Lijuan Huang

Aging-Like Skin Changes Induced by Ultraviolet Irradiation in an Animal Model of Metabolic Syndrome:

Both physiological skin aging and pathologic photo-aging caused by ultraviolet (UV) irradiation are mediated by latent inflammation and oxidative stress. Although numerous animal skin-aging models have used UV irradiation, most require massive doses or long-term irradiation. To establish a more refined skin-aging model, we focused on an animal model of metabolic syndrome (MS) because MS involves damage to various organs via oxidative stress or inflammation, similar to the changes associated with aging. We hypothesized that MS skin might exhibit more aging-like changes after milder, shorter-term UV irradiation than would normal animal skin under similar conditions, thus providing a useful model for skin aging. The authors therefore examined the skin from Tsumura Suzuki obese diabetic (TSOD) mice (MS model) and control Tsumura Suzuki non-obese (TSNO) mice before and after UV irradiation. Skin from TSOD mice had a thinner epidermis and dermis, a thicker fatty layer, reduced density and convolution of the fragmented collagen fibers, and upregulated expression of tumor necrosis factor (TNF)-α, a dual marker for inflammation and aging, compared to the skin from TSNO mice. UV irradiation affected TSOD skin more severely than TSNO skin, resulting in various changes resembling those in aged human skin, including damage to the dermis and subcutaneous fatty tissue, infiltration of inflammatory cells, and further upregulation of TNF-α expression. These results suggest that UV-irradiated TSOD mice may provide a new model of skin aging and imply that skin from humans with MS is more susceptible to UV- or aging-related damage than normal human skin.

Aging-Like Skin Changes in Metabolic Syndrome Model Mice Are Mediated by Mineralocorticoid Receptor Signaling

Aging is accelerated, at least in part, by pathological condition such as metabolic syndrome (MetS), and various molecular pathways such as oxidative stress are common mediators of aging and MetS. We previously developed the aging‐like skin model by single ultraviolet (UV) irradiation on the MetS model mice. Recent studies revealed that mineralocorticoid receptor (MR) signaling plays a pivotal role for various tissue inflammation and damages in MetS. Although previous studies reported that MR is expressed in the skin and that overexpression of MR in the skin resulted in the skin atrophy, the physiological or pathological functions of MR in the skin are not fully elucidated. Here, we show the involvement of MR signaling in the aging‐like skin changes in our own model. Elevations of oxidative stress and inflammation markers were observed in the MetS mice, and the UV‐evoked aging‐like skin damages were attenuated by topical antioxidant. MR expression was higher in the MetS mouse skin, and notably, expression of its effecter gene Sgk1 was significantly upregulated in the aging‐like skin in the UV‐irradiated MetS mice. Furthermore, topical application of MR antagonist spironolactone suppressed Sgk1 expression, oxidative stress, inflammation, and the aging‐like changes in the skin. The 2‐week UV onto the non‐MetS mice, the more usual photoaging model, resulted in the skin damages mostly equivalent to the MetS mice with single UV, but they were not associated with upregulation of MR signaling. Our studies suggested an unexpected role of MR signaling in the skin aging in MetS status.

Aging enhances maceration-induced ultrastructural alteration of the epidermis and impairment of skin barrier function.

BACKGROUND Skin maceration is recognized as a risk factor for the development of certain skin lesions. In health care settings, incontinence-associated skin maceration is highly prevalent in the elderly. However, the effect of senescence on maceration has not been fully elucidated. OBJECTIVE To reveal the enhancement of the maceration-induced ultrastructural alteration and barrier function of the epidermis by aging. METHODS Skin maceration was reproduced by exposure to agarose gel in human and rat. The ultrastructural alterations in human and rat tissue were observed by transmission electron microscopy. The skin barrier function was evaluated by noninvasive methods in human, and by the transdermal penetration of small- and large-fluorescent molecules in rat. In order to reveal the effect of aging on the skin maceration, we compared these parameters between young and aged rats. RESULTS In macerated skin, we observed expansion of the interstices of the stratum corneum, spinosum, and basale of the epidermis; disruption of the intercellular lipid structure in the stratum corneum; a decreased number of cell processes in the stratum spinosum and basale. The transdermal penetration test in the rat using two types of fluorescein indicated that maceration disrupted skin barrier function. Furthermore, senescence-enhanced ultrastructural and functional alterations were revealed in the rodent studies. CONCLUSION This study demonstrates that aging enhances skin maceration. Considering that maceration is a risk factor for the skin damage, the development of technology to promote skin barrier recovery after maceration in the elderly is warranted.

Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in pressure ulcer infection in rats

The impact of quorum sensing (QS) in in vivo models of infection has been widely investigated, but there are no descriptions for ischemic wound infection. To explore the role of QS in Pseudomonas aeruginosa in the establishment of ischemic wound infection, we challenged a pressure ulcer model in rats with the PAO‐1, PAO‐1 derivatives ΔlasIΔrhlI and ΔlasRΔrhlR strains, which cannot induce the virulence factor under QS control, thus the reduced tissue destruction was expended in these mutant strains. However unexpectedly, on postwounding day 3, the inflammatory responses in the three groups were similarly severe and the numbers of bacteria in tissue samples did not differ among the three strains. Biofilm formation was immature in QS‐deficient strains, defined by the absence of dense bacterial aggregates and extracellular polymeric substance, which was confirmed by scanning electron microscopy. The Pseudomonas aeruginosa QS signal, acylated homoserine lactone, was only quantified from wound samples in the PAO‐1 group. The swimming and twitching motilities were significantly enhanced in the ΔlasRΔrhlR group compared with the PAO‐1 group in vitro. A significantly larger wound area was correlated with the bacterial motility. The inflammation in the early phase of bacterial challenge to wounds with immature biofilm formation in the QS‐deficient strains indicated that the role of QS was more crucial for the chronic phase than for the acute phase of infection. The present findings indicate a difference in the importance of QS in ischemic wound infections compared with other infection models.

Changes in serum and exudate creatine phosphokinase concentrations as an indicator of deep tissue injury: a pilot study

Deep tissue injury (DTI) is difficult to detect in the early phase. Creatine phosphokinase (CPK) as a muscle enzyme could represent a promising indicator of DTI. However, serum CPK levels reflect the systemic condition rather than the local wound environment. Wound exudates can be indicative of the local wound environment. This study aimed to investigate the usefulness of CPK levels in wound exudates as an indicator of DTI. Rats were divided into control, 6 hours 10‐kg and 6 hours 20‐kg loading groups. Serum samples were obtained before wounding, and at 8 and 12 hours, and 1, 2 and 3 days after wounding, while exudate samples were obtained on days 2 and 3. Serum CPK levels were markedly increased in the 10‐kg and 20‐kg groups at 8 and 12 hours after loading compared with the baseline value and control group, but decreased to the normal level on day 1. In both loading groups, exudate CPK levels were high on day 2 and decreased on day 3. Muscle necrosis was more severe in the 20‐kg group than in the 10‐kg group by histological examination. This is the first study to indicate the potential of CPK in wound exudates as an indicator of DTI.

Histopathology of Incontinence-Associated Skin Lesions: Inner Tissue Damage Due to Invasion of Proteolytic Enzymes and Bacteria in Macerated Rat Skin.

A common complication in patients with incontinence is perineal skin lesions, which are recognized as a form of dermatitis. In these patients, perineal skin is exposed to digestive enzymes and intestinal bacterial flora, as well as excessive water. The relative contributions of digestive enzymes and intestinal bacterial flora to skin lesion formation have not been fully shown. This study was conducted to reveal the process of histopathological changes caused by proteases and bacterial inoculation in skin maceration. For skin maceration, agarose gel containing proteases was applied to the dorsal skin of male Sprague-Dawley rats for 4 h, followed by Pseudomonas aeruginosa inoculation for 30 min. Macroscopic changes, histological changes, bacterial distribution, inflammatory response, and keratinocyte proliferation and differentiation were examined. Proteases induced digestion in the prickle cell layer of the epidermis, and slight bleeding in the papillary dermis and around hair follicles in the macerated skin without macroscopic evidence of erosion. Bacterial inoculation of the skin macerated by proteolytic solution resulted in the formation of bacteria-rich clusters comprising numerous microorganisms and inflammatory cells within the papillary dermis, with remarkable tissue damage around the clusters. Tissue damage expanded by day 2. On day 3, the proliferative keratinocyte layer was elongated from the bulge region of the hair follicles. Application of proteases and P. aeruginosa induced skin lesion formation internally without macroscopic erosion of the overhydrated area, suggesting that the histopathology might be different from regular dermatitis. The healing process of this lesion is similar to transepidermal elimination.

Wound blotting: A convenient biochemical assessment tool for protein components in exudate of chronic wounds

Because wound exudate includes secreted proteins that affect wound healing, its biochemical analysis is useful for objective assessment of chronic wounds. Wound blotting allows for collection of fresh exudate by attaching a nitrocellulose membrane onto the wound surface. To determine its applicability for several analysis methods and its executability in clinical wound assessment, this study comprised an animal experiment and clinical case reports. In the animal experiment, full‐thickness wounds were created on the dorsal skin of mice, and exudate samples were collected daily by a conventional method and by wound blotting. Extremely small but adequate volumes of exudate were collected by wound blotting for subsequent analysis in the animal experiments. Immunostaining showed the concentration and distribution of tumor necrosis factor (TNF) α. The activity of alkaline phosphatase was visualized by reaction with chemiluminescent substrate. The TNF distribution analysis indicated three different patterns: wound edge distribution, wound bed distribution, and a mostly negative pattern in both the animal and clinical studies, suggesting association between the TNF distribution pattern and wound healing. Our results indicate that wound blotting is a convenient method for biochemical analysis of exudate and a candidate tool with which to predict the healing/deterioration of chronic ulcers.

Novel models for bacterial colonization and infection of full‐thickness wounds in rats

An animal model is needed to study the pathophysiology of wound infections; however, an animal model that is reproducible and clinically relevant has not previously been available. In addition, an animal model of wound colonization generated in a manner similar to the wound infection model would be useful. Here, we describe new animal models of the wound infection continuum for the characterization of essential host–pathogen relationships. We determined the conditions needed to establish rat models of stable wound colonization and infection, without the use of disturbing factors (e.g., foreign bodies or induction of diabetes mellitus). We found that the age of the rats, bacterial inoculum size, and wound location were important elements in generating reproducible, obvious, spreading wound infections. We inoculated approximately 6‐month‐old rats with 2.06 × 109 or 4.12 × 109 colony‐forming units of Pseudomonas aeruginosa to generate the wound colonization and wound infection models, respectively. Wounds were made 2 cm cranial to the greater trochanter. These clinically relevant and highly reproducible animal models can be used to investigate the mechanisms of wound infection and monitor the effect of therapeutic agents in vivo.

Altered Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Matured Rat Adipocytes in Vitro

Obesity is recognized as a risk factor for delayed cutaneous wound healing. The authors hypothesized that the secretion of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) from subcutaneous adipose tissue correlates with disorder of the healing process in obese subjects. Findings from previous studies on the expression of MMPs and TIMPs in obese adipose tissue are inconsistent. Since these conflicting results could be due to the effect of several intrinsic factors, the authors conducted a simple in vitro experiment to clarify the change in profile of MMPs and TIMPs in excessively matured adipocytes. The authors cultured the induced adipocytes under conditions of high or low glucose and with or without insulin supplementation. Oil red O staining and its dye extraction assay revealed excessive lipid accumulation in high glucose and insulin-supplemented adipocytes. Additionally, there was altered expression of adipokines, similar to the change in adipose tissue in obese subjects. Under these conditions, the expression/activity of MMP8 was promoted and the expression of MMP3 and TIMP3 was inhibited. Further studies to determine the effect of other obesity-related factors, such as insulin resistance, on MMPs and TIMPs are required.

Skin blotting: a noninvasive technique for evaluating physiological skin status.

OBJECTIVE: The skin performs important structural and physiological functions, and skin assessment represents an important step in identifying skin problems. Although noninvasive techniques for assessing skin status exist, no such techniques for monitoring its physiological status are available. This study aimed to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted proteins from inside the skin following overhydration in mice. The applicability of this technique was further investigated in a clinical setting. DESIGN: Skin blotting involves 2 steps: collecting proteins by attaching a damp nitrocellulose membrane to the surface of the skin, and immunostaining the collected proteins. The authors implanted fluorescein-conjugated dextran (F-DEX)–containing agarose gels into mice and detected the tissue distribution of F-DEX under different blotting conditions. They also analyzed the correlations between inflammatory cytokine secretion and leakage following ultraviolet irradiation in mice and in relation to body mass index in humans. MAIN RESULTS: The F-DEX in mice was distributed in the deeper and shallower layers of skin and leaked through the transfollicular and transepidermal routes, respectively. Ultraviolet irradiation induced tumor necrosis factor secretion in the epidermis in mice, which was detected by skin blotting, whereas follicular tumor necrosis factor was associated with body mass index in obese human subjects. These results support the applicability of skin blotting for skin assessment. CONCLUSIONS: Skin blotting represents a noninvasive technique for assessing skin physiology and has potential as a predictive and diagnostic tool for skin disorders.