小鼠Ⅲ度烧伤模型的复制及皮肤病理形态改变的动态观察

目的利用近交系小鼠建立方法简便、效果稳定的Ⅲ度烧伤模型,对其皮肤病理改变进行动态观察,为烧伤实验治疗研究提供资料。方法小鼠实验前备毛,乙醚麻醉固定小鼠四肢,苏醒后于备毛部覆盖自制控制烧伤面积硬纸片,滴加95%酒精,点燃计时;烧伤早期和远期取皮肤进行病理检查,用Ki67抗体免疫组化观察细胞增殖。结果按本方法建立小鼠Ⅲ度烧伤模型,面积准确,深度一致,操作简便易行。小鼠烧伤皮肤病理改变与人类基本一致,但有其特点：小鼠Ⅲ度烧伤包括表皮至脂膜肌,烧伤后炎性渗出较晚,约需2 d;创面周围毛发生长活跃,倾向创面,Ki67抗体阳性细胞集中于毛囊底部。结论为利用小鼠烧伤模型进行实验治疗的研究提供了有实用价值的资料。     

Using Raman Spectroscopy in Studying the Effect of Propylene Glycol,Oleic Acid,and Their Combination on the Rat Skin

The permeability enhancement effect of oleic acid (OA) and propylene glycol (PG) as well as their (1:1 v/v) combined mixture was studied using rat skin. The percutaneous drug administration is a challenge and an opportunity for drug delivery. To date, there is limited research that illustrates the mechanism of penetration enhancers and their combinations on the skin. This project aims to explore the skin diffusion and penetration enhancement of PG, OA, and a combination of PG-OA (1:1 v/v) on rat skin and to identify the potential synergistic effect of the two enhancers utilizing Raman spectroscopy. Dissected dorsal skin was treated with either PG or OA or their combination for predetermined time intervals after which the Raman spectra of the treated skin were collected with the enhancer. A spectrum of the wiped and the washed skin were also collected. The skin integrity was tested before and after exposure to PG. The skin histology proved that the skin integrity has been maintained during experiments and the results indicated that OA disrupted rat skin lipid as evident by changes in the lipid peak. The results also showed that PG and OA improved the diffusion of each other and created faster, yet reversible changes of the skin peaks. In conclusion, Raman spectroscopy is a potential tool for ex vivo skin diffusion studies. We also concluded that PG and OA have potential synergistic reversible effect on the skin.     

Muc5ac Mucin Expression During Rat Skin Development

Some mucin genes have been detected during human embryonic and fetal organ development; however, little is known about mucin expression in epidermal development, neither in humans nor in other species. The present research was developed to explore Muc5ac skin expression during pre- and post-natal rat development. Immunohistochemistry (IHC), Western blotting (WB) and RT-PCR were employed. By IHC, Muc5ac protein was found early in embryonic epidermis from day 13 of gestation until seven days after birth when the surface epidermis became negative and the reaction was restricted to secreting sebum cells. In coincidence with IHC findings, WB analysis showed a band at approximately 200KDa at the same periods of development. Results were also confirmed by RT-PCR.Muc5ac expression in rat embryonic epidermis suggests that Muc5ac may play a protective role in embryonic skin previous to birth which may be replaced by pile covering. To our knowledge, this is the first report that confirmed Muc5ac expression during skin development.Key words: Muc5ac, skin, rat, development     

Effect of gramicidin on percutaneous permeation of a model drug

This study investigated the enhancement effect of gramicidin, a cationic ionophore, on percutaneous absorption of a model drug, benzoic acid (BA), through rat abdominal skin. The mechanisms by which gramicidin increased skin permeability to BA were also investigated. Degree of hydration measured by the Karl Fisher method, the concentration gradient measured by cryostat analysis, and lipid concentration measured by the. Fiske-Subbarow method were evaluated and compared. The results showed that BA permeation profiles through rat abdominal skin followed dose- and volume-dependent patterns. The pretreatment of gramicidin increased the permeation rate of BA through rat abdominal skin compared with the untreated control (18.89 vs. 10.86 μ g/cm² hour). Change in skin permeation rate of BA after gramicidin pretreatment was closely correlated with the remaining skin water content. There were no significant differences in the amounts of phospholipid phosphorous between gramicidin pretreated and untreated skin. The enhancing effect of gramicidin on percutaneous absorption of a model drug is mainly a tributed to increasing the diffusivity in the hydration domain of the skin and rearranging the lipid bilayer in the stratum corneum.     

Role of lipid peroxidation and alpha-tocopherol in the conductivity of model membranes from phospholipids of the liver of rats with burns]

The membrane conductivity for K+ and Ca+2 ions was studied on bilayer phospholipid membranes formed by phospholipids extracted from the rat liver 1 hour, 1, 3, 7, and 15 days after burning. A pronounced increase in the membrane conductivity was noted. The most potent effects were seen by the 1t, 3d and 7th day of the experiment and the conductivity value varied depending on the medium pH. The process was accompanied by lipid peroxidation increase. Intraperitoneal administration of alpha-tocopherol at 1 mg/kg dose immediately after burning, followed by injections on the 3d, 7th and 12th day, normalized the characteristics studied. Bilayer membranes formed by healthy rat liver phospholipids previously added with an appropriate amount of methyl oleate or cumylhydroperoxide demonstrated higher conductivity as compared to the normal.     

不同林地清理方式对杉木人工林生态系统的影响

在杉木中心产区福建尤溪建立径流场,进行不同林地清理方式对杉木人工生态系统影响的６ａ定位研究结果表明：炼山清理迹地导致林地严重的水土流失,炼山后的６ａ中要地的水、土、肥人分别达８７６７．３２ｍ＾３／ｈｍ＾２、３８．００ｔ／ｈｍ＾２、５２３．１６ｋｇ／ｈｍ＾２,分别是不炼山林地的３．１０、１９．７０、６．１０倍。两种清理方式林地流失量差异随时间推移逐年缩小,于第６年趋于一致,炼山具有短期激效效应,经雨     

Development of a dermal matrix from glycerol preserved allogeneic skin

Dermal substitutes can be used to improve the wound healing of deep burns when placed underneath expanded, thin autologous skin grafts. Such dermal matrix material can be derived from xenogeneic or human tissue. Antigenic structures, such as cells and hairs must be removed to avoid adverse inflammatory response after implantation. In this study, a cost-effective method using low concentrations of NaOH for the de-cellularization of human donor skin preserved in 85% glycerol is described. The donor skin was incubated into NaOH for different time periods; 2, 4, 6 or 8 weeks. These dermal matrix prototypes were analyzed using standard histology techniques. Functional tests were performed in a rat subcutaneous implant model and in a porcine transplantation model; the prototypes were placed in full thickness excision wounds covered with autologous skin grafts. An incubation period of 6 weeks was most optimal, longer periods caused damage to the collagen fibers. Elastin fibers were well preserved. All prototypes showed intact biocompatibility in the rat model by the presence of ingrowing blood vessels and fibroblasts at 4 weeks after implantation. An inflammatory response was observed in the prototypes that were treated for only 2 or 4 weeks with NaOH. The prototypes treated with 6 or 8 weeks NaOH were capable to reduce wound contraction in the porcine model. In neo-dermis of these wounds, elastin fibers derived from the prototype could be observed at 8 weeks after operation, surrounded by more random orientated collagen fibers. Thus, using this effective low cost method, a dermal matrix can be obtained from human donor skin. Further clinical studies will be performed to test this material for dermal substitution in deep (burn) wounds.     

Changes in human burn fluid biological activity during normal burn healing

The main goal of this work was monitoring the changes occurring in human burn fluid biological activity during normal burn healing. The fluid available in the burn until healing makes a good material for controlling biochemical microenvironment of burn cells. This environment involves factors, such as extracellular matrix proteins and matrix metalloproteinases. In this work our previous studies of the influence of wound and burn fluids on the functional activity of cells were extended to include the effect of burn fluid on fibroblasts and keratinocytes, i. e. human skin cells present in the wound and involved in wound healing. It was shown that human burn fluid biological activity depends on the time that passed after burning, and on the correctness of healing. Migration of human fibroblasts becomes more intensive under the influence of such a fluid independently on the time of fluid sampling. Unlike, keratinocyte migration was inhibited by burn fluid sampled 1-3 days after burning but was enhanced by fluids sampled 6 days following burning. The obtained data are to be necessarily taken into consideration at burn treatment and also at transplantation of cells for healing of wounds of different nature.     

The Sensory Effects of l-Menthol on Human Skin

Psychophysical measurements were made of the sensory effects of l-menthol applied topically to the forearm under controlled thermal conditions. In the first experiment, subjects judged the intensity and quality of sensations produced by warming or cooling the skin in the presence of menthol or the vehicle. During cooling, menthol intensified cutaneous sensations and increased reports of burning. During warming, menthol intensified sensations transiently at low temperatures and weakened them lastingly at higher temperatures; the frequency of reports of burning varied with intensity. A second experiment tested the hypothesis that menthol would lower the threshold for warmth and raise the threshold for heat pain. No change in either threshold was observed. The primary sensory effects of l-menthol on hairy skin are therefore to heighten the perception of cooling and to attenuate the perception of moderate warming. In contrast with other common chemical irritants, menthol's pungent qualities appear to be enhanced by cooling and suppressed by warming; this suggests that its sensory irritancy may be attributable to the stimulation of a population of high-threshold cold fibers or cold-sensitive nociceptors.     

Pannexin 2 is expressed in murine skin and promotes UVB-induced apoptosis of keratinocytes

Pannexins (PANX) are a family of three channel-forming membrane glycoproteins expressed in the skin. Previous studies have focused on the role of PANX1 and PANX3 in the regulation of cellular functions in skin cells while PANX2, the largest member of this protein family, has not been investigated. In the current study, we explored the temporal PANX2 expression in murine skin and found that one Panx2 splice variant (Panx2-202) tends to be more abundant at the protein level and is continuously expressed in developed skin. PANX2 was detected in the suprabasal layers of the mouse epidermis and up-regulated in an in vitro model of rat epidermal keratinocyte differentiation. Furthermore, we show that in apoptotic rat keratinocytes, upon UV light B (UVB)-induced caspase-3/7 activation, ectopically overexpressed PANX2 is cleaved in its C-terminal domain at the D416 residue without increasing the apoptotic rate measured by caspase-3/7 activation. Notably, CRISPR-Cas9 mediated genetic deletion of rat Panx2 delays but does not impair caspase-3/7 activation and cytotoxicity in UVB-irradiated keratinocytes. We propose that endogenous PANX2 expression in keratinocytes promotes cell death after UVB insult and may contribute to skin homeostasis.     

云雾山典型草原火烧不同恢复年限土壤化学性质变化

云雾山典型草原处于黄土高原半干旱地区,也是草原火灾多发区,试验比较了未烧地与新烧地、火烧后3 a和火烧后11 a土壤有机碳(SOC)、全N、全P和速效K含量的变化过程。测量的土壤深度为50 cm,每10 cm一层,比较了4个样地0—10 cm、10—20 cm、20—30 cm、30—40 cm、40—50 cm土壤养分的变化。结果表明:(1)新烧地土壤剖面各层SOC、全N、全P和速效K含量都显著高于未烧地。(2)火烧后3 a样地土壤剖面各层SOC、全N、全P和速效K含量与未烧地差异不显著。(3)火烧后11 a样地土壤剖面各层全N含量都显著高于未烧地,SOC、全P和速效K含量除了0—10 cm层与未烧地差异不显著外,其它土层均显著增加。(4)4个样地的土壤剖面各层从上到下SOC、土壤全N、全P和速效K含量呈递减趋势。(5)3个火烧样地土壤表层(0—10 cm)的pH值和未烧地差异不显著。     

Quantitative determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides in rat skin.

An assay method for determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (ChOOHs) in rat skin using high-performance liquid chromatography (HPLC) with a chemiluminescence detector has been developed. In the assay method, free form and free plus ester forms of ChOOHs could be separately determined by HPLC in combination with the treatment of a tissue extract by cholesterol esterase. Lower limits of quantitation for cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides were 0.2, 0.1, and 0.5 nmol/g skin, respectively. This assay method showed that (i) good absolute recoveries of ChOOHs from rat skin (80-90% of radiolabeled ChOOHs added to rat skin); (ii) negligible autoxidation of cholesterol caused by the assay procedure (<9.4x10(-5)% of radiolabeled cholesterol added to rat skin); and (iii) good correlation between ChOOHs added to rat skin and ChOOHs determined, indicating this assay method is applicable to quantify ChOOHs in rat skin. By using this assay method, we observed that (i) cholesterol 5alpha-hydroperoxide was detected in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation; (ii) concentrations of cholesterol 7-hydroperoxides in skin of rats in an ambient light room were not significantly different from those in a dark room for 12 weeks; and (iii) ultraviolet light B irradiation markedly enhanced the concentrations of cholesterol 7-hydroperoxides in the skin of rats.     

Novel cyclosporin derivatives featuring enhanced skin penetration despite increased molecular weight

Topical cyclosporin A (CsA, 1) is not effective in the treatment of skin diseases, due to its low skin penetration. Following a prodrug strategy, a series of novel derivatives of 1 and of 2-[O-(2-hydroxyethyl)-d-Ser(8)]-CsA (SDZ IMM 125, 5) with potentially enhanced skin penetration properties were synthesized, in order to achieve higher levels of the active parent drugs in the skin. Permeation through skin and prodrug/drug levels in the skin were measured in vitro using rat and human skin. Introduction of a polar side chain, either in the form of a positively charged quaternary amine, a negatively charged phosphate or sulfate, or an amphiphilic phosphocholine moiety, generally increased permeability. Maximal increase in permeability through skin relative to CsA was up to 300-fold with rat skin, and up to 16-fold with human skin. Penetration into skin, as evaluated by measurement of prodrug/drug concentrations in the skin after 48 h, could be enhanced up to 14-fold (rat and human skin). Increases of permeation rates and skin concentrations showed no strict correlation. Using the phosphate 10 as prodrug, a 2.5-fold higher concentration of the active parent compound (5) could be achieved in rat skin as when administering 5 itself. The results demonstrate that in contrast with the '500 Dalton rule', which postulates poor skin penetration of molecules larger than 500 Da, high skin permeation can be achieved also with compounds of a molecular weight in the range between 1200 and 1600 Da. Results also indicate that in principle higher skin levels of active drug can be attained with a prodrug strategy in this class of compounds.     

Tryptophan 2,3-dioxygenase activity in rat skin

We have found an enzyme system that catalyzes the conversion of L-tryptophan to L-kynurenine, presumably via L-formylkynurenine, in soluble and insoluble fractions of rat skin. The enzymatic activity was stimulated by hematin, ascorbate, and catalase, but not by methylene blue. Highest activity was located in the skin of the dorsal posterior region and lowest activity in the abdominal region. The activity in plucked (depilated) skin was only about 25% of that obtained from unplucked (depilated) tissue of the same region. D-Tryptophan, 5-hydroxytryptophan, and tryptamine were not degraded by the skin enzyme and the Km for L-tryptophan determined with the crude enzyme was 1 microM. The decycling activity of rat skin and liver for L-tryptophan began to be stimulated after birth and reached the highest level at 6 weeks. But, 1 week later, most of the skin activity suddenly disappeared and the low level continued at least until 12 weeks. In contrast, the hepatic enzyme did not change so drastically. These findings suggest that an enzyme that catalyzes L-tryptophan to L-kynurenine via L-formylkynurenine is present in rat skin.     

Wildfires disturb the natural skin microbiota of terrestrial salamanders

Environmental change can disturb natural associations between wildlife and microbial symbionts, in many cases to the detriment of host health. We used a North American terrestrial salamander system to assess how the skin microbiota of amphibians responds to wildfires. In northern California's redwood/oak forests, we assessed how recent wildfires affected the skin microbiota of three different salamander species (Taricha sp., Batrachoseps attenuatus, and Ensatina eschscholtzii) over two different sampling seasons in 2018 and 2021. We found species-specific responses to wildfire disturbance on the alpha diversity of the skin microbiota of terrestrial salamanders, although burning in general altered the composition of the skin microbiota. The effect of burning on alpha diversities and body condition indices varied by sampling season, suggesting an additional effect of annual climatic conditions on body condition and skin microbiota response. We tested all salamanders for Batrachochytrium dendrobatidis and found four infected individuals in 2018 and none in 2021. Our study documents correlations in the skin microbiota response to an increasing source of disturbance in western North American ecosystems. In addition, our results highlight the need to consider the effects of increased wildfire regimes/intensities and longitudinal effects on wildlife-associated microbiota and animal health.     

PITFALLS IN THE USE OF RAPID FREEZING FOR STOPPING BRAIN AND SPINAL CORD METABOLISM IN RAT AND MOUSE

The rate at which the metabolism is stopped by means of freezing in Freon-12 (–150°C) was studied in various areas of the rat and mouse CNS, using changes in temperature and levels of glucose and lactate as parameters for this rate. The rat cerebral cortex was frozen after 0.5 min while the hypothalamus reached 0°C after more than 1.5 min. The skin on the skull was found to be the most important temperature isolator for the cortex. Substrate levels can be studied in this area only if this piece of skin is removed previously. In the mouse, the cerebral cortex was frozen after 6 s, the hypothalamus after 0.5 min. The lumbar level of the mouse spinal cord was frozen after 15 s, the cervical level only after 47 s. Liquid nitrogen alone cooled the mouse cerebral cortex at least as fast as did Freon at its melting point. A gradual decrease from dorsal to ventral was observed in the glucose level of the molecular layer of the mouse cerebellum. The existence of a freezing front, moving slowly from dorsal to ventral, and its consequences for the measured levels of biologically labile substrates, are discussed.     

Aboveground plant biomass,carbon, and nitrogen dynamics before and after burning in a seminatural grassland of Miscanthus sinensis in Kumamoto,Japan

Although fire has been used for several thousand years to maintain Miscanthus sinensis grasslands in Japan, there is little information about the nutrient dynamics in these ecosystems immediately after burning. We investigated the loss of aboveground biomass; carbon (C) and nitrogen (N) dynamics; surface soil C change before and after burning; and carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O) fluxes 2 h after burning in a M. sinensis grassland in Kumamoto, Japan. We calculated average C and N accumulation rates within the soil profile over the past 7300 years, which were 58.0 kg C ha^?1 yr^?1 and 2.60 kg N ha^?1 yr^?1, respectively. After burning, 98% of aboveground biomass and litter were consumed. Carbon remaining on the field, however, was 102 kg C ha^?1. We found at least 43% of C was possibly lost due to decomposition. However, remaining C, which contained ash and charcoal, appeared to contribute to C accumulation in soil. There was no difference in the amount of 0–5 cm surface soil C before and after burning. The amount of remaining litter on the soil surface indicated burning appeared not to have caused a reduction in soil C nor did it negatively impact the sub‐surface vegetative crown of M. sinensis. Also, nearly 50 kg N ha^?1 of total aboveground biomass and litter N was lost due to burning. Compared with before the burning event, postburning CO₂ and CH₄ fluxes from soil appeared not to be directly affected by burning. However, it appears the short time span of measurements of N₂O flux after burning sufficiently characterized the pattern of increasing N₂O fluxes immediately after burning. These findings indicate burning did not cause significant reductions in soil C nor did it result in elevated CO₂ and CH₄ emissions from the soil relative to before the burning event.     

The immediate effect of burning and ash-fertilization

Summary Changes in the vegetation and soil store of major nutrients were followed under the local practice of shifting cultivation. Changes in the soil store of N, P, K, Ca, Mg and Na were recorded under recently cleared forest before, 24 hours, and 40 days after the burning of vegetation of known biomass and chemical composition.The immediate increase of K and the delayed increase of P in the upper 50 cm—although equal to the content of the above-ground vegetation before burning—is not to be explained by the ash-fertilization alone. The immediate increase of K after burning was not observed after 40 days at any depth, and less Ca and Mg were found in the topsoil immediately after the burning and after 40 days, than before. Apparently mobilization and/or diffusion due to heat are important factors, even in the deeper horizons.     

Reinnervation of experimental superficial wounds in rats

Sensory reinnervation of a superficial skin wound in the rat was studied by labeling sensory axons with anterogradely transported wheat germ agglutinin-horseradish peroxidase. Reinnervation starts after 3 days from the edge of the wound as well as from beneath the wound. About 2 weeks after the production of the wound, some hyperinnervation appears to be present, but after a few additional weeks, the innervation pattern is essentially normal. The results indicate that structural recovery of sensory axons is rapid and probably complete when skin wounds heal with no or minimal scar formation.