study demonstrated if decreased COX-2 activation could promote the wound healing of pressure ulcers. Therefore, this study investigated the effect of the administration of celecoxib (a selective COX-2 inhibitor) in wound healing of pressure ulcers. MATERIALS AND METHODS: Male mice were treated daily with celecoxib until euthanasia. One day after the beginning of treatment, two cycles of ischemia-reperfusion by external application of two magnetic plates were performed in skin to induce pressure ulcer formation. KEY FINDINGS: Celecoxib administration reduced the protein expression of inducible nitric oxide synthase (iNOS), COX-2 and PGE2. The hydroperoxide levels, neutrophil and macrophage number, and protein elastase and matrix metalloproteinase-1 levels were reduced in celecoxib-treated group when compared to control group. Celecoxib administration increased myofibroblastic differentiation, re-epithelialization and wound contraction, and decreased the skin necrosis and angiogenesis. Celecoxib administration also stimulated the formation of a more organized and mature scar increasing collagen deposition and reducing tenascin-C expression. SIGNIFICANCE: Celecoxib administration improves the wound healing of pressure ulcers through decreased expression of iNOS and COX-2, which reduces wound inflammation and promotes dermal reconstruction and scar formation.

Olive oil-induced reduction of oxidative damage and inflammation promotes wound healing of pressure ulcers in mice.

BACKGROUND: The overproduction of reactive oxygen species (ROS) and exacerbated inflammatory response are the main events that impair healing of pressure ulcers. Therefore, olive oil may be a good alternative to improve the healing of these chronic lesions due to its anti-inflammatory and antioxidant properties. OBJECTIVE: This study investigated the effect of olive oil administration on wound healing of pressure ulcers in mice. METHODS: Male Swiss mice were daily treated with olive oil or water until euthanasia. One day after the beginning of treatment, two cycles of ischemia-reperfusion by external application of two magnetic plates were performed in skin to induced pressure ulcer formation. RESULTS: The olive oil administration accelerated ROS and nitric oxide (NO) synthesis and reduced oxidative damage in proteins and lipids when compared to water group. The inflammatory cell infiltration, gene tumor necrosis factor-α (TNF-α) expression and protein neutrophil elastase expression were reduced by olive oil administration when compared to water group. The re-epithelialization and blood vessel number were higher in the olive oil group than in the water group. The olive oil administration accelerated protein expression of TNF-α, active transforming growth factor-ß1 and vascular endothelial growth factor-A when compared to water group. The collagen deposition, myofibroblastic differentiation and wound contraction were accelerated by olive oil administration when compared to water group. CONCLUSION: Olive oil administration improves cutaneous wound healing of pressure ulcers in mice through the acceleration of the ROS and NO synthesis, which reduces oxidative damage and inflammation and promotes dermal reconstruction and wound closure.

Use of platelet-rich plasma in deep second- and third-degree burns.

Unfortunately burns are a common occurrence, leading to scarring or death. Platelet-rich plasma (PRP) contains many growth factors that can accelerate wound healing. We analyzed the use of PRP in deep second-degree (dSD), deep second-degree associated with diabetes mellitus (dSDD), and third-degree (TD) burns in rats. Sixty syngeneic rats divided into three groups (dSD, dSDD, and TD) were burned, half receiving topical PRP and half being used as control; 10 additional rats per group were used for PRP preparation. On day 21, the animals were sacrificed and skin biopsies were collected. dSD and dSDD wounds treated with PRP showed faster wound closure, reduction in CD31-, CD68-, CD163-, MPO-, and in TGF-ß-positive cells, and an increase in MMP2-positive cells. The neo-epidermis was thinner in the control of both the dSD and dSDD groups and granulation tissue was less reduced in the control of both the dSDD and TD groups. These results indicate that PRP can accelerate the healing process in dSD and dSDD, but not in TD burns.

Blockade of glucocorticoid receptors improves cutaneous wound healing in stressed mice.

Stress is an important condition of modern life. The successful wound healing requires the execution of three major overlapping phases: inflammation, proliferation, and remodeling, and stress can disturb this process. Chronic stress impairs wound healing through the activation of the hypothalamic-pituitary-adrenal axis, and the glucocorticoids (GCs) hormones have been shown to delay wound closure. Therefore, the aim of this study was to investigate the effects of a GC receptor antagonist (RU486) treatment on cutaneous healing in chronically stressed mice. Male mice were submitted to rotational stress, whereas control animals were not subjected to stress. Stressed and control animals were treated with RU486. A full-thickness excisional lesion was generated, and seven days later, lesions were recovered. The RU486 treatment improves wound healing since contraction takes place earlier in RU486-treated in comparison to non-treated mice, and the RU486 treatment also improves the angiogenesis in Stress+RU486 mice when compared to stressed animals. The Stress+RU486 group showed a decrease in inflammatory cell infiltration and in hypoxia-inducible factor-1α and inducible nitric oxide synthase expression; meanwhile, there was an increase in myofibroblasts quantity. In conclusion, blockade of GC receptors with RU486 partially ameliorates stress-impaired wound healing, suggesting that stress inhibits healing through more than one functional pathway.

Psychological stress-induced catecholamines accelerates cutaneous aging in mice.

Psychological stress may be an important extrinsic factor which influences aging process. However, neither study demonstrated the mechanism by which chronic stress participates in skin aging. Aim of this study was to investigate the effects of chronic psychological stress on mice skin. Mice were daily submitted to rotational stress, for 28 days, until euthanasia. After 28 days, mice were killed and normal skin was analyzed. Macroscopically, dorsum skin of chronically stressed mice presented more wrinkled when compared to that of nonstressed mice. In mice skin, chronic stress increased lipid peroxidation, carbonyl protein content, nitrotyrosine levels, neutrophil infiltration, neutrophil elastase, tissue inhibitor of metalloproteinase-1 and metalloproteinase-8 levels. Nevertheless, chronic stress reduced dermis thickness, collagen type I, fibrilin-1 and elastin protein levels in mice skin. In in vitro assays, murine skin fibroblasts were exposed to elevated epinephrine levels plus inhibitors of reactive oxygen species (ROS) and reactive nitrogen species (RNS), fibroblast activity was evaluated in a short time. In skin fibroblast culture, treatment with inhibitors of ROS and RNS synthesis abolished the increase in carbonyl protein content and lipid peroxide accumulation induced by epinephrine. In conclusion, chronic psychological stress may be an important extrinsic factor, which contributes to skin aging in mice.

Exogenous Tryptophan Promotes Cutaneous Wound Healing of Chronically Stressed Mice through Inhibition of TNF-α and IDO Activation.

Stress prolongs the inflammatory response compromising the dermal reconstruction and wound closure. Acute stress-induced inflammation increases indoleamine 2, 3-dioxygenase-stimulated tryptophan catabolism. To investigate the role of indoleamine 2, 3-dioxygenase expression and tryptophan administration in adverse effects of stress on cutaneous wound healing, mice were submitted to chronic restraint stress and treated with tryptophan daily until euthanasia. Excisional lesions were created on each mouse and 5 or 7 days later, the lesions were analyzed. In addition, murine skin fibroblasts were exposed to elevated epinephrine levels plus tryptophan, and fibroblast activity was evaluated. Tryptophan administration reversed the reduction of the plasma tryptophan levels and the increase in the plasma normetanephrine levels induced by stress 5 and 7 days after wounding. Five days after wounding, stress-induced increase in the protein levels of tumor necrosis factor-α and indoleamine 2, 3-dioxygenase, and this was inhibited by tryptophan. Stress-induced increase in the lipid peroxidation and the amount of the neutrophils, macrophages and T cells number was reversed by tryptophan 5 days after wounding. Tryptophan administration inhibited the reduction of myofibroblast density, collagen deposition, re-epithelialization and wound contraction induced by stress 5 days after wounding. In dermal fibroblast culture, the tryptophan administration increased the cell migration and AKT phosphorylation in cells treated with high epinephrine levels. In conclusion, tryptophan-induced reduction of inflammatory response and indoleamine 2, 3-dioxygenase expression may have accelerated cutaneous wound healing of chronically stressed mice.