Characterization of the living skin equivalent as a model of cutaneous re-epithelialization

The living skin equivalent, a three-dimensional organotypic model, has been widely used to investigate many aspects of cutaneous biology. However, there are relatively few studies assessing how faithfully the skin equivalent reproduces normal skin biology. The skin equivalent was fabricated by seeding human epidermal keratinocytes onto the upper surface of a hydrated collagen lattice populated with human dermal fibroblasts and subsequently raised to the air-liquid interface where keratinocyte stratification and differentiation led to the formation of a tissue which showed many common morphological features to that of normal skin. Histology and immunohistochemical detection of keratinocyte integrins and matrix metalloproteinases (MMPs) were used as cytological markers to assess the accuracy of the model during cutaneous re-epithelialization. Analysis of expression of keratinocyte integrins revealed that whilst there were a number of similarities to normal skin, skin equivalent keratinocytes appeared to be 'activated' and hyper-proliferating. Wounding of the skin equivalent, by complete bisection, induced re-epithelialization from both wound edges within 8-12 h, which completely restored the epidermis within 4 days. This migration, like that in vivo, was associated with nascent expression of MMPs and upregulation of certain integrins. However, whilst integrin expression, was similar to in vivo re-epithelialization, there were subtle differences in the level of expression and distribution of certain integrins.     

The effect of anodal/cathodal biphasic electrical stimulation on insulin release

We studied the effects of electrical stimulation on insulin release from rat insulinoma (INS-1) cells. The anodal/cathodal biphasic stimulation (ACBPS) electrical waveform resulted in a voltage- and stimulation duration-dependent increase in insulin release. ACBPS elicited insulin release both in the presence and absence of glucose. Basal and ACBPS-induced insulin secretion could be inhibited by mitochondrial poisons and calcium channel blockers, indicating that insulin release was dependent on adenosine triphosphate (ATP) and the influx of calcium. ACBPS parameters that released insulin caused no detectable plasma membrane damage or cytotoxicity, although temporary morphological changes could be observed immediately after ACBPS. ACBPS did not alter the plasma membrane transmembrane potential but did cause pronounced uptake of MitoTracker Red into the mitochondrial membrane, indicating an increased mitochondrial membrane potential. While the ATP:ADP ratio after ACBPS did not change, the guanosine triphosphate (GTP) levels increased and increased GTP levels have previously been associated with insulin release in INS-1 cells. These results provide evidence that ACBPS can have significant biological effects on cells. In the case of INS-1 cells, ACBPS promotes insulin release without causing cytotoxicity.     

The effect of pulsed electromagnetic fields on secondary skin wound healing: an experimental study

A variety of pulsed electromagnetic fields (PEMFs) have already been experimentally used, in an effort to promote wound healing. The aim of the present study was to investigate the effects of short duration PEMF on secondary healing of full thickness skin wounds in a rat model. Full thickness skin wounds, 2 by 2 cm, were surgically inflicted in two groups of male Wistar rats, 24 animals each. In the first group (experimental group - EG), the animals were placed and immobilized in a special constructed cage. Then the animals were exposed to a short duration PEMF for 20 min daily. In the second group (control group - CG), the animals were also placed and immobilized in the same cage for the same time, but not exposed to PEMF. On days 3, 6, 9, 12, 18, and 22, following the infliction of skin wounds, the size and healing progress of each wound were recorded and evaluated by means of planimetry and histological examination. According to our findings with the planimetry, there was a statistically significant acceleration of the healing rate for the first 9 days in EG, whereas a qualitative improvement of healing progress was identified by histological examination at all time points, compared to the control group.     

The effect of lipoic acid on wound healing in a full thickness uterine injury model in rats

Aim of this study was to investigate the effects of lipoic acid on uterine wound healing by immunohistochemical and biochemical assay in a rat uterine horn model with full thickness injury. Thirty-two female Wistar albino rats were randomised into five groups: Control group, with no intervention; uterine scar group 15days (US15d), uterine scar group 15 days + alpha lipoic acid (ALA) (US15d + ALA), uterine scar group 30 days (US30d) and uterine scar group 30 days + ALA (US30 days + ALA). After uterine incision 100 mg/kg of ALA was administered by oral gavage for either 15 or 30 days. Vascular endothelial growth factor (VEGF) and alpha smooth muscle actin (α-SMA) distribution were evaluated by immunohistochemical methods in tissue and ELISA methods in tissue homogenate. The percentage of α-SMA positive area in US15d + ALA and US30d + ALA groups was significantly higher than US15 and US30d groups. The percentage of VEGF positive area in US15d + ALA group was significantly higher than US15d group and US30d + ALA group was significantly higher than US30d group. Biochemically, α-SMA was significantly higher in the US15d + ALA group when compared to US15d group and higher in US30d + ALA group when compared to US30d group. VEGF was significantly higher in US15d + ALA and US30d + ALA groups when compared to US15 and US30d groups. In conclusion, ALA was found to be effective in enhancing wound healing in uterine full thickness injury.     

Calculation of electric fields and currents induced in a millimeter-resolution human model at 60 Hz using the FDTD method

The finite-difference time-domain (FDTD) method has previously been used to calculate induced currents in anatomically based models of the human body at frequencies ranging from 20 to 915 MHz and resolutions down to about 1.25 cm. Calculations at lower frequencies and higher resolutions have been precluded by the huge number of time steps that would be needed in these simulations. This paper describes a method used to overcome this problem and efficiently calculate induced currents in an MRI-based, 6-mm-resolution model of the human under a high-voltage transmission line. This model is significantly higher resolution than the 1.31-cm-resolution model previously used; therefore, it can be used to pinpoint locations of peak current densities in the body. Proposed safety guidelines would allow external electric fields of 10 kV/m and 25 kV/m for exposure to 60 Hz fields of the general public and workers, respectively. For this external electric field exposure of 10 kV/m, local induced current densities as high as 20 mA/m² are found in the head and trunk with even higher values (above 150 mA/m²) in the legs. These currents are considerably higher than the 4 or even 10 mA/m² that have been suggested in the various safety guidelines, thus indicating an inconsistency in the proposed guidelines. In addition, several ratios of E/H typical of power line exposures were examined, and it was found that the vertical electric field couples strongly to the body, whereas the horizontal magnetic field does not. Bioelectromagnetics 19:293–299, 1998. © 1998 Wiley-Liss, Inc.     

重组融合多肽hEGF-AWRK6的纯化及对小鼠烫伤感染创面愈合的影响

旨在研究重组融合多肽hEGF-AWRK6(EK)对烫伤模型小鼠感染创面的治疗功效。采用大肠杆菌表达系统表达、纯化融合多肽EK,抑菌实验检测EK抑菌活性;构建小鼠II度烫伤和铜绿假单胞菌感染模型,实验组创面滴注EK(30 mg/L),以PBS、庆大霉素(30 mg/L)、烫伤膏(10 mg/L)为对照,计算烫伤后创面愈合率及菌落数;伤后10 d,取各组小鼠的伤口及周边皮肤进行HE染色及胶原蛋白的Western blotting检测,分析创面病理组织结构。实验结果纯化得到了具有抑菌活性的重组表达融合肽EK;伤后6 d始,EK组小鼠创面菌落数少于对照组,差异极显著(P0.01);EK组小鼠烫伤愈合率显著高于PBS对照组(P0.01);与对照组相比,EK组小鼠创面真皮层细胞排列规则,再上皮化较快,毛囊生长较多,Ⅰ型胶原蛋白表达显著增加。结果表明EK具有抑制小鼠烫伤创面感染、促创面愈合功效,具有开发成为治疗烧伤药物的潜力。     

The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.     

The effect of the glycolipoprotein extract (G‐90) from earthworm Eisenia foetida on the wound healing process in alloxan‐induced diabetic rats

Diabetes is now regarded as a major public health problem. The number of patients is estimated to increase to over 439 million cases by 2030. One of the major health clinical problems in patients with diabetes patients is impaired wound healing. Diabetic foot ulcer is a major complication of diabetes mellitus in 12 to 25% of patients, which increases the risk of damage in the limbs or amputation. The earthworm Eisenia foetida glycolipoprotein (as known G‐90) is a blend of macromolecules with some biological properties including mitogenicity, anticoagulation, fibrinolysis, bacteriostatic and antioxidatiaon. Given the biological properties of G‐90, this study was conducted to investigate the effect of extract obtained from the homogenate of Eisenia foetida (G‐90) on the wound healing process in alloxan‐induced diabetic rats. The results of the present study revealed that treatment by using G‐90 can speed up the wound healing process, which is exactly similar to the effect of D‐panthenol treatment in rats. These findings also demonstrated that G‐90 treatment decreases the risk of infection in the wound site compared with D‐panthenol treatment. In addition, histological analysis indicated that a better extracellular matrix formation with increased fibroblast proliferation, neovascularization, collagen synthesis and early epithelial layer formation was observed in G‐90 treated group. Therefore, the G‐90 could be considered as a new wound healing agent introducing promising therapeutic approaches in both human and veterinary medicine. Copyright © 2016 John Wiley & Sons, Ltd.     

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts

BACKGROUND INFORMATION: Different in vitro models, based on co-culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft-tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound-specific conditions. In order to develop a suitable in vitro model, we characterized co-cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co-culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin-1 and ED-A-fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored. RESULTS: The comparison of the HDMEC-NHDF co-culture with the respective mono-cultures revealed that HDMECs showed a lower proliferation activity when co-cultured, but their number was stable throughout a period of 48 h. NHDFs in co-culture were slightly slower at proliferating than in the mono-culture. The MF population was stable for 48 h in the co-culture, as well as in NHDF mono-culture. Co-cultures and HDMEC mono-cultures were characterized by a slower migration rate than NHDF mono-cultures. Hypoxia decreased both cell proliferation and migration in the mono-cultures, as well as in the co-cultures, indicating the general suitability of the assay. Exclusively, in co-cultures well-defined cell clusters comprising HDMECs and MFs formed at the edges of the in vitro wounds. CONCLUSIONS: On the basis of these results, the CCSWMA developed using co-cultures, including HDMECs, NHDFs and MFs, proved to be an effective tool to directly visualize cellular interaction. Therefore, it will serve in the future to evaluate the influence of wound-healing-related factors in vitro, as shown for hypoxia in the present study.     

Test of a digestion optimization model: effect of variable-reward feeding schedules on digestive performance of a migratory bird

Birds on migration often alternate between feeding and nonfeeding periods, in part because food resources may be patchily distributed and in part because birds on migration may adopt a risk-prone foraging strategy characterized by selection of variable rather than constant food rewards. Optimal digestion models predict that increases in intermeal interval like those encountered by some migratory birds should result in longer retention time of digesta and higher digestive efficiency if birds are maximizing their rate of energy intake. We tested these predictions by comparing residence time of digesta and extraction efficiency of lipid for captive yellow-rumped warblers (Dendroica coronata) feeding adlibitum and when we added intervals of time when the birds received no food. We increased the likelihood that the warblers were maximizing their rate of energy intake by increasing light levels during spring to induce hyperphagia (treatment birds (16L:8D light: dark cycle) ate 2.13 ± 0.14 g dry food day⁻¹ (n = 8) while control birds (10L:14D) ate 1.25 ± 0.03 g dry food day⁻¹ (n = 6)). Treatment birds offered food only every other 2–3 h ate 50% more during the 4-h test period than when they were always feeding adlibitum. Despite these differences in food intake, extraction efficiency of glycerol trioleate remained high and constant (93%), and mouth-to-anus total mean retention time (TMRT) did not change (overall mean: 54.8 ± 6.0 min). Residence time of lipid in the stomach increased whereas residence time of lipid in the intestine decreased when birds fed only every other 2–3 h compared to when birds always fed ad libitum. None of the results were consistent with the predictions of the optimal digestion model unless we assume that birds were minimizing their feeding time rather than maximizing their rate of energy gain. Furthermore, the ability of yellow-rumped warblers to maintain high extraction efficiency with no change in TMRT suggests some spare digestive capacity when food intake increases by as much as 50%. Received: 14 June 1997 / Accepted: 20 November 1997     

Simulation of the effect of an external GHz electric field on the potential energy profile of Ca2+ ions in the selectivity filter of the CaVAb channel

Ca_V channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca²⁺ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion‐selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the Ca_VAb channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca²⁺ ions in the Ca_VAb channels in presence and absence of an external field. Exposing Ca_VAb channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca²⁺ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca²⁺ ions binding in the selectivity filter may interrupt the mechanism of Ca²⁺ ion conduction, and leads to a reduction of Ca²⁺ ion permeation through the Ca_VAb channel.     

连翘酯苷对拟AD复合动物模型小鼠学习记忆的改善作用及其机制研究

目的以β淀粉样蛋白损伤自然衰老小鼠建立一种新的复合式老年痴呆(AD)小鼠模型,观察连翘酯苷对复合模型学习记忆障碍的改善作用,并对其机制进行初步探讨。方法采用14月龄C57BL/6小鼠侧脑室注射Aβ25-35形成拟AD复合模型;Morris水迷宫实验观察小鼠学习记忆能力,实验结束取小鼠脑组织用放射免疫分析法检测TNF-α及IL-1的含量;Western blot方法检测GFAP蛋白表达,化学比色法测定ChAT、AchE、SOD酶活性及MDA的含量。结果水迷宫实验中连翘酯苷组可显著改善小鼠的学习记忆能力(P<0.05)。作用机制研究发现:连翘酯苷能降低TNF-α、IL-1的含量(P<0.05),抑制GFAP蛋白表达。提高ChAT、SOD酶活力,降低AchE活性及MDA的含量(P<0.05,P<0.01)。结论连翘酯苷对拟AD复合动物模型学习记忆的改善作用可能与抑制脑内炎症反应,调节胆碱能系统,抗氧化作用等有关。     

The use of an aeration system to prevent thermal stratification of a freshwater impoundment and its effect on downstream fish assemblages

Warm-water riverine fish assemblages were investigated downstream of an impoundment before and after thermal stratification and the associated cold-water pollution was prevented using an aeration system. Temperatures below the dam significantly increased after installation of the aeration system and this correlated with an increased abundance and greater number of species downstream. Overall, aeration appeared to be beneficial for both the lake (upstream) and the downstream riverine environments.     

Lack of a co-promotion effect of 60 Hz rotating magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in F344 rats

The present study was conducted to investigate the possible effect of 60 Hz magnetic fields as promoters of brain tumors initiated transplacentally by ethylnitrosourea (ENU) in F344 rats. One hundred twenty mated animals were divided into six different groups and exposed in utero on day 18 of gestation to a single intravenous dose of either Saline (vehicle control, Group I), or ENU 10 mg/kg (Groups II-VI). In the present study, a total of 480 offspring was used. The offspring in group II were given no further treatment while the offspring in Groups III-VI were exposed to four different intensities of magnetic fields. Animals received exposure to 60 Hz magnetic field at field strengths of 0 Tesla (sham control, T1, Group III), 5 muT (T2, Group IV), 83.3 muT (T3, Group V), or 500 muT (T4, Group VI), for 21 h/day from the age of 4 weeks to the age of 32 or 42 weeks. At histopathological examination, tumors of the nervous system were seen in all the ENU-treated groups. The tumor incidence of the ENU group at 32nd and 42nd week necropsy was higher than that of the vehicle control group. The incidence of glial tumors at 42nd week necropsy was higher than the 32nd week necropsy. However, there were no differences in the tumor incidence between the sham control (T1) and ENU + magnetic field exposure groups (T2-T4). In conclusion, there was no evidence that exposure of offspring to 60 Hz at magnetic field strengths up to 500 muT to the age of 32 or 42 weeks promoted ENU-initiated brain tumors in rats.     

The influence of continuous exposure to 50 Hz electric field on nerve regeneration in a rat peroneal nerve crush injury model

The effect of power frequency electric field (EF) on nerve regeneration was investigated on a rat peroneal nerve crush injury model. The animals were assigned to three groups: 50 Hz EF and Static EF groups were exposed at 10 kV/m. The sham group was kept in the same setting without any EF applications. EF was uninterruptedly applied for 21 days postoperatively. Repeated measures analysis of daily walking tracks during EF exposure demonstrated lower toe spread recovery (TSR) in the 50 Hz EF group. Significant difference across the groups was found only at days 7, 8, 12, 16, 17, 20, and 21 when TSR was analyzed for each measurement time. Print length recovery and peroneal function index did not differ across the groups. Walking track parameters were found to recover to their baseline values by day 28 in all groups. Day 14 but not day 21 measurements revealed smaller nerve cross-sectional area, lower total regenerating axon area, and higher mean myelin debris area in 50 Hz EF group. Both day 14 and 21 measurements revealed higher total myelin debris area, lower EDL muscle weight, and lack of significant enlargement in nerve cross-section distal to the injury, compared to the normal counterpart in 50 Hz EF group. All differences were in keeping with lower rates of Wallerian degeneration and nerve regeneration in 50 Hz EF group. When walking track, histomorphometry and muscle weight are considered individually, their differences across the groups may appear to be subtle to derive a conclusion for a 50 Hz EF effect. However, their concordance with each other in direction of effect suggests that continuous 50 Hz EF exposure has a weak effect that is detrimental mostly to the rate of early nerve regeneration in this axonotmetic injury model. Recovery of walking tracks was not different between Static EF and Sham groups. This suggests that the surface charges that may indirectly affect walking behaviors of the rats, do not account for the lower recovery of TSR in 50 Hz EF group. Differences in nerve regeneration between 50 Hz EF and Static EF groups suggests that electric induction may be required for pure EF effects even though the estimated density of induced fields is not above the endogenous background level for the 50 Hz EF exposure in this study.     

Effect of photoperiod and host distribution on the horizontal transmission of Isaria fumosorosea (Hypocreales: Cordycipitaceae) in greenhouse whitefly assessed using a novel model bioassay

A model bioassay was used to evaluate the epizootic potential and determine the horizontal transmission efficiency of Isaria fumosorosea Trinidadian strains against Trialeurodes vaporariorum pharate adults under optimum conditions (25±0.5°C, ~100% RH) at two different photoperiods. Untreated pharate adults were arranged on laminated graph paper at different distributions to simulate varying infestation levels on a leaf surface. Four potential hosts were located 7, 14 and 21 mm away from a central sporulating cadaver simulating high, medium and low infestation levels, respectively. Percent hosts colonized were recorded 7, 12, 14 and 21 days post-treatment during a 16- and 24-h photophase. After 21 days, mean percent hosts colonized at the highest, middle and lowest infestation levels were 93 and 100%, 22 and 58%, 25 and 39% under a 16- and 24-h photophase, respectively. From the results, it was concluded that the longer the photophase, the greater the percentage of hosts colonized, and as host distance increased from the central sporulating cadaver, colonization decreased. The use of this novel model bioassay technique is the first attempt to evaluate the epizootic potential and determine the horizontal transmission efficiency of I. fumosorosea Trinidadian strains under optimal environmental conditions at different photoperiods. This bioassay can be used to assess horizontal transmission efficiency for the selection of fungi being considered for commercial biopesticide development.